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A) Oligonucleotide synthesis
1. What kind of chemistry does Thermo Scientific use?
Thermo Scientific routinely uses solid phase synthesis and performs phosphoramidite chemistry.
repeat cleavage
deprotection
Tr off
oxidation
2%
5. Why does Thermo Scientific sell oligos in OD amounts rather than nmol?
The reason is that the same amount of reagents is needed in order to produce 1 OD260nm of an
oligonucleotide – whether it is a 20mer or a 100mer. In contrast to this, we would need 5 times the
reagents for 1 nmol of a 100mer than for a 20mer.
So in order to keep our base prices independent from the actual oligo length, we have to sell
“length-independent” scales.
Please refer to the following tables for information on oligo yields in OD (Optical Density):
synthesis scale
purification oligo type primer 0.02 0.04 0.2 1.0 10
HPLC unmodified 2 2 4 10 30 300
single modification --- 1 2 5 15 150
double modification --- 0,5 1 2,5 7,5 75
PAGE unmodified --- 0,5 1 3 10 100
single modification --- 0,3 0,5 1,5 5 50
double modification --- 0,2 0,3 0,5 2,5 25
synthesis scale
purification oligo type 0.02 0.04 0.2 1.0 10
HPLC unmodified 1 2 5 15 150
single modification 0,5 1 2,5 7,5 75
PAGE unmodified 0,3 0,5 1,5 5 50
single modification 0,2 0,3 0,7 2,5 25
The amount of oligonucleotide is determined from its base composition and the measured OD
value with the following formula (formula 1):
Formula 1
100 * n [OD]
n [nmol ] =
(1.54 * A +1.17 * G + 0.75 * C + 0.92 * T )
n [OD]: OD value
n [nmol]: amount in nmol
A,G,C,T: number of the respective bases in the oligo
1.54; 1,17; 0,75;0,92: extinction coefficients for each type of base
Multiplying this value by the molecular weight of the oligo reveals the amount in nanogram
(formula 2). 1 OD equals approximately 33 µg ss-DNA (equimolar mixed sequence of all 4
bases).
Division by the volume used for dissolving displays the concentration (formula 3):
Formula 2 Formula 3
n [nmol ]* MW [ g / mol ] 1000 * n [nmol ]
m [µ g ] = c [ pmol / µ l ] = c =
1000 v [µ l ]
m [µg]: amount in µg
n [nmol]: amount in nmol
MW [g/mol]: molecular weight (see certificate)
c [pmol/µl]: concentration
v [µl]: volume of oligo solution
8. How does Thermo Scientific calculate the molecular weight (MW) of an oligonucleotide?
The molecular weight MW of your oligo is calculated from the base composition and its
modifications (formula 4). This formula is used for calculation of the amount in µg and as a
reference value for MALDI –TOF analysis.
Formula 4
Please note that it is not possible to calculate molecular weights for oligos with mixed
bases as such oligos represent a mixture of different individual oligos!
Please note that it is not possible to calculate melting temperatures for oligos with mixed
bases as such oligos represent a mixture of different individual oligos!
For oligos longer than 15 bases, Thermo Scientific use the „Nearest Neighbour“ method (Rychlik et
al, Nucl Acid Res 1990, 18, 6409-6413)
to calculate Tm (formula 5). This method is considered to be one
of the more accurate calculations of Tm for a wide range of oligonucleotide lengths. It takes into
account each pair of neighbouring bases in a sequence, as well as oligonucleotide and salt
concentration:
Formula 5
1000 * ∆H
Tm = − 273.15 +16.6 * log c( K + )
A + ∆S + R * ln(C / 4)
∆H [kcal/mol]: sum of the nearest neighbour enthalpy changes
∆S [cal/mol]: sum of the nearest neighbour entropy changes
A = -10,8 cal: constant entropy factor for helix initiation
R = 1.98 cal/(°C*mol): universal gas constant
C = 250 pmol/l: concentration of oligonucleotide
c(K+) = 50 mmol/l: concentration of monovalent cations
For oligos shorter than 15 bases Thermo Scientific use the simple but therefore more accurate
„Wallace rule“ (formula 6). This equation was developed for hybridising short oligonucleotides to
membrane-bound DNA.
Formula 6
Td = 2 °C * ( A + T ) + 4 °C * (G + C )
Td: temperature at which 50 % of an oligo and its perfect surface-bound complement are in duplex
(salt concentration 0.9 M)
A,G,C,T: number of the respective bases in the oligo
To use this equation for solution based experiments you should add 8 °C to the result.
All current formulae do not take into account any type of modification such as dyes, haptens or
anchor groups. These modifications tend to decrease the Tm value (internal modifications have
higher grade of influence than terminal ones).
If your browser allows JavaScript, you can calculate the Tm value of your oligos using our
Physical parameter calculator on our online ordering page www.thermo.com/oligos.
After dissolving, oligonucleotide solutions have to be stored at temperatures below –20 °C and
stay stable for some months in general. Repeated freeze-thaw cycles have to be avoided to
prevent untimely degradation and contamination of stock solutions. Oligonucleotides with dye
modifications have to be protected from light at all times to prevent bleaching of the dye.
The volume to prepare a 100 µM (= 100 pmol/µl) oligonucleotide solution can be found in the
quality certificate that accompanies every oligonucleotide delivery. In general, oligonucleotides
will dissolve in this volume - when vortexed - within a few minutes. If a standard oligonucleotide
is reluctant to dissolve, careful warming of the solution for 10-30 minutes up to 37°C is
recommended.
For special purity requirements Thermo Scientific offers Ion Exchange Chromatography (IEC), too.
full-length product
(DMT on)
capped
sequences
(DMT off)
At first, capped sequences (by-products during synthesis) and protection groups are being eluted.
The full-length product peak usually appears after 1.5 – 2 minutes.
The black mark in above profile denotes the part of the product peak, that is being collected during
HPLC purification. Shorter products (so-called n-x products) usually follow the main product in the
“tail” of the peak. These aren’t collected.
Oligos with fluorescent or DIG-label can display the following type of HPLC profile:
unmodified
oligo
dye-labelled oligo
desired product
oligo with
degraded dye
The black mark denotes the fraction of the crude product that is being collected. If several dye-
labelled peaks appear, they are collected separately and are analysed by MALDI-TOF Mass
Spectrometry to identify the fraction that includes the desired product with the correct molecular
weight.
The standard collection of modifications is shown in our price list. A broad spectrum of special
modifications is available on request by e-mail to services.oligos@thermo.com
Application
functionalisation assays
Immobilisation on solid
Coupling of additional
Structure-relationship
Fragment Analysis
Cloning / Ligation
Antisence assays
Gene synthesis
Gene silencing
Real-time PCR
modifications
Hybridisation
Mutagenesis
Microarrays
Sequencing
Biological
surfaces
assays
FRET
PCR
Enhanced purification
PAGE x x x
Functional Groups
NH2 (Aminolink) x x x
SH (Thiollink) x x x
PO4 (Phosphate) x x
Biotin x x x
DIG (Digoxigenin) x x
HRP (Horseradish
x x
Peroxidase)
Fluorescence Dyes
Fluorescent dyes x x x x x x
Double-labelled
x x x
fluorescent probes
Dye-dT x x x x x
Dabcyl x x x x
Internal Modifications
PTO (Phosphothioate-
x x x
Oligo)
Methyl-Phosphonate-
x x
Oligo
2´ O-Methyl-RNA x x
2´deoxy Inosine x x x
2´deoxy Uridin x
Biotin-dT x x x x X
Amino-dT x x
Dye-dT x x x
dSpacer x x
halogenated bases
(5-Br-dC, 5´Br-dU, x x
5-I-dC, 5-I-dU)
Special Modifications
Carbon Chain Spacers x x
Ethylenglycol Spacer x x
3´Block (C3-Spacer) x x x x
Inverted End
x x x
(3´-3-linkage)
Asymmetric doubler x x
Symmetric doubler x x
RNA
RNA x x x
si-RNA x
3‘-terminal
Rhodamine Green
Rhodamine Red
Oregon Greens
Bodipy™ dyes
Aminolink C6
Inverted end
Digoxigenin
Marina Blue
Pacific Blue
Fluorescein
Thiolink C3
Phosphate
Texas Red
Cy™ dyes
TAMRA
Dabcyl
6-FAM
AMCA
Biotin
ROX
JOE
5‘-terminal
Aminolink C6
Thiolink C6
Phosphate
Biotin
Digoxigenin
Fluorescein
6-FAM
HEX
TET
TAMRA
JOE
ROX
Cy™dyes
IR™dyes
AMCA
Bodipy™ dyes
Marina Blue
Pacific Blue
Oregon Greens
Rhodamine Green
Rhodamine Red
Texas Red
HRP
If your assay procedure does not otherwise require, Thermo Scientific recommends to attach
modifications to the 5´-end of your oligonucleotide (p.e. most PCR applications are still possible if
the modification is attached to the 5'- end).
In contrast, modifications at the 3'- end usually block this end for further enzymatic reactions
(nevertheless, sensitive applications might detect elongation even of 3'-modified oligonucleotides.
For complete blocking we recommend inverted end or C3-Spacer modification).
6. What does Thermo Scientific charge for wobble bases (degenerate primers)?
Standard wobbles (equimolar ratio of the respective bases) are offered free of charge – even at
the 3’-end of your oligo.
If you need a special mixture (p.e.: 70% A and 30% G), a special handling fee for individual
mixtures applies (price list).
Please order degenerate primers using the official IUB-code (please refer to table on our web site.
An oligo with wobbles represents a mixture of many different oligonucleotides. Thus, no molecular
weight or Tm value can be calculated for these.
Please note: when using degenerate oligos for PCR, please keep in mind that the 100%
homologue to your template is only a small fraction of the total oligo mixture – so an increase of
primer concentration might be necessary to obtain results.