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PNA Chemistry

for the Expedite 8900 Nucleic Acid Synthesis System

Users Guide

Copyright 2001, Applied Biosystems. All rights reserved.


Printed in the United States of America. This book or parts thereof may not be reproduced in any form without the written
permission of the publishers.
For Research Use Only. Not for use in diagnostic procedures.
NOTICE
Information in this document is subject to change without notice and does not represent a commitment by Applied
Biosystems. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This manual is
believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental
or consequential damages in connection with or arising from the use of this manual.
U.S and foreign patents pending.
Applied Biosystems grants the customer of synthons in the accompanying package a license to manufacture PNA oligomers
for internal use only. Sale of PNA oligomers manufactured with the synthons, or resale of the synthons, is prohibited and
may constitute patent infringement.
Applied Biosystems is a registered trademark, and Expedite is a trademark, of Applera Corporation or its subsidiaries in the
U.S. and certain other countries.
Waters and Delta-Pak are trademarks of Waters Corporation. Millipore is a trademark of Millipore Corporation. YMC is a
trademark of YMC, Inc.

WARNING
For continued protection against fire hazard, replace fuses with those of the same
type and rating.

AVERTISSEMENT
Remplacez les fusibles par ceux de mme type et puissance pour viter les risques
dincendie.

WARNING
Most of the reagents and solvents used in PNA and nucleic acid synthesis are
hazardous. Wear a lab coat, gloves and eye protection when handling reagents.
Adequate ventilation is essential and working under a fume hood is recommended.
Consult Appendix A, Reagent Safety, for reagent safety considerations.

AVERTISSEMENT
La plupart des ractifs et solvants employs en synthse PNA en d'acides
nucliques sont dangereux. Portez une blouse, des gants et des lunettes de
protection lorsque vous manipulez des ractifs. Une ventilation adquate est
ncessaire et il est recommand de travailler sous une hotte. Consultez la partie
Appendix A, Reagent Safety, de ce manuel.

US Safety and EMC (Electromagnetic Compliance) Standards


Safety
This instrument has been tested to and complies with standard ANSI/UL 1262, Electrical
Equipment for Laboratory Use; Part 1: General Requirements, 1st Edition. It is an ETL Testing
Laboratories listed product.

EMC
This device complies with Part 15 of the FCC Rules. Operation is subject to the following two
conditions: (1) This device may not cause harmful interference, and (2) this device must accept
any interference received, including interference that may cause undesired operation.

Warning: Changes or modifications to this unit not expressly approved by the party responsible
for compliance could void the users authority to operate the equipment.
Note: This equipment has been tested and found to comply with the limits for a Class A digital
device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable
protection against harmful interference when the equipment is operated in a commercial
environment. This equipment generates, uses, and can radiate radio frequency energy and, if not
installed and used in accordance with the instruction manual, may cause harmful interference to
radio communications. Operation of this equipment in a residential area is likely to cause harmful
interference in which case the user will be required to correct the interference at his own expense.
Note: Shielded cables must be used with this unit to ensure compliance with the Class A FCC
limits.

Canadian Safety and EMC (Electromagnetic Compliance) Standards


Safety
This instrument has been tested to and complies with standard C22.2 No. 151, Safety
Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use; Part 1:
General Requirements. It is an ETL Testing Laboratories listed product.

Scurit
Cet instrument a t vrifi avec la norme C22.2 No. 151, Spcifications de scurit du matriel
lectrique utilis pour les mesures, les contrles et dans les laboratoires ; Partie 1 : Spcifications
gnrales, et il est conforme cette norme. Cest un produit homologu par les ETL Testing
Laboratories.

EMC
This Class A digital apparatus meets all requirements of the Canadian Interference-Causing
Equipment Regulations.
Cet appareil numrique de la classe A respecte toutes les exigences du Rglement sur le
materiel brouilleur du Canada.

European Safety and EMC (Electromagnetic Compliance) Standards


Declaration of Conformity

Application of Council Directive(s):

73/23/EEC Low Voltage


89/336/EEC Electromagnetic Compatibility

Standard(s) to which conformity is


declared:

IEC 1010-1 Safety Requirements for Electrical Equipment for


Measurement, Control and Laboratory Use
EN55011:1991, Group 1, Class B Radiated Emissions
EN50082-1:1991 Generic Immunity

Manufacturers Name:

Applied Biosystems

Manufacturers Address:

500 Old Connecticut Path


Framingham, Massachusetts 01701 USA

Type of Equipment:

Laboratory Instrumentation

Model Name & Number:

Expedite Nucleic Acid Synthesis System, Models 8905 and 8909

Part Number:

GEN600041 or GEN600042

Serial Number:

FX???8200?
Question marks represent date and manufacturing codes that are part of the serial number.

Year of Manufacture:

1995 and later

Table of Contents

Table of Contents
How to Use This Guide ............................................................................ ix
Chapter 1 Introduction to PNA Synthesis
1.1
1.2
1.3
1.4

......................................... 1-2
PNA Synthesis on the Expedite 8900 System ................................ 1-5
Characteristics of PNA Oligomers ............................................... 1-7
Guidelines for Sequence Design of PNA Oligomers ........................ 1-10

Peptide Nucleic Acid (PNA) Synthesis

1.4.1

General Guidelines ...............................................................1-10

1.4.2

Triplex Formation ..................................................................1-13

1.4.3

Strand Invasion .....................................................................1-14

Chapter 2 Installing the PNA Software


.............................................................................. 2-2

2.1

Overview

2.2

Installing Instrument Software .................................................... 2-3

Chapter 3 Performing a Synthesis


.............................................................................. 3-2
3.2
Powering Up the System .......................................................... 3-3
3.3
Selecting the Chemistry ............................................................ 3-3
3.4
Entering the Sequence ............................................................. 3-5
3.5
Preparing PNA Reagents ......................................................... 3-6
3.6
Flushing the System When Switching Chemistries .......................... 3-8
3.7
Loading PNA Reagents ........................................................... 3-11
3.8
Priming ................................................................................ 3-15
3.9
Installing Reaction Columns ..................................................... 3-19
3.10 Performing the Synthesis ......................................................... 3-19
3.1

Overview

Expedite 8900 PNA Chemistry Users Guide

vii

Table of Contents

Chapter 4 Post-Synthesis Procedures


..................................
Drying the Resin .....................................................................
Cleavage and Deprotection .......................................................
Analysis, Purification, Handling, and Quantitation ...........................

4.1

Removing the Final Fmoc Protecting Group

4.2
4.3
4.4

4-2
4-2
4-3
4-6

4.4.1

PNA Analysis ......................................................................... 4-6

4.4.2

Reversed-Phase Purification of PNA Oligomers ..................... 4-8

4.4.3

PNA Handling ......................................................................4-10

4.4.4

PNA Quantitation .................................................................. 4-11

Labeling PNA Oligomers .......................................................... 4-12

4.5

4.5.1

Labeling of Support-bound PNA ............................................4-12

4.5.2

Labeling PNA Oligomers in Solution .....................................4-15

Chapter 5 Troubleshooting and Maintenance


5.1

Troubleshooting ...................................................................... 5-2

5.2

Maintenance .......................................................................... 5-4

Appendix A Reagent Safety ............................................................. A-1


Appendix B Ordering Information ............................................... B-1
Appendix C PNA Reference Information ................................. C-1
Appendix D Technical Support and Training ....................... D-1
Index

viii

Applied Biosystems

How to Use This Guide


Purpose of this
guide
Structure of this
guide

The Applied Biosystems PNA Chemistry for Expedite 8900


Nucleic Acid Synthesis System Users Guide describes the
synthesis of peptide nucleic acid (PNA) oligomers on the
Expedite 8900 Nucleic Acid Synthesis System.
Applied Biosystems PNA Chemistry for Expedite 8900 Nucleic
Acid Synthesis System Users Guide is divided into chapters.
Each chapter page is marked with a tab and a header to help
you locate information within the chapter.
The table below describes the material covered in each
chapter.

Chapter 1,
Introduction to PNA
Synthesis

Describes peptide nucleic acid (PNA)


synthesis and gives reference information on
PNA synthesis.

Chapter 2,
Installing the PNA
Software

Describes how to install the software.

Chapter 3,
Performing a
Synthesis

Describes how to set up and run a synthesis.

Chapter 4,
Post-Synthesis
Procedures

Provides procedures for analysis, purification,


handling, quantitation, and labeling.

Chapter 5,
Troubleshooting and
Maintenance

Provides information for troubleshooting and


maintenance.

Appendix A,
Reagent Safety

Includes chemical handling precautions.

Appendix B,
Ordering Information

Lists part numbers you need to order parts and


accessories from Applied Biosystems.

Expedite 8900 PNA Chemistry Users Guide

ix

How to Use This Guide

Appendix C,
PNA Reference
Information

Includes background information for optical


density conversion, HPLC and MALDI-TOF
methods, and waste composition.

Appendix D,
Technical Support
and Training

Describes how to contact Technical Support,


obtain technical documents and obtain
customer training information.

Related
documents

Use this document in conjunction with the Expedite 8900


Nucleic Acid Synthesis System Users Guide. This document
refers you to the Expedite 8900 Nucleic Acid Synthesis
System Users Guide for procedures that are common to
standard nucleotide synthesis and PNA synthesis.

Conventions

This guide uses the following conventions to make text easier


to understand.

General
conventions

Bold indicates user action:


Type 0 and press Enter for the remaining
fields.

Italic text denotes new or important words, and is also


used for emphasis:
Before analyzing, always prepare fresh
matrix.

Notes, Cautions,
and Warnings

A note calling out important information to the operator


appears as:

NOTE: Record your result before proceeding with the next


step.

Applied Biosystems

A caution calling out information to avoid damage to the


system or equipment appears as:

CAUTION
Changing reagent bottles during a synthesis is not
recommended. Check the reagent resources prior to
initiating a synthesis to make sure that there is a sufficient
supply.
A warning calling out information essential to the safety of the
operator appears as:

WARNING
The Expedite Cabinet weighs 102 pounds (46 kg). Two
people are required to safely lift the instrument cabinet.

Send us your
comments

We welcome your comments and suggestions for improving


our manuals. You can send us your comments in two ways:

Use the Technical Publications Customer Survey at:


www.appliedbiosystems.com/techsupport

Send e-mail to:


support@appliedbiosystems.com

Expedite 8900 PNA Chemistry Users Guide

xi

1Introduction to PNA
Synthesis

This chapter contains the following sections:


1.1 Peptide Nucleic Acid (PNA) Synthesis......... 1-2
1.2 PNA Synthesis on
the Expedite 8900 System ............................ 1-5
1.3 Characteristics of PNA Oligomers................ 1-7
1.4 Guidelines for Sequence Design of
PNA Oligomers .........................................1-10

Expedite 8900 PNA Chemistry Users Guide

1-1

Chapter 1

Introduction to PNA Synthesis

1.1 Peptide Nucleic Acid (PNA)


Synthesis
PNA oligomers

Peptide Nucleic Acid (PNA) oligomers are analogs of DNA in


which the phosphate backbone is replaced with a peptide-like
backbone. Figure 1-1 compares the structure of PNA and
DNA molecules.
5'-end

N-terminal

HO

H3N

O
P

N
H

-O

N
O

O
O

N
H

O
P

n
-O

N
O

NH2

HO

C-terminal
3'-end

PNA

PB100697

DNA

PB100698

Figure 1-1 Structure of PNA and DNA Molecules


The achiral backbone is made from N-(2-aminoethyl)-glycine
units linked by amide bonds. The backbone is uncharged. The
four standard monomers A, C, G, and T are attached to the
backbone by methylene carbonyl linkages.
Refer to Section 1.3, Characteristics of PNA Oligomers, for
additional information.

1-2

Applied Biosystems

Peptide Nucleic Acid (PNA) Synthesis

Expedite PNA
monomers

The Expedite PNA monomers (Figure 1-2) are protected for


synthesis in the following way:

The primary amino group in the monomer backbone is


protected with the 9-fluorenylmethoxycarbonyl (Fmoc)
group. The Fmoc protecting group is base-labile, and is
typically removed with 20% piperidine in DMF.

The exocyclic amino group of the monomers A, G, and


C is protected with the benzhydryloxycarbonyl (Bhoc)
group. The Bhoc group is removed at the end of the
synthesis (cleavage step) with trifluoroacetic acid (TFA)
in the presence of the scavenger m-cresol.
B=see below
O
O

N
H

OH
PB100692

Monomer

O
O

HN
N

HN
N

PB100694

PB100693

N
C monomer

A monomer

MW=701.73

MW=724.75

O
O HN
O N N
H

HN

O
PB100695

N
PB100696

G monomer

T monomer

MW=741.76

MW=506.51

Figure 1-2 Fmoc/Bhoc PNA Monomers


Expedite 8900 PNA Chemistry Users Guide

1-3

Chapter 1

Introduction to PNA Synthesis

1
O
O

N
H

O-spacer monomer
MW=384.41

OH
O
O

PB100691

Figure 1-3 Fmoc-protected O-spacer Monomer

Activation and
coupling

Fmoc monomer activation and coupling (acylation) must be


maximally efficient to ensure quality PNA products. Optimal
conditions are highly dependent on the nature of the activation
reagent and the monomer-activated species.
Expedite PNA chemistry uses HATU
(O-(7-azabenzo-triazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) as the coupling reagent. Activation is
accomplished by mixing monomer, HATU, and base solution
containing DIEA (diisopropylamine) and lutidine. HATUactivated carboxylic acids have demonstrated enhanced
coupling efficiency in peptide synthesis and are especially
suited for hindered amino acid and other difficult couplings.

1-4

Applied Biosystems

PNA Synthesis on the Expedite 8900 System

1.2 PNA Synthesis on the Expedite


8900 System
The PNA option for the Expedite 8900 Nucleic Acid Synthesis
System is software that allows you to synthesize PNA
oligomers on an Expedite Model 8909 system.

NOTE: The Expedite 8905 Nucleic Acid Synthesis System


does not have enough reservoir positions on the B-train to
support the PNA option.
The Expedite 8909 Nucleic Acid Synthesis System allows
simultaneous synthesis on two columns.
Expedite Workstation software version 2.2 or higher supports
the PNA option.
Operation during PNA synthesis is very similar to standard
operation of the Expedite Nucleic Acid Synthesis System. This
document refers you to the Expedite 8900 Nucleic Acid
Synthesis System Users Guide for procedures that are
common to PNA synthesis and DNA/RNA synthesis.

Protocol

Fmoc PNA synthesis can be performed at a 2 mole or


25 mole scale. Additional protocols may be supported in the
future.

Expedite 8900 PNA Chemistry Users Guide

1-5

Chapter 1

Introduction to PNA Synthesis

Overview of PNA
synthesis

PNA synthesis on the Expedite 8909 Nucleic Acid Synthesis


System uses the Fmoc peptide synthesis method.
This method provides PNA oligomers in high yield and allows
conjugation/continued synthesis to many other biomolecules,
in particular peptides.
The PNA synthesis cycle is outlined in Figure 1-4.
Place column
on instrument

Wash
DMF

Cleave and
deprotect

Cap
acetic anhydride
lutidine in DMF
3 minutes

Fmoc deblock
20% piperidine
in DMF
2.5 minutes

Wash
DMF

Wash
DMF
Activate and Couple
5 eq DIPEA 7.5 eq lutidine
5 eq monomer 4.5 eq HATU
in NMP/DMF (1/2)
8.25 minutes

Figure 1-4 Expedite PNA Synthesis Cycle


(2 mol Scale Protocol)

1-6

Applied Biosystems

Characteristics of PNA Oligomers

1.3 Characteristics of PNA


Oligomers
Characteristics of PNA oligomers discussed in this section
include:

Thermal stability

Thermal stability
Stronger binding independent of salt concentration
Greater sequence specificity
Strand invasion
Resistance to nucleases and proteases

PNA oligomers have properties favorable to many molecular


biology applications. One of the most impressive is the higher
thermal stability of PNA/DNA and PNA/RNA duplexes
compared to DNA/DNA and DNA/RNA duplexes. This
stronger binding is attributed to the lack of charge repulsion
between the neutral PNA strand and the DNA or RNA strand.
Typically, the Tm for a 15-mer PNA/DNA duplex is about 70C,
where the corresponding DNA/DNA duplex exhibits a Tm of
approximately 55C (pH7, 100mM NaCl). PNA also forms very
stable duplexes with RNA with a similar increase in Tm. As a
general rule, the Tm for a PNA/DNA duplex is 1C higher per
base pair as compared to the Tm of the corresponding
DNA/DNA duplex (in 100 mM NaCl).

Expedite 8900 PNA Chemistry Users Guide

1-7

Chapter 1

Introduction to PNA Synthesis

Table 1-1 shows the greater stability (reflected in the higher


Tm values) of PNA/DNA or PNA/RNA duplexes over the
corresponding DNA/DNA or DNA/RNA duplexes.

Table 1-1 Stability and Tm*


Duplex

Tm

15-mer PNA/DNA

69C

15-mer DNA/DNA

54C

15-mer PNA/RNA

72C

15-mer DNA/RNA

50C

* Reprinted with permission of Nature (1993, 365, 565567). Studies


conducted in 10mM phosphate buffer, 0.1 mM EDTA, 100mM NaCl, pH 7.0.

Stronger binding
independent of
salt concentration

Another important consequence of the neutral backbone is


that the Tm of PNA/DNA duplexes are practically independent
of salt concentration. This is in sharp contrast to the Tm of
DNA/DNA duplexes, which are highly dependent on ionic
strength.

Table 1-2 Salt Concentration and Tm*


Salt (NaCl)
Concentration

1-8

Applied Biosystems

PNA/DNA
Tm

DNA/DNA Tm

0 mM

72C

38C

140 mM

69C

56C

1000 mM

65C

65C

Reprinted with permission of Nature (1993, 365, 565567). PNA and DNA
15-mers were hybridized to a complementary DNA 15-mer in
10mM phosphate buffer, pH7, with varying salt concentrations.

Characteristics of PNA Oligomers

At low ionic strength, PNA can bind to a target sequence at


temperatures where DNA hybridization is strongly inhibited.
Hybridization of PNA can also proceed in the absence of
Mg++, a factor that further inhibits DNA/DNA duplex formation.
Adjusting the ionic strength can, therefore, be a very useful
parameter in designing procedures where competing DNA or
RNA is present in the sample or where the nucleic acid being
probed contains a high level of secondary structure.

Greater
specificity of
interaction

PNA also shows greater specificity in binding to


complementary DNA. A PNA/DNA mismatch is more
destabilizing than a mismatch in a DNA/DNA duplex. A
single mismatch in mixed PNA/DNA 15-mers lowers the Tm
by 8 to 20C (15C average). In the corresponding DNA/DNA
duplexes, a single mismatch lowers the Tm by 4 to 16C
(11C average).

Strand invasion

PNA oligomers that only contain thymine and cytosine (the


pyrimidines) often bind with a 2PNA/1DNA stoichiometry
resulting in PNA/DNA/PNA triplex formation. The triplex is
comprised of a PNA/DNA double helix (formed by WatsonCrick hydrogen bonds) with a second PNA strand lying in the
major groove of the duplex (held by Hoogsteen hydrogen
bonds).
The stability of these triplexes is so great (Tm >70C for a
10-mer) that strand invasion of DNA/DNA is possible;
binding of the PNA results in formation of a D-loop in the
double stranded DNA, where the PNA displaces one of the
DNA strands. This characteristic may potentially be used to
manipulate gene expression at the transcriptional level.

Resistance to
nucleases and
proteases

A polyamide backbone with purine and pyrimidine base side


chains is not a combination that is recognized by either
nucleases or proteases. As a result, PNA oligomers are not
susceptible to degradation by enzymes, and the lifetime of
these compounds will be extended both in vivo and in vitro. In
addition, PNA is stable over a wide pH range.

Expedite 8900 PNA Chemistry Users Guide

1-9

Chapter 1

Introduction to PNA Synthesis

1.4 Guidelines for Sequence Design


of PNA Oligomers
This section presents considerations for:

General guidelines
Triplex formation
Strand invasion

1.4.1 General Guidelines


Orientation

PNAs can form duplexes in either orientation, but the


anti-parallel orientation is strongly preferred and forms the
most regular duplex. Anti-parallel is the preferred configuration
for antisense and DNA probe type applications. By
convention, the amino-terminus of a PNA oligomer is
considered equivalent to 5' end of the DNA.

DNA 5'
PNA

3'
H2NOC

NH2

C-terminal

N-terminal

Figure 1-5 Duplex in the Anti-Parallel Orientation

Because the PNA strand is uncharged, a PNA/DNA duplex


has a higher Tm than the corresponding DNA/DNA duplex.
There will typically be an increase in Tm of about 1C per base
pair at 100 mM NaCl.

1-10

Applied Biosystems

Guidelines for Sequence Design of PNA Oligomers

Length of PNA
oligomer

For most applications an oligomer length of 12 to 17 units is


optimal. Length is primarily determined by the sequence
uniqueness required for a particular application. Applications
that require more than 25 oligonucleotides can be routinely
performed with much shorter PNA oligomers.
Long PNA oligomers (for example, 25 to 40 units typical of an
oligonucleotide probe) are rarely required. Longer PNA
oligomers, depending on the sequence, tend to aggregate and
are difficult to purify and characterize.
However, the shorter a sequence, the more specific it is.
Consequently, the impact of mismatch is greater for a shorter
sequence than a longer sequence.

Purine content

Purine-rich PNA oligomers tend to aggregate, with G-rich


oligomers having the greatest tendency. This often results in
reduced aqueous solubility.
Do not include more than six purines in any stretch of 10 units,
and do not include more than three G residues in a row.
GAT TAG CAG TCT ACG
Good design (fewer than 6 of 10 are purines)

ATT AGG GGC ATC TAC


Poor design (more than three G residues in a row)

CTA GAT AGC AGG TTC*


Poor design (8 of 10 bases are purines)

*Notice that the complement, the probe for the other strand
includes a good design sequence:
GAA CCT GCT ATC TAG

Figure 1-6 Examples of PNA Sequence Design

Expedite 8900 PNA Chemistry Users Guide

1-11

Chapter 1

Introduction to PNA Synthesis

Labels

Labels, such as fluorescein and biotin, decrease PNA


oligomer solubility, especially with purine-rich sequences.
When using labeled sequences, pay particular attention to the
purine content.
When labeling PNA oligomers, note the following:

Incorporate two spacer monomers (Fmoc-AEEA-OH)


between the label and the PNA oligomer.

If you are labeling with biotin, fluorescein, rhodamine, or


other acid resistant reporter groups, PNA oligomers can
be labeled on the resin before cleavage/deprotection.

If you are labeling with acid-sensitive compounds,


labels must be attached in solution after
cleavage/deprotection. C-terminal labels may be
similarly prepared.

The spacer moiety is typically represented as an O. See


table below for examples of N-terminal and C-terminal labeled
PNA oligomers.

Label

Example

Labeled at N-Terminal
Fluorescein

Flu-O-O-PNA

Biotin

Bio-O-O-PNA

Label at C-Terminal
Fluorescein

Acetyl-PNA-O-Lys(Flu)

Labels at Both Terminals


Fluorescein and Biotin

1-12

Applied Biosystems

Bio-O-O-PNA-O-Lys(Flu)

Guidelines for Sequence Design of PNA Oligomers

Sequences to
avoid

Avoid self-complementary sequences such as inverse


repeats, hairpin-formations, and palindromic sequences
because PNA/PNA interactions are stronger than PNA/DNA
interactions.
For example, the AATT sequence is acceptable, but CCGG or
ATTAAT sequences are not acceptable. You can perform the
synthesis with no problems, but the resulting sequences
self-hybridize and are difficult to characterize and purify.

Homo thymine
PNA oligomers

Homo thymine PNA oligomers (T7 and higher) also readily


aggregate. The phenomenon is not well-understood, but
appears to be reduced by incorporating lysines at both
terminals.

1.4.2 Triplex Formation


PNA oligomers that contain only thymines and cytosines often
prefer to bind with a 2PNA/1DNA stoichiometry. In these
cases, a 1:1 mixture of the PNA and the DNA target consist of
an equal mixture of the 2PNA/1DNA-triplex and the
DNA-target. The mixture contains no PNA/DNA-duplex.

Stability

The stability of 2PNA/1DNA-triplexes is extremely high.


A 10-mer homopyrimidine PNA will typically bind to its target
with a Tm in excess of 70C.
The most stable triplex is formed when two different PNAs are
used, that is, one PNA strand is anti-parallel to the target and
the other PNA strand is parallel. However, stable triplexes are
also formed, even when both PNA strands are parallel to the
target.

NH2

CONH2
3'

5'
CONH2

NH2

Figure 1-7 Stable Triplex Configuration

Expedite 8900 PNA Chemistry Users Guide

1-13

Chapter 1

Introduction to PNA Synthesis

Cytosine content

The second PNA-strand binds through Hoogsteen base


pairing, which requires protonated cytosines in the second
strand. Avoid many cytosines in a row if you want to form
triplexes. A triplex cannot accommodate many
closely-situated, positively-charged cytosines in the second
strand.
Applied Biosystems offers a base analog that aids in triplex
formation and does not require a low pH. Contact Applied
Biosystems Technical Support for more information.

Unanticipated
triplex formation

Triplexes can also be formed when not anticipated. A PNA


oligomer designed for duplex formation can also, in rare
cases, form a triplex. Conditions that contribute to triplex
formation include:

Presence of many pyrimidines in the sequence


A parallel target sequence
Low pH

The target recognized will still be the Watson-Crick


complementary target. Therefore, unwanted specificity is not
an issue.

1.4.3 Strand Invasion


The extremely high stability of the 2PNA/1DNA triplexes
explains why homopyrimidine PNA-oligomers can displace the
non-complementary strand when they are aimed at
complementary homopurine targets in double stranded DNA.

Ionic strength

To observe strand displacement, keep the ionic strength as


low as possible. The buffer should be the only contributing
factor. Avoid divalent metal ions.
The low salt concentration is necessary for the formation of
the strand invasion complex. After it is formed, the salt
concentration can be increased (for example, to carry out
enzymatic reactions).

1-14

Applied Biosystems

Guidelines for Sequence Design of PNA Oligomers

PNA clamps

By linking two strands together, PNA clamps are obtained


(Figure 1-8).

PNA-clamp

dsDNA
PB100656

Figure 1-8 Strand Invasion by PNA Clamp

In PNA clamps, the Watson-Crick and Hoogsteen strands are


synthesized consecutively in the same synthesis with spacer
units in between the two strands.1 Not only does this place the
PNA strands in correct relative orientation, the PNA clamps
also exhibit higher Tms and faster strand invasion. It has
furthermore been shown that incorporating lysines in the PNA
clamps accelerate the invasion into double stranded DNA.2, 3
Whenever strand invasion is the objective PNA clamps are
almost exclusively used in current applications.

1. Egholm, M. et al. Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Research 23, 217222 (1995).
2. Griffith, M.C. et al. Single and Bis Peptide Nucleic Acids as triplexing agents: Binding and
stoichiometry. Journal of the American Chemical Society 117, 831832 (1995).
3. Veselkov, A.G., Demidov, V.V., Nielsen, P.E. & Frank-Kamenetskii, M.D. A new class or
genome rare cutters. Nucleic Acids Research 24, 24832487 (1996).

Expedite 8900 PNA Chemistry Users Guide

1-15

2Installing the PNA


Software

This chapter contains the following sections:


2.1 Overview ........................................................ 2-2
2.2 Installing Instrument Software ...................... 2-3

Expedite 8900 PNA Chemistry Users Guide

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Chapter 2

Installing the PNA Software

2.1 Overview
The PNA instrument boot disk includes:

Version 2.4.2 Expedite instrument software

PNA protocols (2 mole and 25 mole) and files to


support the functions called out by the protocol

Version 2.4.2 of the Expedite PNA-enabled software adds


PNA synthesis functionality to your standard Expedite system.
With 2.4.2 software, you can run the following chemistries:

PNA
DNA
RNA
Thio
User

CAUTION
Follow the proper flushing procedures when switching
between PNA reagents and standard nucleic acid reagents.
Switching reagents without proper flushing can damage the
instrument. See Section 3.7, Loading PNA Reagents, for
more information.

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Installing Instrument Software

2.2 Installing Instrument Software


Installing
instrument
software

To install the PNA Option Instrument boot disk:


1.

Turn the instrument off (the on/off switch is located at the


lower left rear of the unit).

2.

Remove the old instrument disk from the drive (the disk
drive is located at the upper left rear of the instrument).

3.

Insert the PNA option instrument disk in the drive.

4.

Press and hold the right-most key on the instrument


keypad (Key #8).

5.

Turn on the instrument. You will hear a single beep.


Continue pressing Key #8.

6.

When you hear more than two consecutive beeps


(approximately 30 seconds), release the key.
The software is loaded and the system is initialized.
When the instrument passes all diagnostics, the Main
menu (Figure 2-1) is displayed.

Figure 2-1 Main Menu with PNA-Enabled


Version 2.4.2 Software

The PNA profile name is displayed in the upper left portion of


the screen.

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Chapter 2

Installing the PNA Software

NOTE: If Fmoc PNA is not displayed in the upper left of the


screen, the PNA software has not been properly loaded.
Repeat the installation, making sure that you press and
hold Key #8 as the software loads.
The PNA profile differs from the default profile by only the
chemistry type (PNA instead of DNA) and the selection of the
universal support option. A universal support can be used in
the synthesis of any PNA sequence, because the 3' monomer
(carboy terminus) is not pre-attached to the support.

Expedite
workstation
software

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The Expedite Workstation software (version 2.4.2 or higher)


supports the PNA chemistry option and provides the benefits
of multiple instrument control, sequence and protocol editing,
and data management.

3Performing a Synthesis

This chapter contains the following sections:


3.1 Overview ........................................................ 3-2
3.2 Powering Up the System .............................. 3-3
3.3 Selecting the Chemistry ................................ 3-3
3.4 Entering the Sequence ................................. 3-5
3.5 Preparing PNA Reagents.............................. 3-6
3.6 Flushing the System When Switching
Chemistries ................................................ 3-8
3.7 Loading PNA Reagents............................... 3-11
3.8 Priming ......................................................... 3-14
3.9 Installing Reaction Columns ....................... 3-18
3.10 Performing the Synthesis............................ 3-18

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Chapter 3

Performing a Synthesis

3.1 Overview
Key steps in a
synthesis

The key steps to perform a PNA synthesis are:


1.

Power up the system. See the Expedite 8900 Nucleic


Acid Synthesis System Users Guide, Section 2.2,
Powering Up the System.

2.

Select the chemistry.

3.

Enter the sequence.

4.

Prepare the reagents.

5.

Flush the system if you are switching between


chemistries.

6.

Load the reagents.

7.

Prime the fluidic system.

8.

Install the reaction columns.

9.

Perform the synthesis.

10.

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Perform post-synthesis procedures:

Deprotect and cleave the product from the resin.

Analyze, purify, and quantitate the product.

Powering Up the System

3.2 Powering Up the System


See the Expedite 8900 Nucleic Acid Synthesis System Users
Guide, Section 2.2, Powering Up the System.

3.3 Selecting the Chemistry


If PNA chemistry is not selected:
1.

In the Main menu, select Tools.

2.

In the Tools menu, select Config.

3.

In the Configuration menu, select Profil.

4.

In the Profile menu select Read to select an existing


profile, or select Edit to make changes to the current
profile.

The first User Profile screen is displayed.


5.

Select More two times.


The screen shown in Figure 3-1 is displayed.

Figure 3-1 Chemistry Selection Screen

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Chapter 3

Performing a Synthesis

6.

Press the soft key under the Default Chemistry option


to cycle through the available options:

DNA

RNA

THIOATE

USER

PNA

7.

Select the PNA chemistry.

8.

Press More two times until the following screen is


displayed.

Figure 3-2 User ProfileFifth Screen

9.

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Applied Biosystems

Select Yes under Universal Support to specify that the


3' base will be coupled to the support on the instrument in
the first synthesis cycle.

10.

Press Exit followed by ok to save the new profile to


disk.

11.

Press Exit repeatedly until the Main menu is displayed.

Entering the Sequence

3.4 Entering the Sequence


The procedure for entering a PNA sequence is the same as
the procedure for entering standard oligonucleotide synthesis
sequences.

Direction of
synthesis

In a PNA sequence, the amino-terminus is analogous to the


5'-terminus in a DNA sequence. The Expedite instrument
synthesizes in the 3' (carboxy-terminus) to 5' (amino-terminus)
direction. For example, for a sequence of AGT CCA TTG C, C
is C-terminus (3') and A is the amino-terminus (5'). This
terminology is consistent with DNA and peptide chemistry.

NOTE: You can set the instrument to enter a sequence from


the 3' or 5' end. The default is to enter in the 5' to 3'
direction.
Before entering the sequence, read Section 1.4, Guidelines
for Sequence Design of PNA Oligomers.
See the Expedite 8900 Nucleic Acid Synthesis System Users
Guide, Section 2.3, Entering the Sequence, for additional
information.

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Chapter 3

Performing a Synthesis

3.5 Preparing PNA Reagents


CAUTION
Follow the proper flushing procedures when switching
between PNA reagents and standard nucleic acid reagents.
Switching reagents without proper flushing can damage the
instrument. See Section 3.7, Loading PNA Reagents, for
more information.

Reagent kit

The PNA Reagent Kit contains columns and the following


reagents:

Wash A (DMF)

Wash B (DMF)

Activator (requires preparation)

Deblocking solution

Capping solution

Base solution

Four monomers (require preparation)

Monomer diluent (NMP)

Each kit contains reagents for approximately 150 coupling


cycles at the 2 mole scale.
Prepare activator and monomers as described below.
Remaining reagents do not require preparation.
Fmoc-AEEA-OH spacer is available separately. The O-spacer
is used to separate labeling agents like fluorescein and biotin
from the PNA oligomer. Two spacers are recommended for all
labels. See Labels on page 1-12, for more information.

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Applied Biosystems

Preparing PNA Reagents

Preparing
activator

Add 13.5 mL of DMF to the activator. The activator dissolves


in 20 to 30 seconds.

Preparing
monomers

1.

Add 3.25 mL of PNA monomer diluent to each vial.

2.

Shake the monomers until completely dissolved. Final


volume after dissolution is approximately 3.5 mL.

Preparing amino
acids

1.

Calculate the milligrams of the amino acid:

Preparing
Fmoc-AEEA-OH
spacer
Columns

m [mg] = 0.2M * 4 mL * MWaa


2.

Dissolve in 3.8 mL of diluent (NMP).

3.

Shake until completely dissolved. Final volume after


dissolution is approximately 4 mL.

1.

Add 2.4 mL of diluent (NMP) to the Fmoc-AEEA-OH vial.

2.

Shake the vial until spacer is completely dissolved. Final


volume is approximately 2.5 mL.

The PNA chemistry kit includes 2 mole prepacked columns


containing Fmoc-XAL-PEG-PS support. The resin is a
universal support. It does not have the first monomer attached
to it and can be used for any PNA sequence. Upon cleavage,
the support yields a C-terminal amide.
The columns do not have a specific orientation so they can be
connected in either direction.

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Chapter 3

Performing a Synthesis

3.6 Flushing the System When


Switching Chemistries
If PNA reagents are already installed on your system, skip this
section.

Flushing before
switching to PNA
reagents

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Applied Biosystems

If nucleic acid reagents are currently installed on your system,


flush the system before loading PNA reagents.
If your instrument is new or has already been thoroughly
cleaned with acetonitrile and dried out using Shutdown, you
may start with step 7.
1.

Choose the PNA profile or edit the current profile to


select PNA chemistry and default parameters (see
Section 3.3, Selecting the Chemistry).

2.

Select Prime from the Main menu.

3.

Choose 7 - SHUTDOWN and column 1. Put a union in


both column positions and press Ok. The instrument
performs a short term shutdown routine (washes each
reagent train with acetonitrile from the Wash reservoirs
and blows gas through the reagent passages for
60 seconds to dry them out) and beeps five times.

4.

Press Ok to continue with the long term shutdown.


Remove all the bottles, blot the end-line filters dry with a
lint-free tissue, and replace all bottles on the instrument
with clean bottles containing acetonitrile.

5.

Press Ok to continue the shutdown routine. The


instrument performs a thorough washout of all reagent
positions and beeps five times.

6.

Remove all the bottles, replace with clean dry bottles and
press Ok. At the end of the dryout routine, the instrument
beeps ten times to indicate the shutdown procedure is
finished.

7.

Remove all the bottles and replace with clean bottles


containing dimethylformamide (DMF).

Flushing the System When Switching Chemistries

8.

Select 2 - Prime All from the Prime menu and column 1.


Press Ok to prime all reagent positions. Repeat this step
two more times for column 1 and three times for column 2.

9.

Remove all bottles, replace the end-line filters on each


line with fresh filters.

10.

Flushing before
switching to
standard nucleic
acid reagents

Load reagents and prime as described in


Section 3.7, Loading PNA Reagents.

If PNA reagents are currently installed on your system, flush


the system before loading nucleic acid reagents, and prime
after loading reagents.
1.

Choose a new profile and edit it for the appropriate


chemistry and default parameters (see Section 3.3,
Selecting the Chemistry).

2.

Select Prime from the Main menu.

3.

Choose 7 - SHUTDOWN and column 1. Put a union in


both column positions and press Ok. The instrument
performs a short term shutdown routine (washes each
reagent train with acetonitrile from the Wash reservoirs
and blows gas through the reagent passages for
60 seconds to dry them out) and beeps five times.

4.

Press Ok to continue with the long term shutdown.


Remove all the bottles, blot the end-line filters dry with a
lint-free tissue, and replace all bottles on the instrument
with clean bottles containing DMF.

5.

Press Ok to continue the shutdown routine. The


instrument performs a thorough washout of all reagent
positions and beeps five times.

6.

Remove all the bottles, replace with clean dry bottles and
press Ok. At the completion of the dryout routine, the
instrument beeps ten times to indicate it is done with the
shutdown procedure.

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Chapter 3

Performing a Synthesis

7.

Select 6 - STARTUP from the Prime menu and column 1.


Remove all the bottles and replace with clean bottles
containing acetonitrile and press Ok. Press Ok to
acknowledge that a union is in both column positions and
to initiate the startup routine.

8.

At the completion of the first phase of the startup (after


5 beeps), remove all B-train bottles (Wsh, Act, monomers
A, C, G, T/U, and 5-9) and fill with fresh dry acetonitrile.

9.

Press Ok to continue the startup routine. The instrument


completes the second phase of the startup and beeps
5 times.

10.

Remove all bottles, replace the end-line filters on each


line with fresh filters, and install all the nucleic acid
reagents.

11.

Press Ok to complete the startup routine. The


instrument beeps 10 times when it is finished.

The instrument is now ready for nucleic acid synthesis.

NOTE: Select the proper profile before performing a


synthesis. See Section 3.3, Selecting the Chemistry.

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Applied Biosystems

Loading PNA Reagents

3.7 Loading PNA Reagents


Install PNA reagents and monomers on the Expedite Model
8909 as listed below.

Turning off
blanket gas

Bottle locations

Before you unscrew a bottle, turn off blanket gas:


1.

Select Tools, Bottle.

2.

Press More. Doing so turns off the Automatic Deblanket


gas for all bottles. (When you press Exit, the Automatic
Deblanket gas is turned on.)

Figure 3-3 illustrates bottle locations. Locations are described


in Table 3-1.

Top
rack

A
6

G
7

WshB
1

T
8

Cap
2

9
WshA
WA7*

Dblk
3

Bottom
rack

AuxC
DB8*
AuxB
4

AuxA
5

AuxA
6

External caddy
*Labels on reagent lines

Figure 3-3 Reagent Positions

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Chapter 3

Performing a Synthesis

NOTE: All of the bottles are pressurized at the same time.


Therefore a bottle (even if it is empty) must be attached to
each position for the synthesizer to function properly.

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Applied Biosystems

Loading PNA Reagents

Table 3-1 PNA Reagent Location


Position

Reagent

Bottom row
1

Wash solvent B

Capping solution

Deblocking solution

46

Not used for standard PNA


protocols, but bottles must be
installed to allow pressurization

Top row
A, C, G, T

Monomers

Fmoc-AEEA-OH (O-spacer)

67

Additional amino acids

Base solution

Activator solution

External reagents
2 L bottle
(rear)

Wash solvent A (DMF)

1 L bottle
(front)

Optional end of synthesis


dichloromethane (DCM) wash of the
support. If not used, bottle must still
be installed to allow pressurization.

2 L bottle
(not shown)

Waste container

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Chapter 3

Performing a Synthesis

Resetting bottle
volumes

After loading reagents, use the Bottle Change Tool to reset the
reagent bottle volume:
1.

In the Tools menu, select Bottle.


The screen shown in Figure 3-4 is displayed.

Figure 3-4 Bottle Change ToolFirst Screen

The reagents (B-train), the reagent kit size


(150 cycles), and the current volume in each bottle are
displayed on the screen.

2.

Press the appropriate keys to reset the volumes of the


bottles that have been changed or refilled.

3.

Press More and reset remaining bottles.

NOTE: You may press the same key a second time to


recall the volume the system believes is currently left in
the bottle.
When you press Exit after resetting all bottles, blanket
gas is turned on again.

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Priming

3.8 Priming
Prime the fluidics system after:

Loading reagents
The instrument has been idle for more than 48 hours

If you have just primed after switching from nucleic acid


reagents, skip this section.

NOTE: During the priming procedure, watch the waste line


to ensure that every bottle is delivering liquid.
Table 3-2 Prime Volumes

Command

Reagent

Pulses*/Volume

Prime
Individual

Selected
Reagent

20 pulses (320 L)

Prime All

Monomers

10 pulses (160 L)

Activator, Caps
and Auxiliary

20 pulses (each 320 L)

Internal Wash

30 pulses (480 L)

Wash A and
Deblock

120 pulses (each


1.92 mL) and a gas
flush

Prime
Reagents

Each of the ancillary reagents and


Activator in varying amounts as described
for Prime All and a gas flush.

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Chapter 3

Performing a Synthesis

Command
Prime
Monomers

Reagent
Monomers

Pulses*/Volume
10 pulses from each
monomer bottle
followed by 20 pulses of
Activator, 30 pulses of
Internal Wash and a gas
flush.

* Each pulse delivers 16 L

Priming after
loading reagents

1.

Install a union in place of the reaction columns


(Figure 3-5).

CAUTION
Do not install the column before you prime the reagent
passages. The priming procedure pumps all the
reagents through the column to waste, and would
damage the column.

Union

Figure 3-5 Install a Union in Place of the Column

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Priming

2.

In the Main menu, select Prime.


The Prime menu, shown in Figure 3-6, is displayed.

Figure 3-6 Prime Menu


3.

Select 3 - Prime Reagents from the Prime menu and


column 1. Press Ok to prime all but the monomer
positions. Repeat this step one more time for column 1
and two times for column 2.

NOTE: If you replaced individual reagents, you can use


Prime Individual instead of Prime Reagent.
4.

Select 4 - Prime Monomers from the Prime menu and


column 1. Press Ok to prime the monomer positions.
Repeat this step one more time for column 2.

NOTE: Each priming cycle supplies 10 pulses


(~160 L) of monomer to the fluid passages. You must
prime both column positions one time, for a total of
20 pulses (~320 L).
The instrument is ready for PNA synthesis.

NOTE: Select the PNA profile before performing a


synthesis.

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Chapter 3

Performing a Synthesis

Priming after
48-hour idle
period

1.

Install a union in place of the reaction columns


(Figure 3-5).

CAUTION
Do not install the column before you prime the reagent
passages. The priming procedure pumps all the
reagents through the column to waste, and would
damage the column.
2.

In the Main menu, select Prime.


The Prime menu, shown in Figure 3-7, is displayed.

Figure 3-7 Prime Menu


3.

Select 2 - Prime all.

NOTE: The Prime menu is described in the Expedite


8900 Nucleic Acid Synthesis System Users Guide,
Section 3.5, Prime Menu.
4.

Select Column 1.
A message is displayed instructing you to replace the
column with a union.

5.

Press OK to continue after you install the union.


All the reagent passages are primed sequentially.

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Applied Biosystems

6.

Press Cancel to return to the Prime menu.

7.

Select column 2 and repeat step 5 and step 6.

8.

Select Exit to return to the Main menu.

Installing Reaction Columns

3.9 Installing Reaction Columns


See the Expedite 8900 Nucleic Acid Synthesis System Users
Guide, Section 2.5.3, Installing the Column, for information.

3.10Performing the Synthesis


During a synthesis run, the software monitors the progress
(see Figure 3-8) and displays the time left until the synthesis is
completed.

3
Stop
Instrument Stop Button

Figure 3-8 Screen During a Run

Stop button

You may press the Instrument Stop button, shown in


Figure 3-8, at any time. When you press this button, all
synthesis operations are halted immediately and remain
halted until you press the Start key in the Main menu.

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Chapter 3

Performing a Synthesis

NOTE: Pausing a synthesis for extended periods in the


middle of a cycle may be detrimental to the synthesis. If you
use the Stop Button, restart the synthesis as soon as
possible. If you wish to pause a synthesis to replenish
reagents, use the Hold option (see Interrupting the
synthesis on page 3-20).

Monitoring the
synthesis

During the synthesis, the Main menu displays the status of


both columns and the estimated time
(hours:minutes:seconds), until completion of the synthesis.
The Stat option on the Main menu displays more detailed
information on the synthesis. When you select Stat, the screen
shown in Figure 3-9 is displayed.

3
Figure 3-9 Synthesis Status Screen

Interrupting the
synthesis

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Applied Biosystems

To interrupt the synthesis on both columns, select Hold under


the Status: Combined menu (see Figure 3-9). The instrument
halts the synthesis on each column at the end of the current
cycle (usually within 15 minutes). This is the safest point to
interrupt the synthesis. The PNA column is washed with DMF
before the instrument halts. The synthesis remains halted until
you press No Hold to restart the synthesis.

4Post-Synthesis
Procedures

This chapter contains the following sections:


4.1

Removing the
Final Fmoc Protecting Group ................... 4-2

4.2

Drying the Resin ...................................... 4-2

4.3

Cleavage and Deprotection ...................... 4-3

4.4

Analysis, Purification,
Handling, and Quantitation....................... 4-6

4.5

Labeling PNA Oligomers ........................ 4-12

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Chapter 4

Post-Synthesis Procedures

4.1 Removing the Final Fmoc


Protecting Group
Perform a final deblock to remove the Fmoc protecting group.
To deblock the PNA:
1.

In the Main menu, select Prime.

2.

Select 5 - Final Deblock.

3.

Repeat for column 2.

The final deblock takes about 5 minutes.


If you are labeling the PNA oligomer, see Section 4.5.1,
Labeling of Support-bound PNA, for information.

4.2 Drying the Resin


1.

Remove the column from the instrument.

2.

If you installed DCM in the Aux C bottle for automatic


washing, skip to step 3.
If DCM is not installed DCM in the Aux C bottle, rinse
the resin by pushing DCM through the column.

3.

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Applied Biosystems

Blow nitrogen or argon through the column for 3 to


5 minutes to remove residual DCM. This shrinks the resin
and makes it easier to remove the resin from the column.

Cleavage and Deprotection

4.3 Cleavage and Deprotection


Cleavage and deprotection are performed in one step using a
mixture of trifluoroacetic acid (TFA) and m-cresol.

WARNING
TFA is a highly corrosive acid. TFA may be fatal if
swallowed.

AVERTISSEMENT
Lacide trifluoractique (TFA) est un acide hautement
corrosif. Le TFA peut tre mortel sil est aval.

Preparing
cleavage mixture

Make a stock solution of TFA:m-cresol (4:1).

Scale

Volume of cleavage
mixture required for
each cleavage

2 mole

400 L

25 mole

2.5 mL

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Chapter 4

Post-Synthesis Procedures

Cleaving and
deprotecting

After drying the resin:


1.

Remove the crimp seal from one end of the column.

2.

Transfer the resin to a Millipore Ultrafree-MC PTFE


0.2 m filtered microcentrifuge tube for the cleavage and
deprotection (see Appendix B, Ordering Information, for
part number), or similar apparatus.

3.

Add 200 L of the TFA:m-cresol (4:1) cleavage mixture to


the filtered microcentrifuge tube (2 mol scale). Allow to
sit at room temperature for:

PAL resin90 minutes

XAL resin5 minutes

4.

Centrifuge the resin at 2,000 RPM for 5 minutes.

5.

Repeat step 3 and step 4.

6.

Remove the filter insert from the microcentrifuge tube.

7.

Add 1 mL of diethyl ether to the microcentrifuge tube.


Shake or vortex for about 1 minute.

8.

Centrifuge at 2,000 RPM for 5 minutes. The crude PNA


forms a pellet at the bottom of the microcentrifuge tube.

NOTE: If PNA does not precipitate, see below.

9.

Decant the supernatant. Save the supernatant until


cleavage and deprotection is complete, and you have
successfully recovered the PNA.

10. Repeat step 7 through step 9 two more times.


11. Dry the PNA by doing one of the following:

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Applied Biosystems

Remove the cap from the tube and leave open


for about an hour to allow excess diethyl ether
to evaporate.

Put in 55C heat block for 5 to 7 minutes.

Cleavage and Deprotection

If PNA does not


precipitate

If PNA does not precipitate, it indicates a problem with the


synthesis.
Check that reagent and monomer bottles contain adequate
volumes.
If volumes are adequate, check the volume of WashA and
WashB delivered during the fluidic diagnostic test:

Run a Fluid diagnostic test and measure the volume


dispensed from the waste tube with a graduated
cylinder. See the Expedite 8900 Nucleic Acid Synthesis
System Users Guide, Fluid Test on page 3-74.

Perform a Prime All and make sure each reagent


position is delivering to the column.

If the volume of WashA and WashB delivered is less


than 1.6 mL, or if all reagent positions do not deliver
during a Prime All, it indicates a blockage. Call Applied
Biosystems Technical Service.

Expedite 8900 PNA Chemistry Users Guide

4-5

Chapter 4

Post-Synthesis Procedures

4.4 Analysis, Purification, Handling,


and Quantitation
This section describes:

PNA analysis
Reversed-phase purification of PNA oligomers
PNA handling
PNA quantitation

4.4.1 PNA Analysis


Preparing PNA
stock solution

1.

Add 200 L of 0.1% aqueous TFA to the dried PNA.

2.

Vortex for 1 to 2 minutes to dissolve.

3.

Place in a 55C heating block for 10 minutes.


If PNA does not dissolve, refer to Section 1.4,
Guidelines for Sequence Design of PNA Oligomers, for
information on factors that contribute to low solubility.

Determining the
crude yield

Before analyzing, make sure that your stock solution contains


the expected amount of PNA oligomer:
1.

Dilute 4 L of the PNA stock solution with 36 L of


Milli-Q water.

2.

Dilute 4 L from step 1 (1:10) into 996 L of Milli-Q water.

3.

Measure the absorbance at 260 nm.

4.

Calculate the total OD for the measured absorbance:


ODtotal

AU260 x 500

The yield should be approximately 8 OD per base. For


example, the crude yield of a 10-mer should be approximately
80 OD.
If you observe a lower OD, it may indicate that the system is
not functioning properly.

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Analysis, Purification, Handling, and Quantitation

Setting the HPLC


system for
analysis

Set your HPLC system to the following:

NOTE: Section C.2, HPLC Methods, includes an alternative


HPLC method.
Table 4-1 Settings for PNA Analysis

Condition

Setting

Column

YMC AQ C18, 3, 120, 4 mm x 50 mm

Eluent A

0.1% TFA in water

Eluent B

0.08% TFA in acetonitrile

Gradient

5-35% B over 13 minutes


35 to 95% B over 2 minutes

Flow

1.2 mL/min

Temperature

50C (column heater required)


NOTE: If you do not perform the analysis with a
column heater, peaks may be poorly resolved.

Detector

260 nm

Injection
volume

25 L

Reequilibration
between
runs

5 minutes in 95% A, 5% B

HPLC analysis

(Dilute 2 L of crude PNA in 100 L of Eluent A, then


inject 25 L)

2 mL/min

The retention time for the test 10-mer sequence AGT CCA
TTG C is 4 to 6 minutes.

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Chapter 4

Post-Synthesis Procedures

4.4.2 Reversed-Phase Purification of PNA


Oligomers
Setting the HPLC
system for
purification

Set your HPLC system to the following:

Table 4-2 Settings for PNA Purification (2 mole Scale)


Condition

Setting

Column

YMC AQ C18, 3, 120, 10 mm x 30 mm

Eluent A

0.1% TFA in water

Eluent B

0.08% TFA in acetonitrile

Gradient

0 to 5% B over 3 minutes
5 to 20% B over 15 minutes
20 to 95% B over 3 minutes

Flow

7.5 mL/min

Temperature

60C (column heater required)


NOTE: If you do not perform the purification with
a column heater, peaks may be poorly resolved.

4-8

Detector

300 nm (290 nm for biotinylated oligomers)

Injection
volume

Remaining stock solution prepared on page 4-6.

Sample loop

500 L or larger

Applied Biosystems

Analysis, Purification, Handling, and Quantitation

Purification

1.

Monitor at 300 nm (not 260 nm; 290 nm for biotinylated


oligomers) and collect fractions.

2.

Freeze the collected fractions.

3.

Dry collected fractions in a lyophilizer.

NOTE: Do not use the same speed vac for PNA and
DNA or RNA. The small amount of TFA in the PNA
solutions can destroy the DNA or RNA.
4.

Determining
amount of PNA in
fractions

Dissolve each fraction in 200 L of 0.1% TFA and analyze


on the analytical HPLC system using the settings
described in Table 4-1, Settings for PNA Analysis, on
page 4-7.

To determine the amount of PNA in each fraction:


1.

Dilute 4 L of the PNA stock solution with 36 L of


Milli-Q water.

2.

Dilute 4 L from step 1 (1:10) into 996 L of Milli-Q water.

3.

Measure the absorbance at 260 nm.

4.

Calculate the total OD for the measured absorbance:


ODtotal

AU260 x 500

Expedite 8900 PNA Chemistry Users Guide

4-9

Chapter 4

Post-Synthesis Procedures

4.4.3 PNA Handling


When working with sub-micro molar concentrations of PNA, a
majority of the PNA may become bound to the glass.
Whenever possible, use polypropylene or polyethylene
materials during handling and storage of PNA oligomers.
After purification, the pure fractions can be:

Used directly
Divided into aliquots and used or dried on a speed vac

NOTE: Store the dried samples at 4C until they are used.

Using purified
PNA

4-10

Applied Biosystems

Dissolve dried PNA material in one of the following ways:

Dissolve in water or 0.1% aqueous TFA. The resulting


solution is acidic, even when dissolved in water,
because the purification step provides the PNA as its
TFA salt. However, the acid level does not affect the
typical application. Dissolving in water or TFA is the
preferred method.

Dissolve in the buffer you are going to use for your


application. Occasionally, the PNA will not dissolve
directly in a buffer. If this happens, decant the buffer
and dissolve the PNA in 0.1% aqueous TFA. If the
PNA does not dissolve in TFA, you can add up to
30% acetonitrile.

Analysis, Purification, Handling, and Quantitation

4.4.4 PNA Quantitation


Quantitation

Extinction coefficients have been determined for three of the


four PNA monomer units:

C=6.6
T=8.6
A=13.7 mLmol-1cm-1

These values are very close to those determined for the


corresponding natural nucleotides. Therefore, assume a value
of 11.7 mLmol-1cm-1 for the G monomer until the actual
value is established.
Hypochromic effects must be considered when quantifying
PNA oligomers based on UV absorption at 260 nm at room
temperature. Single-stranded PNA is highly stacked, leading
to significant reduction in observed extinction coefficients of
the oligomers relative to the sum of the extinctions of the
individual bases that comprise the oligomer. Melts performed
on single-stranded PNAs show a sigmoidal curve with a Tm
of 40 to 45C and an observed hyperchromicity of 15 to 25%
almost independent of the sequence and length of the PNA.
For most purposes, include a 20% hypochromicity factor when
calculating PNA concentration. When a more accurate
determination of concentration is necessary, measure
absorption at 260 nm at a temperature higher than 50C.

Melting
experiments

The above considerations have an impact on UV behavior in


connection with hybridization. Hypochromicity associated with
PNA/DNA duplex formation at room temperature is small,
typically only 3 to 5%, and is primarily due to an increase in
the stacking of the DNA upon binding. This has little impact,
however on the magnitude of the melting curves of PNA/DNA
duplexes because they typically melt at temperatures above
45C (that is, above the melting temperature of the single
stranded PNA) and typically display the same net gain in
absorbance as DNA/DNA duplexes upon melting.

Expedite 8900 PNA Chemistry Users Guide

4-11

Chapter 4

Post-Synthesis Procedures

4.5 Labeling PNA Oligomers


This section includes:

Labeling of support-bound PNA


Labeling PNA oligomers in solution

4.5.1 Labeling of Support-bound PNA


Labeling of PNA oligomers with a reporter group such as
biotin, fluorescein or rhodamine can easily be carried out.
Support-bound PNA oligomers are manually labeled by
introducing the reporter group in solution to the column using a
dual syringe method as follows:

NOTE: The N-terminal amine protecting group must be


removed prior to labeling. See Section 4.1, Removing the
Final Fmoc Protecting Group.

Preparing the
reporter group
solution

1.

Weigh out dry reporter group as indicated in Table 4-3.


This is a 5 to 10 fold excess.

Table 4-3 Preparing Reporter Group Solution


(2 mol Synthesis)
Reporter Group
N-Hydroxy-succinimido
Biotin
5&6
Carboxyfluorescein,
succinimidyl ester

4-12

Applied Biosystems

Amount

DMF

DIEA

7.7 mg

300 L

6.6 L

10.7 mg

300 L

11 L

Labeling PNA Oligomers

Table 4-3 Preparing Reporter Group Solution


(2 mol Synthesis) (Continued)
Reporter Group
5 & 6 Tetramethyl
Rhodamine,

Amount

DMF

DIEA

12.0 mg

300 L

11 L

succinimidyl ester

NOTE: Diisopropylethylamine (DIEA) is added to keep the


amino group in the free base form (non-protonated). Biotin
is less acidic, therefore, less base is needed.
Table 4-4 Table of Weights
Reporter Group

Molecular Wt.

Residue
Mass

N-Hydroxysuccinimido
Biotin

341.40

227.31

5 & 6 Carboxyfluorescein,
succinimidyl ester

473.39

359.3

5 & 6 Tetramethyl
Rhodamine,

527.53

414.46

succinimidyl ester
2.

Add the DMF. Vortex.

3.

Add the diisopropylethylamine (DIEA).

4.

Vortex the mixture until all the solid is in solution. This


solution is approximately 0.07 M.

Expedite 8900 PNA Chemistry Users Guide

4-13

Chapter 4

Post-Synthesis Procedures

Labeling the PNA


oligomer

1.

Attach a 1 mL Luer slip-tip syringe to one end of the


column.

2.

Draw the reporter group solution into a second 1 mL Luer


slip-tip syringe and attach it to the other end of the
column.

3.

Slowly push the solution back and forth through the


column (approximately 10 times).

4.

Allow the reaction to proceed at room temperature for the


following times, pushing the solution back and forth
through the column every 10 minutes:

Biotinylated oligomers30 minutes

Fluorescein or rhodamine-labeled
oligomers60 minutes

5.

Wash the labeled PNA oligomer three times with a


1:1 mixture of DCM:DMF.

6.

Dry the column by passing nitrogen or argon over it for


several minutes.

NOTE: For photosensitive reporter groups, cover the


syringe/column setup with foil.

Cleavage of the
labeled PNA
oligomer from the
support

Follow the standard cleavage and deprotection methods for


PNA oligomers as described in Section 4.3, Cleavage and
Deprotection.

TFA cleavage and deprotection can cause oxidation of


biotinylated PNA.
To avoid oxidation of biotinylated PNA:

4-14

Applied Biosystems

Store the TFA in the freezer.

Use Applied Biosystems TFA. Old or poor-quality TFA


contains peroxides which oxidize biotin.

Labeling PNA Oligomers

Hint: A rapid method for removing peroxide from TFA


is to partially freeze the TFA and retain the liquid
portion, which is relatively free of peroxide.

Buy only small bottles and use up completely before


opening new bottles.

4.5.2 Labeling PNA Oligomers in Solution


Labeling of PNA is accomplished by attaching a reporter
group, such as biotin or fluorescein, to the PNA oligomer via a
free amine. There are two ways to generate a free amine:

At the N-terminal
Via a lysine incorporated into the sequence

Free amines at the N-terminal are generated when the final


Fmoc group is removed. Labeling of this amine is most easily
accomplished when the PNA oligomer is still attached to the
solid support. See Section 4.5.1, Labeling of Support-bound
PNA, for more details. This is the preferred method of labeling
using biotin, fluorescein or rhodamine, for coupling of these
dyes is difficult in solution, resulting in low yields of labeled
product.

NOTE: The above procedure cannot be used for


acid-sensitive dyes such as Amersham Cy3 or Cy5,
because after labeling, the support-bound oligomer is
cleaved from the support using a solution of trifluoroacetic
acid and m-cresol (4:1).
Labeling can also be accomplished via the epsilon-amino
group of an incorporated lysine. During synthesis, this group is
protected with a Boc group. The Boc group is removed during
cleavage of the oligomer from the support. Labeling via a
lysine group is useful for introducing a C-terminal label or a
second label to the oligomer.

Expedite 8900 PNA Chemistry Users Guide

4-15

Chapter 4

Post-Synthesis Procedures

NOTE: If you have removed the final Fmoc group and have
not labeled it with another label, labeling will occur at this
position as well as at the lysine side-chain. Avoid this by
capping the N-terminal amino group.
It is important to note that labels at the N-terminal require two
spacer molecules to separate the label from the PNA
oligomer. When a label is to be introduced via a lysine, the
lysine side-chain itself acts as one spacer. The spacer
molecule(s) allows for more efficient attachment of the label,
resulting in higher yields.

Solution-phase
labeling of PNA
oligomers

This procedure is only for monofunctional labels, such as


digoxigenin, Oregon Green, Texas Red, Bodipy FLX, Bodipy
TRX, Cy3 and Cy5.
1.

Prepare the following solutions:

Solution A1 mg of label dissolved in 110 L


of DMF

Solution B10 to 15 OD260 of HPLC-purified


PNA dissolved in 100 L of Milli-Q-grade
water

Solution C25 L 10% N- Methylmorpholine


(aq)

Controls for HPLC analysis:


Unlabeled PNA: 2 L of Solution B in 100 L
of 0.1% TFA (aq.)
Unreacted labeling reagent: 2 L of Solution A
in 100 L of 0.1% TFA (aq.)

4-16

Applied Biosystems

2.

Combine the remaining 98 L of Solution B and 25 L of


Solution C. Vortex.

3.

Add 100 L of Solution A and vortex. Let the reaction


proceed for 1 hour in the dark at room temperature.

Labeling PNA Oligomers

4.

5.

Thiol-specific
labels

To confirm the labeling reaction was successful, analyze


the samples and controls by HPLC and the samples by
MALDI-TOF mass spectrometry:

HPLC analysisAdd 2 L of the sample to


100 L of 0.1% TFA (aq.)

MALDI-TOF analysisMake 1:10, 1:100, and


1:1,000 dilutions of the sample. The 1:1,000
usually provides the best data.

Upon confirming the labeling reaction was successful do


one of the following:

Purify the labeled PNA immediately

Freeze the solution

Lyophilize (in the dark), and store the dried


material at -20C until purification can be
performed

Thiol-specific reporter groups can be attached to PNA


oligomers by incorporating a cysteine into the sequence.

Expedite 8900 PNA Chemistry Users Guide

4-17

5Troubleshooting and
Maintenance

This chapter contains the following sections:


5.1

Troubleshooting ....................................... 5-2

5.2

Maintenance ............................................ 5-4

Expedite 8900 PNA Chemistry Users Guide

5-1

Chapter 5

Troubleshooting and Maintenance

5.1 Troubleshooting
Table 5-1 PNA Troubleshooting
Symptom
PNA does not
precipitate

PNA does not dissolve

Mass spectrometry
analysis data shows
the expected MW
+222.2 Da, and HPLC
shows a late eluting
peak

5-2

Applied Biosystems

Possible Cause

Corrective Action

Reagent empty

Check for empty reagent and


monomer bottles.

Insufficient gas
pressure

Check the gas pressure.

Blockage in a line

Perform the fluidic diagnostic


test. See page 3-64 in the
Expedite 8900 Nucleic Acid
System Users Guide.

Fmoc protecting
group not removed

Resynthesize the PNA


remembering to perform a final
deblock (Prime #5).

Sequence
aggregates

Place in heating block. If PNA


still does not dissolve, add up
to 30% acetonitrile.

Sequence is
insoluble

Redesign sequence. See


Section 1.4.1, General
Guidelines.

Fmoc protecting
group not removed

Resynthesize the PNA


remembering to perform a final
deblock (Prime #5).

Troubleshooting

Table 5-1 PNA Troubleshooting (Continued)


Symptom
On-support labeling
reaction fails

Possible Cause

Corrective Action

Fmoc protecting
group not removed

Resynthesize the PNA


remembering to perform a final
deblock (Prime #5).

Base was not added


to the labeling
reaction

Resynthesize the PNA. See


Section 4.5.1, Labeling of
Support-bound PNA.

5
Expedite 8900 PNA Chemistry Users Guide

5-3

Chapter 5

Troubleshooting and Maintenance

5.2 Maintenance
Proper attention to maintenance will result in increased
instrument life and performance. Regular preventive
maintenance will help the Expedite Nucleic Acid System
perform to specifications.

Daily

Do the following once a day:

Weekly

Do the following once a week:

Monthly

As needed

Check reagent bottles


Check gas tanks

Empty waste bottles


Perform leak test
Change O spacer (bottle position 5) and clean out
bottle 5 with DMF

Once a month, clean the synthesizer with DMF:

Fill all bottles with DMF

Run a mock synthesis containing ACGT567 repeated 4


times

Perform a fluid test in all bottle positions

Change the following filters and O-rings as needed, or every


three months as indicated:

Endline FilterWhen bottle is changed


O-Ring Kit Amidite3 months or as needed
O-Ring Kit Reagent3 months or as needed
O-Ring Kit Caddy3 months or as needed

See Appendix B, Ordering Information, for part numbers.

General
guidelines

5
5-4

Applied Biosystems

Note the following when performing maintenance procedures:

Clean lines with DMF and replace endline filters when


changing reagents.

Collect and measure waste when priming to check that


the instrument is working properly. Measure prime
volumes either by volume or weight.

AReagent Safety

WARNING
Most reagents and solvents used in PNA synthesis are
hazardous. Wear a lab coat, gloves, and eye protection
when handling reagents. Adequate ventilation is
essential and working under a fume hood is
recommended. Flammable reagents should be kept in
an appropriate flameproof cabinet.

AVERTISSEMENT
La plupart des ractifs et solvants employs en
synthse PNA sont dangereux. Portez une blouse, des
gants et des lunettes de protection lorsque vous
manipulez des ractifs. Une ventilation adquate est
ncessaire et il est recommand de travailler sous une
hotte.

This section describes the handling of specific chemicals.

Waste disposal

Collect the waste generated from PNA synthesis and


dispose of it in accordance with local, state, and federal
regulations pertaining to toxic waste removal.

Expedite 8900 PNA Chemistry Users Guide

A-1

Appendix A

Reagent Safety

Acetic Anhydride

Acetic anhydride is a colorless liquid with a strong odor. It is


harmful if swallowed, inhaled, or absorbed through the skin. It
is extremely destructive to the tissue of the mucous
membranes and upper respiratory tract, eyes and skin.
Inhalation may be fatal as a result of spasm, inflammation and
edema of the larynx and bronchi, chemical pneumonitis and
pulmonary edema. Symptoms of exposure may include
burning sensation, coughing, wheezing, laryngitis, shortness
of breath, headache, nausea, and vomiting.

First Aid: Remove contaminated clothing and shoes. Flush


eyes or skin with plenty of water for at least 15 minutes. If
inhaled, remove to fresh air. If not breathing, give artificial
respiration. If breathing is difficult, give oxygen. Call a
physician. Discard contaminated shoes. Wash contaminated
clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a cool
dry place.
Fire and Explosion Data: Extinguish with CO2, dry chemical
powder, alcohol or polymer foam. Wear-self contained
breathing apparatus, rubber boots and heavy rubber gloves to
prevent contact with the skin and eyes.
Spill or Leak Procedures: Evacuate area. Wear
self-contained breathing apparatus, rubber boots, and heavy
rubber gloves. Cover with activated carbon adsorbent, take up
and place in closed containers. Transport outdoors. Ventilate
area and wash spill site after material pickup is complete.

A-2

Applied Biosystems

Activator (ACT)

This powder contains O-(7-azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HATU).


When reconstituted, also contains dimethylformamide (DMF).
See individual reagents for details and safety precautions.

WARNING
Activator may cause allergic skin reaction. Depending on
the intensity of exposure, effects may vary from mild to
severe irritation. Avoid all contact. Use only in a fume hood.
Wear gloves when handling and wash thoroughly after
handling.

AVERTISSEMENT
Lactivateur peut entraner des ractions allergiques sur la
peau. Lirritation peut tre soit lgre, soit svre, selon
lintensit de lexposition. viter tout contact. Utilisez
seulement avec une hotte. Portez des gants lors de la
manipulation et lavez-vous entirement aprs.
A white powder. Should be considered hazardous as the
toxicological properties are unknown. Do not breathe the
powder.

Recommended Storage: Keep tightly closed. Store at 4C in


a desiccator.

Base solution
(Base)

This solution contains N,N-diisopropylethylamine (DIEA),


2,6-lutidine, and dimethylformamide (DMF). See individual
reagents for details and safety precautions.

Recommended Storage: Keep tightly closed. Store in a dark


cool dry place at 4C.

Capping solution
(CAP)

This solution contains acetic anhydride, 2,6-lutidine, and


dimethylformamide (DMF). See individual reagents for details
and safety precautions.

Recommended Storage: Keep tightly closed. Store in a dark


cool dry place at 4C.

Expedite 8900 PNA Chemistry Users Guide

A-3

Appendix A

Reagent Safety

Cleavage and
deprotection
solution (AP)

This solution contains scavengers and trifluoroacetic acid


(TFA). See individual reagents for details and safety
precautions.

Recommended Storage: Keep tightly closed when not in use.


Prepare fresh reagent before use. Store at -20C.

Deblock Solution
(Dblk)

This solution contains piperidine and dimethylformamide


(DMF). See individual reagents for details and safety
precautions.

Recommended Storage: Keep tightly closed. Store in a dark


cool dry place at 4C.

Dichloromethane
(DCM) (Methylene
Chloride)

A colorless, clear liquid with mild chloroform-like odor. Harmful


if inhaled, irritating to skin, eyes, and mucous membranes.
May cause nausea, vomiting, light-headedness, or headache.
Inhalation of vapors in high concentration causes narcosis.

First Aid: If inhaled, remove to fresh air. If not breathing, give


artificial respiration. If breathing is difficult, give oxygen. If
ingested and if conscious, induce vomiting with water. Keep
airway clear. Call a physician. Flush eyes with plenty of water
for at least 15 minutes. Wash skin with soap and water. Wash
contaminated clothing before reuse.
Recommended Storage: Store at room temperature, away
from light in a solvent storage cabinet.
Fire and Explosion Data: Fire involving DCM is unlikely. In
the event of fire use extinguishing media appropriate for
surrounding fire.
Spill or Leak Procedures: Wear self-contained breathing
apparatus and full protective clothing. Use water spray to
reduce vapors. Take up with sand or vermiculite and place in
closed container. Flush spill area with water.

A-4

Applied Biosystems

N,N-diisopropylethylamine (DIEA)

A colorless liquid. Harmful if swallowed, inhaled, or absorbed


through the skin. Material is extremely destructive to the tissue
of the mucous membranes and upper respiratory tract, eyes
and skin. Inhalation may be fatal as a result of spasm,
inflammation and edema of the larynx and bronchi, chemical
pneumonitis and pulmonary edema. Symptoms of exposure
may include burning sensation, coughing, wheezing,
laryngitis, shortness of breath, headache nausea and
vomiting.

First Aid: In case of contact, immediately flush eyes or skin


with copious amounts of water for at least 15 min while
removing contaminated clothing and shoes. Assure adequate
flushing of the eyes by separating the eyelids with fingers. If
inhaled, remove to fresh air. If not breathing give artificial
respiration. If breathing is difficult, give oxygen. If ingested,
wash out mouth with water. Call a physician. Wash
contaminated clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a cool
dry place at 4C.
Fire and Explosion Data: Flash point: 10C. Dangerous fire
hazard when exposed to heat or flame. Vapor-air mixtures are
explosive. Extinguish with carbon dioxide, dry chemical
powder, alcohol or polymer foam. Use water to cool fire
exposed containers. Vapors may travel a considerable
distance to a source of ignition and flash back. Closed
containers exposed to heat may explode.
Spill or Leak Procedures: Evacuate the area. Shut off all
sources of ignition. Wear self-contained breathing apparatus
and full protective clothing. Cover with dry lime, sand, or soda
ash. Place in covered containers using non-sparking tools and
transport outdoors. Ventilate are and wash spill site after
pickup is complete.

Expedite 8900 PNA Chemistry Users Guide

A-5

Appendix A

Reagent Safety

Dimethylformamide (DMF)

A colorless hygroscopic liquid with a faint amine-like odor.


Harmful if swallowed, inhaled, or absorbed through the skin.
Vapor or mist is irritating to the eyes, mucous membranes, and
upper respiratory tract. Causes skin irritation. Symptoms of
exposure can include stomach pains, vomiting, diarrhea and
nausea, dizziness and headache. Intolerance for alcohol can
occur up to 4 days after exposure. Chronic effects include
damage to the liver and kidneys. Considered to be a potent
liver toxin.

First Aid: In case of contact, immediately flush eyes or skin


with copious amounts of water for at least 15 min while
removing contaminated clothing and shoes. Assure adequate
flushing of the eyes by separating the eyelids with fingers. If
inhaled, remove to fresh air. If not breathing give artificial
respiration. If breathing is difficult, give oxygen. If ingested,
wash out mouth with water. Call a physician. Wash
contaminated clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a dark
cool dry place at 4C.
Fire and Explosion Data: Flash point: 58C. Moderate fire
hazard when exposed to heat or flame. Vapor-air mixtures are
explosive above flash point. Extinguish with carbon dioxide,
dry chemical powder, alcohol or polymer foam. Use water to
cool fire exposed containers. Vapors are heavier than air and
may travel a considerable distance to a source of ignition and
flash back. Closed containers exposed to heat may explode.
Spill or Leak Procedures: Evacuate the area. Shut off all
sources of ignition and ventilate the area. Wear self-contained
breathing apparatus and full protective clothing. Take up with
sand or vermiculite and place in a closed container.

2,6-Lutidine

A-6

Applied Biosystems

A colorless liquid. May be harmful if swallowed, inhaled, or


absorbed through the skin. Vapor or mist is irritating to the
eyes, mucous membranes, and upper respiratory tract.
Causes skin irritation. The toxicological properties have not
been thoroughly investigated.

First Aid: In case of contact, immediately flush eyes or skin


with copious amounts of water for at least 15 min while
removing contaminated clothing and shoes. Assure adequate
flushing of the eyes by separating the eyelids with fingers. If
inhaled, remove to fresh air. If not breathing give artificial
respiration. If breathing is difficult, give oxygen. If ingested,
wash out mouth with water. Call a physician. Wash
contaminated clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a dark
cool dry place at 4C.
Fire and Explosion Data: Flash point: 33C. Moderate fire
hazard when exposed to heat or flame. Extinguish with carbon
dioxide, dry chemical powder, alcohol or polymer foam.
Spill or Leak Procedures: Evacuate the area. Wear selfcontained breathing apparatus and full protective clothing.
Cover with dry lime or soda ash, take up and place in a closed
container. Transport outdoors. Ventilate and wash spill site
after material pickup is complete.

1-Methyl-2pyrrolidinone
(NMP)

A colorless hygroscopic liquid. Harmful if swallowed, inhaled,


or absorbed through the skin. Causes severe eye irritation.
Prolonged exposure can cause stomach pains, vomiting, and
diarrhea. Overexposure may cause reproductive disorders
based on tests with laboratory animals.

First Aid: In case of contact, immediately flush eyes or skin


with copious amounts of water for at least 15 min while
removing contaminated clothing and shoes. Assure adequate
flushing of the eyes by separating the eyelids with fingers. If
inhaled, remove to fresh air. If not breathing give artificial
respiration. If breathing is difficult, give oxygen. If ingested,
wash out mouth with water. Call a physician. Wash
contaminated clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a cool
dry place at 4C.
Fire and Explosion Data: Flash point: 86C. Moderate fire
hazard when exposed to heat or flame. Vapor-air mixtures are
explosive above flash point. Extinguish with water spray,
carbon dioxide, dry chemical powder, alcohol or polymer foam.
Use water to cool fire exposed containers.

Expedite 8900 PNA Chemistry Users Guide

A-7

Appendix A

Reagent Safety

Spill or Leak Procedures: Evacuate the area. Wear selfcontained breathing apparatus and full protective clothing.
Take up with sand or vermiculite and place in a closed
container.

A
Monomer Diluent

This solution contains 1-methyl-2-pyrrolidinone (NMP). See


NMP listing for details and safety precautions.

Recommended Storage: Keep tightly closed. Store in a cool


dry place at 4C.

Monomers
(A,C,G,T)

White powders. Should be considered hazardous as the


toxicological properties are unknown. Do not breathe the
powders.

Recommended Storage: Keep tightly closed. Store at 4C in


a desiccator.

Piperidine

A colorless liquid with a strong amine-like odor. May be fatal if


swallowed, inhaled, or absorbed through the skin. Material is
extremely destructive to the tissue of the mucous membranes
and upper respiratory tract, eyes and skin. Inhalation may be
fatal as a result of spasm, inflammation and edema of the
larynx and bronchi, chemical pneumonitis and pulmonary
edema. Symptoms of exposure may include burning
sensation, coughing, wheezing, laryngitis, shortness of breath,
headache, nausea, and vomiting.

First Aid: In case of contact, immediately flush eyes or skin


with copious amounts of water for at least 15 min while
removing contaminated clothing and shoes. Assure adequate
flushing of the eyes by separating the eyelids with fingers. If
inhaled, remove to fresh air. If not breathing give artificial
respiration. If breathing is difficult, give oxygen. If ingested,
wash out mouth with water. Call a physician. Wash
contaminated clothing before reuse.
Recommended Storage: Keep tightly closed. Store in a dark
cool dry place at 4C.

A-8

Applied Biosystems

Fire and Explosion Data: Flash point: 4C. Dangerous fire


hazard when exposed to heat or flame. Vapor-air mixtures are
explosive above flash point. Extinguish with carbon dioxide,
dry chemical powder, alcohol or polymer foam. Use water to
cool fire exposed containers. Vapors may travel a
considerable distance to a source of ignition and flash back.
Closed containers exposed to heat may explode.
Spill or Leak Procedures: Evacuate the area. Shut off all
sources of ignition. Wear self-contained breathing apparatus
and full protective clothing. Cover with dry lime, sand, or soda
ash. Place in covered containers using non-sparking tools and
transport outdoors. Ventilate area and wash spill site after
material pickup is complete.

Trifluoroacetic
Acid (TFA)

A corrosive colorless hygroscopic liquid. Harmful if swallowed,


inhaled, or absorbed through the skin. Material is extremely
destructive to the tissue of the mucous membranes and upper
respiratory tract, eyes and skin. Inhalation may be fatal as a
result of spasm, inflammation and edema of the larynx and
bronchi, chemical pneumonitis and pulmonary edema.
Symptoms of exposure may include burning sensation,
coughing, wheezing, laryngitis, shortness of breath, headache
nausea, and vomiting.

First Aid: Contamination of the eyes should be treated by


immediate and prolonged irrigation with copious amounts of
water. Assure adequate flushing of the eyes by separating the
eyelids with fingers. In case of contact, immediately wash skin
with soap and copious amounts of water. If inhaled, remove to
fresh air. If not breathing give artificial respiration. If breathing
is difficult, give oxygen. If ingested, wash out mouth with
water. Call a physician. Wash contaminated clothing before
reuse.
Recommended Storage: Keep tightly closed. Store in a dark
cool dry place at -20C.
Fire and Explosion Data: Flash point: none.
Noncombustible. Use extinguishing media appropriate to
surrounding fire conditions.

Expedite 8900 PNA Chemistry Users Guide

A-9

Appendix A

Reagent Safety

Spill or Leak Procedures: Evacuate the area. Shut off all


sources of ignition. Wear self-contained breathing apparatus
and full protective clothing. Cover with dry lime, sand, or soda
ash. Place in covered containers using non-sparking tools and
transport outdoors. Ventilate area and wash spill site after
material pickup is complete.

A
Wash Solutions
(WshA, WshB)

These solutions contain dimethylformamide (DMF). See DMF


listing for details and safety precautions.

Recommended Storage: Keep tightly closed. Store in a dark


cool dry place at 4C.

A-10

Applied Biosystems

BOrdering Information

Table B-1 PNA Reagent Kit


Description

Quantity

Part Number

1 kit

GEN063018

PNA Fmoc-A-(Bhoc)-OH (MW=724.75)

0.508 g

GEN063014

PNA Fmoc-C-(Bhoc)-OH (MW=701.73)

0.493 g

GEN063015

PNA Fmoc-G-(Bhoc)-OH (MW=741.76)

0.519 g

GEN063016

PNA Fmoc-T-(Bhoc)-OH (MW=506.51)

0.355 g

GEN063017

Diluent, monomer (NMP)

15 mL

GEN063104

PNA Activator (HATU)

0.932 g

GEN063080

PNA Base Solution (0.2M DIEA, 0.3M 2,6-Lutidine)

15 mL

GEN063100

PNA Deblocking Solution (20% piperidine in DMF)

200 mL

GEN063101

Reagent kit, Expedite PNA, 150 cycle, includes one of


each of the following monomers, and reagents, and 3
packages of 2 mol XAL columns (4/pkg)
Monomers

Reagents

Expedite 8900 PNA Chemistry Users Guide

B-1

Appendix B

Ordering Information

Table B-1 PNA Reagent Kit (Continued)


Description

Quantity

Part Number

PNA Capping Solution (5% Acetic anhydride,


6% 2,6-Lutidine in DMF)

200 mL

GEN063102

Wash A Solution (DMF)

1L

GEN910003

Wash B Solution (DMF)

200 mL

GEN063103

3 pkg
(4/pkg)

GEN063053

Columns
Column, 2 mol Fmoc-XAL PEG-PS

Table B-2 Additional PNA Reagents Columns


Description

Quantity

Part Number

PNA O-spacer Fmoc-AEEA-OH (MW=385.40)

0.193 g

GEN063032

Trifluoroacetic acid (TFA)

100 mL

GEN910004

Dichloromethane (DCM)

1 liter

GEN902017

Column, 2 mol Fmoc-PAL PEG-PS

4/pkg

GEN063051

Column, 25 mol Fmoc-XAL PEG-PS

1/pkg

GEN063054

Table B-3 Miscellaneous


Description

Quantity

Part Number

Filters, endline

set of 19

GEN210168

O-Ring Kit Amidite

1 kit

GEN210165

O-Ring Kit Reagent

1 kit

GEN210164

O-Ring Kit Caddy

1 kit

GEN210167

B-2

Applied Biosystems

Recommended
vendors for
additional
materials

Documentation

The following materials and vendors are recommended:

Millipore Ultrafree-MC PTFE 0.2 m Microcentrifuge


tube, 25 tubes per box, Millipore part number
SE3P230J3

Waters Delta-Pak C18 column, 5, 300,


3.9 mm x 150 mm, Waters part number 11793

Waters Delta-Pak C18 column, 5, 300,


7.8 mm x 300 mm, Waters part number 11803

YMC Analytical Column, 4.0 x 50 mm, S3 um, 120


(one column)AQ12S03-0504WTA

YMC 2 umol Preparative Column, 10 x 30 mm, S3 um,


120A (one column)AQ12S03-0310WT

YMC 20 umol Preparative Column 20x50 mm, S5 um,


120A (one column)AQ12S05-0520WT

YMC Starter Kit (3 columns and one set of end


fittings)XPMDKIT3 (specify 3x(AQ12S03-0504WTA)

YMC End Fittings, setXPEF43WT1

m-CresolAldrich C8572-7

Diethyl ether (anhydrous)Aldrich or VWR

N-Hydroxysuccinimido BiotinSigma part


number H1759

5 & 6 Carboxyfluorescein, succinimidyl ester


Molecular Probes Catalog No. C1311

5 & 6 Tetramethyl Rhodamine, succinimidyl ester


Molecular Probes Catalog No. C1171

GEN601318

Documentation set containing Expedite


8900 Nucleic Acid Synthesis System Users
Guide and Expedite 8900 Nucleic Acid
Synthesis System Quick Reference Card

GEN601308

PNA Chemistry for the Expedite 8900


Nucleic Acid Synthesis System Users
Guide

Expedite 8900 PNA Chemistry Users Guide

B-3

CPNA Reference
Information

This appendix contains the following sections:


C.1

PNA Optical Density Conversion .............. C-2

C.2

HPLC Methods......................................... C-4

C.3

MALDI-TOF Methods ............................... C-5

C.4

Waste Composition for


Fmoc PNA Synthesis (2.0 mol Scale) ..... C-8

Expedite 8900 PNA Chemistry Users Guide

C-1

Chapter C

PNA Reference Information

C.1 PNA Optical Density


Conversion
Calculations

To convert from OD260 to mole or g:


1.

Calculate the Molar Extinction Coefficient () using the


following residue values:

A = 13.7 mL/(mole x cm)

C = 6.6 mL/(mole x cm)

G = 11.7 mL/(mole x cm)

T = 8.6 mL/(mole x cm)

NOTE: Values for the A,C,G and T monomers only are


used in the calculation because other residues, such as
amino acids and labels, do not add to the OD260.

C
Example

2.

To calculate the mole amount, divide the OD260 by the


Molar Extinction Coefficient.

3.

To calculate the g amount, multiply the mole amount by


the average mass.

Sequence:

(N-C) Bio-O-O-AGT-CCA-TTG-C

Average Masses:

MW3220.21 Da
(M+H)+3221.21 Da

C-2

Applied Biosystems

Volume of crude:

0.20 mL

OD:

100

PNA Optical Density Conversion

Composition

MW

275.2720

251.2468

291.2586

266.2586

Total Bases

1.

10

O-spacer

145.16

Amino Acids

varies

Labels

varies

Given the composition above, calculate the Molar


Extinction Coefficient:

2.

Number of
Residues

= 13.7 x 2 + 6.6 x 3 + 11.7 x 2 + 8.6 x 3


= 96.4 mLmol-1cm-1

To calculate the mole amount, divide the OD260 by the


Extinction Coefficient:
mole = 100/96.4 = 1.04 mole

3.

To calculate the g amount multiply the mole amount by


the average mass:
1.04x 3220.21 = 3341 g (3.34 mg)

NOTE: Final amount is not corrected for hypochromic


effects.

Expedite 8900 PNA Chemistry Users Guide

C-3

Chapter C

PNA Reference Information

C.2 HPLC Methods


Table C-1 Analytical Methods

Column

Gradient

YMC AQ C18, 3, 120,


4 mm x 500 mm

535% B over 13 minutes

Delta-Pak C18, 5, 300,


3.9 mm x 150 mm

55% B over 5 minutes

Flow
(ml/min)

Reequilibration
Time (min)

1.2

1.0

15

3595% B over 2 minutes

535% B over 30 minutes


3595% B over 1 minute

Eluents: A: 0.1% TFA (aq), B: 0.08% TFA in ACN

Table C-2 Purification Methods (2 mole Scale)

Column

YMC AQ C18, 3, 120,


10 mm x 30 mm

Gradient

05% B over 3 minutes

ReFlow
equilibration
(ml/min)
Time (min)
7.5

1.5

10

520% B over 15 minutes


2095% B over 3 minutes

Delta-Pak C18, 5, 300,


7.8 mm x 300 mm

55% B over 5 minutes,


535% B over 30 minutes,
35100% B over 5 minutes

Eluents: A: 0.1% TFA (aq) B: 0.08% TFA in ACN

C-4

Applied Biosystems

MALDI-TOF Methods

C.3 MALDI-TOF Methods


Matrix and
standard

Preparing
standard and
sample

For MALDI-TOF analysis of PNA, use the following:

Sinnapinic acid matrix10 mg/mL in 2:1 of


0.1% TFA (aq):acetonitrile

Insulin internal standard1 mg/mL 0.1% TFA (aq)

1.

For a 1:20 dilution of the insulin internal standard, add


25 L of the insulin solution to 475 L of the matrix.

2.

With 1 L of crude PNA, make 1:10, 1:100, and 1:1,000


dilutions in 9 L aliquots of insulin/matrix solution
(prepared above).

3.

Spot 1 L of 1:100 and 1:1,000 dilutions on the sample


plate. Dry the sample plate before analyzing.

NOTE: The 1:1,000 dilution usually provides the best


data.
Table C-3 Mass Spectrum Peaks
Ion

Description

M+1

Product peak

M+23

Sodium adduct

M+63

Copper adduct

M+222.2

Fmoc-On

M-251

C-deletion

M-266

T-deletion

M-275

A-deletion

M-291

G-deletion

Expedite 8900 PNA Chemistry Users Guide

C-5

Chapter C

PNA Reference Information

Table C-3 Mass Spectrum Peaks (Continued)


Ion

Description

M+43

Capped sequence

M+16

Oxidation of biotin

Table C-4 Mass Differences Caused by Sequence


Entry Errors
Mass Difference (Da)

C-6

Applied Biosystems

Entry Error

+9

A entered instead of T

-9

T entered instead of A

+15

T entered instead of C

-15

C entered instead of T

+16

G entered instead of A

-16

A entered instead of G

+24

A entered instead of C

-24

C entered instead of A

+25

G entered instead of T

-25

T entered instead of G

+40

G entered instead of C

-40

C entered instead of G

MALDI-TOF Methods

Table C-5 Common Masses


Residue

Mass (Da)

A residue

275.27

C residue

266.25

G residue

291.27

T residue

251.24

O Spacer residue

145.16

NH2 (Terminal Amide)

16

Fmoc

223

Acetyl

43

Expedite 8900 PNA Chemistry Users Guide

C-7

Chapter C

PNA Reference Information

C.4 Waste Composition for Fmoc


PNA Synthesis (2.0 mol Scale)
Table C-6 lists information you need to prepare and budget for
the disposal of wastes produced during synthesis.

Table C-6 Waste Composition for Fmoc PNA Synthesis


(2.0 mol Scale)
Chemical

N,N-Dimethylformamide
(DMF)

92.6

N-Methylpyrolidinone (NMP)

1.6

Piperidine

4.9

N,N-Diisopropylethylamine
(DIEA)

0.04

2,6-Lutidine

0.25

Acetic Anhydride

0.2

HATU

0.2

Fmoc PNA monomer

0.2

Total volume per cycle = 5.54 mL

C-8

% Composition

Applied Biosystems

DTechnical Support and


Training

This appendix contains the following sections:


D.1

Contacting Technical Support .......................... D-2

D.2

Obtaining Technical Documents ...................... D-9

D.3

Obtaining Customer Training Information........ D-11

Expedite 8900 PNA Chemistry Users Guide

D-1

Appendix D

Technical Support and Training

D.1 Contacting Technical Support


Overview

You can contact Applied Biosystems for technical support:


By e-mail
By telephone or fax
Through the Applied Biosystems Technical Support
web site

NOTE: For information on obtaining technical documents


such as Applied Biosystems user documents, MSDSs, and
certificates of analysis, see Obtaining Technical
Documents on page -9.

By E-mail

You can contact technical support by e-mail for help in the


product areas listed below.

Product/Product Area

E-Mail Address

Genetic Analysis (DNA Sequencing)

galab@appliedbiosystems.com

Sequence Detection Systems and PCR

pcrlab@appliedbiosystems.com

Protein Sequencing, Peptide, and DNA Synthesis corelab@appliedbiosystems.com

Biochromatography
PerSeptive DNA, PNA and Peptide Synthesis
systems
FMAT 8100 HTS System
CytoFluor 4000 Fluorescence Plate Reader
Mariner Mass Spectrometers
Voyager Mass Spectrometers
Mass Genotyping Solution 1 (MGS1) System

tsupport@appliedbiosystems.com

LC/MS
(Applied Biosystems/MDS Sciex)

support@sciex.com

Chemiluminescence (Tropix)

tropix@appliedbiosystems.com

D-2

Applied Biosystems

Contacting Technical Support

By telephone or
fax (North
America)

To contact Applied Biosystems Technical Support in North


America, use the telephone or fax numbers in the table below.

NOTE: To schedule a service call for other support needs,


or in case of an emergency, dial 1.800.831.6844, then
press 1.

Product/Product Area

Telephone

Fax

ABI PRISM 3700 DNA Analyzer

1.800.831.6844, then
press 8a

1.650.638.5981

DNA Synthesis

1.800.831.6844, press 2,
then press 1a

1.650.638.5981

Fluorescent DNA Sequencing

1.800.831.6844, press 2,
then press 2a

1.650.638.5981

Fluorescent Fragment Analysis


(including GeneScan applications)

1.800.831.6844, press 2,
then press 3a

1.650.638.5981

Integrated Thermal Cyclers


(ABI PRISM 877 and Catalyst 800
instruments)

1.800.831.6844, press 2,
then press 4a

1.650.638.5981

ABI PRISM 3100 Genetic Analyzer

1.800.831.6844, press 2,
then press 6a

1.650.638.5981

Peptide Synthesis
(433 and 43 x Systems)

1.800.831.6844, press 3,
then press 1a

1.650.638.5981

Protein Sequencing
(Procise Protein Sequencing
Systems)

1.800.831.6844, press 3,
then press 2a

1.650.638.5981

Expedite 8900 PNA Chemistry Users Guide

D-3

Appendix D

Technical Support and Training

Product/Product Area
PCR and Sequence Detection

Telephone
1.800.762.4001, then
press:

Fax
1.240.453.4613

1 for PCRa
2 for TaqMan applications
and Sequence Detection
Systems including ABI
Prism 7700, 7900, and
5700a
6 for the 6700 Automated
Sample Prep Systema
or
1.800.831.6844, then
press 5a
Voyager MALDI-TOF
Biospectrometry Workstations

1.800.899.5858, press 1,
then press 3b

1.508.383.7855

Biochromatography
(BioCAD, SPRINT, VISION, and
INTEGRAL Workstations and
POROS Perfusion Chromatography
Products)

1.800.899.5858, press 1,
then press 4b

1.508.383.7855

Expedite Nucleic Acid Synthesis


Systems

1.800.899.5858, press 1,
then press 5b

1.508.383.7855

Peptide Synthesis (Pioneer and


9050 Plus Peptide Synthesizers)

1.800.899.5858, press 1,
then press 5b

1.508.383.7855

Mariner ESI-TOF Mass


Spectrometry Workstations
Mass Genotyping Solution 1 (MGS1)
System

D-4

Applied Biosystems

Contacting Technical Support

Product/Product Area

Telephone

Fax

PNA Custom and Synthesis

1.800.899.5858, press 1,
then press 5b

1.508.383.7855

FMAT 8100 HTS System

1.800.899.5858, press 1,
then press 6b

1.508.383.7855

Chemiluminescence (Tropix)

1.800.542.2369
(U.S. only),
or 1.781.271.0045c

1.781.275.8581

LC/MS
(Applied Biosystems/MDS Sciex)

1.800.952.4716

1.508.383.7899

CytoFluor 4000 Fluorescence Plate


Reader

a. 5:30 A.M. to 5:00 P.M. Pacific time.


b. 8:00 A.M. to 6:00 P.M. Eastern time.
c. 9:00 A.M. to 5:00 P.M. Eastern time.

By telephone or
fax (outside North
America)

To contact Applied Biosystems Technical Support or Field


Service outside North America, use the telephone or fax
numbers below.

Region

Telephone

Fax

Africa and the Middle East


Africa (English Speaking; Fairlands,
South Africa)

27 11 478 0411

27 11 478 0349

Africa (French Speaking; Courtaboeuf 33 1 69 59 85 11


Cedex, France)

33 1 69 59 85 00

South Africa (Johannesburg)

27 11 478 0349

27 11 478 0411

Expedite 8900 PNA Chemistry Users Guide

D-5

Appendix D

Technical Support and Training

Region
Middle Eastern Countries and North
Africa (Monza, Italia)

Telephone

Fax

39 (0)39 8389 481

39 (0)39 8389 493

Australia (Scoresby, Victoria)

61 3 9730 8600

61 3 9730 8799

China (Beijing)

86 10 64106608 or
86 800 8100497

86 10 64106617

Hong Kong

852 2756 6928

852 2756 6968

India (New Delhi)

91 11 653 3743/3744

91 11 653 3138

Korea (Seoul)

82 2 5936470/6471

82 2 5936472

Malaysia (Petaling Jaya)

60 3 79588268

603 79549043

Singapore

65 896 2168

65 896 2147

Taiwan (Taipei Hsien)

886 2 2358 2838

886 2 2358 2839

Thailand (Bangkok)

66 2 719 6405

662 319 9788

Austria (Wien)

43 (0)1 867 35 75 00

43 (0)1 867 35 75 11

Belgium

32 (0)2 532 4484

32 (0)2 582 1886

Eastern Asia, China, Oceania

Europe

Czech Republic and Slovakia (Praha) 420 2 35365189

420 2 35364314

Denmark (Naerum)

45 45 58 60 00

45 45 58 60 01

Finland (Espoo)

358 (0)9 251 24 250

358 (0)9 251 24 243

France (Paris)

33 (0)1 69 59 85 85

33 (0)1 69 59 85 00

Germany (Weiterstadt)

49 (0) 6150 101 0

49 (0) 6150 101 101

Hungary (Budapest)

36 (0)1 270 8398

36 (0)1 270 8288

Italy (Milano)

39 (0)39 83891

39 (0)39 838 9492

Norway (Oslo)

47 23 12 06 05

47 23 12 05 75

D-6

Applied Biosystems

Contacting Technical Support

Region

Telephone

Fax

Poland, Lithuania, Latvia, and Estonia 48 22 866 4010


(Warszawa)

48 22 866 4020

Portugal (Lisboa)

351.(0)22.605.33.14

351.(0)22.605.33.15

Russia (Moskva)

7 502 935 8888

7 502 564 8787

South East Europe (Zagreb, Croatia)

385 1 34 91 927/838

385 1 34 91 840

Spain (Tres Cantos)

34.(0)91.806.1210

34.(0)91.806.12.06

Sweden (Stockholm)

46 (0)8 619 4400

46 (0)8 619 4401

Switzerland (Rotkreuz)

41 (0)41 799 7777

41 (0)41 790 0676

The Netherlands (Nieuwerkerk a/d


IJssel)

31 (0)180 392400

31 (0)180 392409 or
31 (0)180 392499

United Kingdom (Warrington,


Cheshire)

44 (0)1925 825650

44 (0)1925 282502

All other countries not listed


(Warrington, UK)

44 (0)1925 282481

44 (0)1925 282509

81 3 5566 6230

81 3 5566 6507

Caribbean countries, Mexico, and


Central America

52 55 35 3610

52 55 66 2308

Brazil

0 800 704 9004 or


55 11 5070 9654

55 11 5070 9694/95

Argentina

800 666 0096

55 11 5070 9694/95

Chile

1230 020 9102

55 11 5070 9694/95

Uruguay

0004 055 654

55 11 5070 9694/95

Japan
Japan (Hacchobori, ChuoKu, Tokyo)
Latin America

Expedite 8900 PNA Chemistry Users Guide

D-7

Appendix D

Technical Support and Training

Through the
Technical Support
web site

To contact Technical Support through the Applied Biosystems


web site:
T

1.

Access the Applied Biosystems Technical Support web


site at www.appliedbiosystems.com/techsupp.

2.

Under the Troubleshooting heading, click Support


Request Forms, then select the support region for the
product area of interest.

3.

In the Personal Assistance form, enter the requested


information and your question, then click Ask Us RIGHT
NOW.

4.

In the Customer Information form, enter the requested


information, then click Ask Us RIGHT NOW.

Within 24 to 48 hours, you will receive an e-mail reply to your


question from an Applied Biosystems technical expert.

D-8

Applied Biosystems

Obtaining Technical Documents

D.2 Obtaining Technical Documents


Overview

Ordering
documents by
telephone

You can obtain technical documents, such as Applied


Biosystems user documents, MSDSs, certificates of analysis,
and other related documents for free, 24 hours a day. You can
obtain documents:
By telephone
Through the Applied Biosystems Technical Support web
site
To order documents by telephone:
1.

From the U.S. or Canada, dial 1.800.487.6809, or from


outside the U.S. and Canada, dial 1.858.712.0317.

2.

Follow the voice instructions to order documents (for


delivery by fax).

NOTE: There is a limit of five documents per fax request.

Obtaining
documents
through the
web site

To view, download, or order documents through the Applied


Biosystems Technical Support web site:
1.

Access the Applied Biosystems Technical Support web


site at www.appliedbiosystems.com/techsupp.

2.

Under the Resource Libraries heading, select the type of


technical document you want.

3.

In the search form, enter or select search criteria, then


click Search.

Expedite 8900 PNA Chemistry Users Guide

D-9

Appendix D

Technical Support and Training

4.

In the results screen, do any of the following:


Click

to view a PDF version of the document.

Right-click
, then select Save Target As to
download a copy of the PDF file.
Select the Fax check box, then click Deliver
Selected Documents Now to have the document
faxed to you.
Select the Email check box, then click Deliver
Selected Documents Now to have the document
(PDF format) e-mailed to you.

NOTE: There is a limit of five documents per fax request,


but no limit on the number of documents per e-mail request.

D-10

Applied Biosystems

Obtaining Customer Training Information

D.3 Obtaining Customer Training


Information
The Applied Biosystems Training web site at
www.appliedbiosystems.com/techsupp/training.html
provides course descriptions, schedules, and other
training-related information.

Expedite 8900 PNA Chemistry Users Guide

D-11

Index
A
Acetic anhydride, safety
precautions A-2
Activation and coupling, Fmoc 1-4
Activator (ACT), safety precautions A-3
Activator, preparing 3-7
Aggregation, cause of 1-11, 1-13
Amino acid, preparing 3-7
Analysis of PNA 4-6
Applied Biosystems
world wide web address xi

priming when switching 3-8, 3-9


selecting 3-3
Clamps, PNA 1-15
Cleavage 4-3
Cleavage and deprotection solution,
safety precautions A-4
Columns
installing 3-19
provided in kit 3-7
removing before priming 3-16, 3-18
Customer training, information D-11
Cystosine 1-14

Base solution, safety precautions A-3


Binding specificity 1-9
Biotin
and purine content 1-12
labeling PNA with 4-15
removing from oligomer 3-6
Blanket gas 3-11
Bottle changing tool 3-14
Bottle locations 3-11
Bottle volume, resetting 3-14

Deblock Solution (Dblk), safety


precautions A-4
Deprotection 4-3
Dichloromethane (DCM) (methylene
chloride), safety
precautions A-4
Dimethylformamide (DMF), safety
precautions A-6
DNA structure 1-2

C
Capping solution (CAP), safety
precautions A-3
Certificates of analysis
obtaining D-9
Chemicals, ordering B-1
Chemistry
available in version 2.2 2-2
default, changing 3-4

E
E-mail
contacting technical support D-2
E-mail address, Technical
Publications xi
Extinction coefficients, PNA 4-11

Expedite 8900 PNA Chemistry Users Guide

Index-1

I
N
D
E
X

I
N
D
E
X

F
Field Service in North America,
contacting D-3
Fluorescein
and purine content 1-12
labeling PNA with 4-15
removing from oligomer 3-6
Flushing requirements 3-8
Fmoc
definition 1-3
removing 4-2
Fmoc-AEEA-OH spacer
function 3-6
number required for labeling 1-12
preparing 3-7

H
Handling PNA 4-10
HATU, safety precautions A-3
Hazard information, reagents A-1
Holding a synthesis 3-20
Homo thymine oligomers 1-13
How to Use This Guide ix
HPLC methods C-4

I
Installing software 2-2
Instrument stop key 3-19
Interrupting a synthesis 3-20
Ionic strength 1-14

L
Labeling
guidelines 1-12
number of Fmoc-AEEA-OH spacer

Index-2

Applied Biosystems

required 1-12
PNA in solution 4-15
sequences, and purine
content 1-12
support-bound PNA 4-12
Length, PNA oligomer 1-11
Loading reagents 3-11
Lutidine, safety precautions A-6

M
Maintenance 5-4
MALDI-TOF methods C-5
Melting experiments 4-11
Molecular weights of monomers 1-3,
1-4
Monomers
diluent, safety precautions A-8
Expedite PNA 1-3
extinction coefficients 4-11
molecular weights 1-3, 1-4
ordering B-1
preparing 3-7
safety precautions A-8
MSDSs, obtaining D-9

N
N,N- Diisopropylethylamine, safety
precautions A-5
NMP, safety precautions A-7

O
Oligomer, PNA, see PNA
Optical density, conversion to
concentration or weight C-2
Ordering information B-1
Orientation 1-10
O-spacer, see Fmoc-AEEA-OH spacer

P
Part numbers B-1
Piperidine, safety precautions A-8
PNA
analysis 4-6
characteristics of 1-7
clamps 1-15
cleavage and deprotection 4-3
cytosine content 1-14
definition 1-2
drying 4-2
extinction coefficients 4-11
handling 4-10
homo thymine 1-13
labeling in solution 4-15
labeling support-bound 4-12
labels, effect of 1-12
oligomer length 1-11
orientation 1-10
purification 4-8
purine content 1-11
quantitation 4-11
sequence design 1-10
sequences to avoid 1-13
stability 1-13
strand invasion 1-9, 1-14
structure 1-2
thermal stability 1-7
triplex formation 1-13
PNA synthesis
see also Synthesis
cycle 1-6
monomers 1-3
on Expedite instrument 1-5
overview 1-2
protocol available 1-5
troubleshooting 5-2
waste composition C-8
Post-synthesis procedures
analysis 4-6
cleavage and deprotection 4-3

drying the resin 4-2


labeling 4-12
PNA handling 4-10
purification 4-8
quantitation 4-11
Powering up 3-3
Preface ix
Preparing reagents 3-6
Prime
after 48 hour idle period 3-15, 3-18
after loading reagents 3-15, 3-16
before running synthesis 3-15
fluidics system 3-15
monomers, volume used 3-17
removing column before 3-16, 3-18
when switching chemistries 3-8,
3-9
Protecting group, fmoc
definition 1-3, 4-2
Protocol scale, PNA 1-5
Purification of PNA 4-8
Purine content
and effect of labels 1-12
recommended 1-11

Q
Quantitation of PNA 4-11

R
Reagents
contents of kit 3-6
flushing requirements 3-8
loading 3-11
location 3-13
ordering B-1
preparing 3-6
resetting volume 3-14
safety information A-1
Related documents x

Expedite 8900 PNA Chemistry Users Guide

Index-3

I
N
D
E
X

I
N
D
E
X

Resetting bottle volume 3-14


Resin, drying 4-2
Resistance 1-9

S
Safety information A-1
Sequence
design of PNA 1-10
direction 3-5
entering 3-5
structures to avoid 1-13
Software
if PNA not displayed 2-3
installing 2-2
version number 2-2
Spacer, Fmoc-AEEA-OH, see FmocAEEA-OH spacer
Specificity of binding 1-9
Stoichiometry
duplex 1-10
triplex 1-13
Stop button 3-19
Stopping a synthesis 3-19
Strand invasion 1-9, 1-14
Synthesis
see also PNA synthesis
entering sequence 3-5
flushing the system 3-8
interrupting 3-20
key steps 3-2
loading reagents 3-11
monitoring 3-19, 3-20
overview 3-2
post-synthesis procedures 4-2
preparing reagents 3-6
priming 3-8, 3-9, 3-15
running 3-19
selecting chemistry 3-3
starting 3-19
steps in 3-2

Index-4

Applied Biosystems

stopping 3-19

T
Technical documents, obtaining D-9
Technical support, contacting
Africa and the Middle East D-5
Eastern Asia, China, Oceania D-6
e-mail D-2
Europe D-6
Japan D-7
Latin America D-7
telephone or fax in North
America D-3
TFA, safety precautions A-9
Thermal stability of PNA 1-7
Thiol-specific labels 4-17
Triplex formation 1-13
Troubleshooting 5-2

U
Users guide structure x
Users guide structure ix

V
Version number of software 2-2
Volume, resetting reagent 3-14

W
Wash solutions (WshA, WshB), safety
precautions A-10
Waste composition C-8
Waste disposal A-1
World wide web address,Applied
Biosystems xi

Headquarters
850 Lincoln Centre Drive
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Phone: +1 650.638.5800
Toll Free: +1 800.345.5224
Fax: +1 650.638.5884
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support and applications personnel, reaches
into 150 countries on six continents. For
international office locations, please call our
local office or refer to our web site at
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