Basics of Gene Cloning

&
Cloning Enzymes

Lecture 1 BT-620

What is DNA cloning?

DNA sequencing has depended on advances in technology, starting with
making recombinant DNA
In recombinant DNA, nucleotide sequences from two different sources, often
two species, are combined in vitro into the same DNA molecule
Methods for making recombinant DNA are central to genetic engineering, the
direct manipulation of genes for practical purposes
Sequencing of the human genome was largely completed by 2003

Bacterium

Why Clone DNA?

Gene inserted into

plasmid

A particular gene can be isolated

and its function determined
Sequences of DNA
identified & analyzed

can

Bacterial
chromosome

Plasmid
Recombinant
DNA (plasmid)

Gene of
interest
Plasmid put into
bacterial cell

be

Organisms can be engineered for

specific purposes, e.g. insulin
production, insect resistance, etc.
The concept of Pharmanimals
Basic Research

DNA of
chromosome

Recombinant
bacterium

Protein/enzyme/RNA function can

be investigated
Mutations can be identified, e.g.
gene defects related to specific
diseases

Cell containing gene

of interest

Host cell grown in culture

to form a clone of cells
containing the cloned
gene of interest
Gene of
interest

Protein expressed
by gene of interest

Copies of gene

Basic
research
on gene

Gene for pest

resistance inserted
into plants

Protein harvested
Basic research and
various applications

Gene used to alter

bacteria for cleaning
up toxic waste

Protein dissolves
blood clots in heart
attack therapy

Basic
research
on protein

Human growth hormone treats stunted

growth

Recombinant DNA Products

Human Insulin
Human growth factor
Hepatitis B virus vaccine

Streptokinase
Recombinant antibodies
Recombinant Enzymes

Safflower

Cloning: Genes or Organisms

Genes, Plants &

Whole Organisms

Cloning Organisms

Dolly the sheep

Snuppy

Natures Cloning Experiment!!

Dionne quintuplets

ETHICS REGARDING RECOMBINANT DNA TECHNOLOGY

Regulations to monitor rDNA

While some animals have been cloned Human cloning is banned

International and National rules that regulate rDNA organisms
Asilomar Recommendation (1975)
Recombinant DNA Advisory Committee (RAC) , USA
Genetic Manipulation Advisory Group (GMAG) , UK
Modified restrictions (1978)
Special permissions, facilities and supervisory bodies to oversee & regulate
the use rDNA organisms and products

Bioterrorism

Anthrax (Bacillus anthracis)

1993 (Japan)
2001 (USA)

Botulinism
(Clostridium botulinum)

Plague
(Yersinia pestis)

Smallpox
(Variola major, Variola Minor)

Viral hemorrhagic fevers, tularemia (bacterial)

DNA

DNA (deoxyribonucleic acid) was first isolated in 1869 by the Swiss

chemist Johann Frederick Miescher.
He separated nuclei from the cytoplasm of cells, and then isolated an acidic
substance from these nuclei that he called nuclein.
Miescher showed that nuclein contained large amounts of phosphorus and
no sulphur, characteristics that differentiated it from proteins.

In what proved to be a remarkable insight, he suggested that :

if one wants to assume that a single substance. . . is the specific cause of
fertilization then one should undoubtedly first of all think of nuclein.

In 1926, based on the idea that DNA contained approximately equal

amounts of four different groups, called nucleotides, and by
determining the type of linkage that joined the nucleotides together,
Levene and Simms (1926) proposed a tetranucleotide structure to
explain the chemical arrangement of nucleotides within nucleic
acids.

In 1928, Frederick Griffith performed demonstrated that heat killed virulent strains of
the bacterium Streptococcus pneumoniae could transform avirulent strains.

Avery, MacLeod and McCarty,

1944
showed
that
DNA was
responsible for this change
Avery,
MacLeod
and
McCarty,
1944
Additional proof that DNA was the genetic material came from

Alfred Hershey and Martha Chase 1952 and their experiments with T2 bacteriophage

Curiously, however, Hershey and Chase seemed somewhat skeptical about their
findings, concluding their paper by saying that :
. . . protein has no function in phage multiplication, and that DNA has some function

In the early 1950s, Chargaffs rule proposed that for any species :

A = T and G = C
sum of the purines = sum of the pyrimidines
Adenine (6-amino purine) + Guanine ( 2-amino-6-oxy purine) =
Thymine (2,4-dioxy-5-methyl pyrimidine) + Cytosine (2-oxy-4-amino pyrimidine)
the percentage of (C + G) does not necessarily equal the percentage of (A +
T).

As early as 1938 William Astbury applied the Xray crystallography

to fibres of DNA. By 1947, he had detected a periodicity (or
repeating unit) within DNA of 0.34 nm.
Between 1950 and 1953, Rosalind Franklin obtained improved X-ray
data from highly purified samples of DNA. Her work confirmed the
0.34 nm periodicity, and suggested that the structure of DNA was
some sort of helix.
Linus Pauling and Robert Corey used Franklins data, together with
that of others, to propose that DNA was a triple helix with the
phosphates near the centre of the axis and the bases on the outside
(Pauling and Corey, 1953).

James Watson and Francis Crick(1953)

(a) Two long polynucleotide chains coiled around a central axis, forming a righthanded double helix.
(b) The two chains are antiparallel; that is, each chain has a specific
orientation, and these run in opposite directions.
(c) The bases of both chains are flat structures, lying perpendicular to the axis.
They are stacked on one another, 0.34 nm apart, and are located on the
inside of the helix.
(d) The nitrogenous bases of opposite strands are paired to one another by
hydrogen bonds.
(e) Each complete turn of the helix is 3.4 nm long. This means that just over ten
bases from each strand (10.4 bp) form one complete turn of the helix.

(f) Along the molecule, alternating larger major grooves and smaller minor
grooves are apparent.
(g) The double helix measures approximately 2 nm in diameter.

The joining of nucleotides. The joining of an adenine and a guanine nucleotide. The
phosphates on the sugar ring of guanine are designated as , or . In the formation of the
dinucleotide, pyrophosphate (representing the and phosphates) is lost and the
phosphodiester bond links the 3 hydroxyl to the phosphate on the 5 carbon atom of the
sugar. DNA molecules invariably have a free 5 phosphate and 3 hydroxyl

Reversible Denaturation of DNA, Hyperchromic Effect

Heating DNA to almost boiling temperatures will break the hydrogen bonding
between bases but not the backbone..resulting in the separation of the two
strands of the helix.
Slow cooling can renature the two strands back to the original helix.
DNA absorbs light at a wavelength of 260 nm due to the presence of alternating
single and double bonds in the DNA bases.
A solution of double-stranded, native DNA, with a concentration of 0.05 mg/mL,
has an absorbance (or optical density, OD) of about 1.0 at the 260 nm peak.
If the same solution is denatured to single strands the OD will be 1.5.
This is known as the Hyperchromic Effect

The stacking of the base pairs in double stranded DNA has the effect of dampening
the absorption of individual nucleotides due to the competing interactions of adjacent
base pairs in the stack.
In the single-stranded form, no such competition occurs and consequently singlestranded DNA absorbs light to greater extent than its double-stranded counterpart.

Commonly used Techniques

1. DNA is extracted
from blood/tissue

2. PCR/RE digestion of DNA & vector

3. Ligation in the vector

How is DNA cloned ?

6. Expression in a
suitable host

5. Screening
for positives

4. Transformation into a host

Genomic DNA Libraries

cDNA libraries
Expression libraries

Genomic DNA

Genomic DNA is the entire genomic DNA content of an organism

Suitable for determining genomic DNA sequence: Looking at regulatory

elements or entire genes (exons & introns)
Requires chromosomal DNA isolation.

1. GENOMIC DNA: Is suitable for cloning genes of bacterial/viral origin

For studying gene regulatory elements
Limitations:

Looking for a Needle in the Haystack?!! The frequency of the required

gene is a minute part of the genomic DNA.
&
Cloning the Elephant?!! Eukaryotic DNA has intervening (intron)
sequences.

Genomic DNA libraries

A Genomic DNA library basically contains the entire genetic information in
pieces cloned within a suitable vector!

Genomic DNA Isolation

A source of cells
Lysis by buffered solution containing detergent

Digestion of cellular proteins using Proteinase K

Removal of these proteins by either Phenol, Chloroform or salt
Precipitation of Genomic DNA by Isopropanol
Removal of any excess salts by Ethanol wash
Resuspension in a suitable buffer

mRNA / complementary DNA (cDNA): The entire coding region of

eukaryotic genes can be obtained from mRNA .

Limitations:
Cells/tissues that specifically express the required mRNA are needed.

cDNA and cDNA libraries

(Complementary DNA)
Why is it needed?
What does this involve?

Purification of mRNA
Synthesis of first strand (cDNA)
Synthesis of second strand
Cloning

Transcription
Exons

Exons

Exons

Exons

Introns

Introns

Introns

DNA

AAAA mRNA

AAAA Intron splicing

AAAA

Processed mRNA

Purification of RNA
AAAA

AAAA

AAAA

Cell Lysis

AAAA

AAAA

Total RNA
t, r& m

TTTT
TTTT
AAAA
TTTT
TTTT
TTTT
AAAA
TTTT
TTTT
TTTT
TTTT
TTTT

Oligo dT sepharose

AAAA

AAAA

Protein pptn.

AAAA

Removal/
pptn of DNA
AAAA
TTTT
AAAA
TTTT
AAAA
TTTT
AAAA

TTTT
AAAA
TTTT
AAAA
TTTT
AAAA

TTTT

AAAA

TTTT

AAAA

tRNA, rRNA

TTTT

AAAA

TTTT

mRNA

Purification of RNA
A cell expressing the required protein
A clean environment. Sterile glassware, plasticware free of nucleases, RNA inhibitors
Purification involves the lysis of the cell
Removal of contaminating proteins by Guanidinium isothiocyanate and subsequently
chloroform extraction
Removal of contaminating DNA by extraction with acid phenol and Rnase free DNase
Total RNA prepared by such a method may now be used to purify mRNA

The fact that eukaryotic mRNA has a poly A tail is exploited.

A oligo dT sequence attached to sepharose / magnetic particles is used to fish out the
cellular mRNA
This mRNA can now be used to prepare cDNA

C G
A T A G T C G A A T T C C A A T C C C GA A A A
RNA
Pol

G C T A T C A G C T T C C G G T T A G G G C T T T T

5
3

CRNA
G A U A G U C G A A G G C C A A U C C CRev.G A A A A
Pol

3 mRNA

Transcr.

G CC T A T C A G C TT T CC C GG G T TT AA GG G GG C T T T TT 5 cDNA

5
3

cDNA-mRNA hybrid

3 AAAA
TTTT
5

mRNA to cDNA

5
3 AAAA
5 TTTT

ds cDNA

Rnase H
3 endonuclease

DNA Polymerase I
5 to 3 polymerase activity
3 to 5 exonuclease activity

DNA Ligase

Commonly used Vectors

Plasmid Vectors: Circular, ds DNA that is capable of independent

replication.
It is widely used in rDNA techniques
Phage based vectors: These are suitable for cloning larger
fragments of DNA.

Isopycnic CsCl gradient centrifugation of DNA molecules

The DNA sample is mixed with a substance (caesium chloride, CsCl) that is capable of
forming a self-generating uniform density gradient during the centrifugation process.
DNA molecules will sediment only to the position in the centrifuge tube at which the
gradient density is equal to its own density, and they will remain there to form a band.
After the DNA band has formed, it is removed from the centrifuge tube and the CsCl
removed by precipitating the DNA with alcohol.
Genomic DNA may be further purified by using positively charged matrices such as
silica or DEAE in the presence of high salt that permits DNA binding but prevents the
binding of contaminants.

Isopycnic centrifugation of DNA molecules

The inclusion of ethidium bromide in the centrifugation mix

results in its binding to DNA. The topological constraints of
supercoiled DNA mean that closed-circular DNA molecules (e.g.
plasmids) are able to bind only limited amounts of ethidium
bromide.
Nicked DNA circles, and linear DNA fragments, are able to bind
much more ethidium bromide and are extensively unwound in
its presence.
This results in nicked and linear DNA being less dense than
their supercoiled equivalent, and consequently being separated
in a different location on a CsCl/EtBr gradient.
The main drawback is the length of the procedure

Plasmid DNA Isolation

Plasmid isolation is based on the alkaline lysis method

developed by Birnboim and Doly (1979)
The initial method utilized phenol:chloroform,
purifications and ethanol precipitation
Current spin column based protocols depend upon DNA
binding to positively charged matrices.

Current spin column based protocols depend upon DNA binding

to positively charged matrices such as silica.

DNA Binding
High Salt Wash
Elution

Analysis of Nucleic Acids

Electrophoresis

DNA is negatively charged, when subjected to an constant electric field fragments will move
to the anode or positive terminal.
Size differences are distinguished by friction, smaller fragments will move faster through the gel.

The gel as a Sieve.

Analysis of Oligonucleotides
PAGE is a common method used for the separation of proteins but it is also used for the
separation of nucleic acids (Raymond and Weintraub, 1959).
The pore size of the gel may be varied by the concentration of the Acrylamide used.
This method is suitable for separating fragments of upto 1000 bp, differences of upto 1bp
can be detected using this method
Larger fragments cannot be separated by this method because they cannot enter the
pores of the gel.

Analysis of larger DNA fragments

Agarose gel electrophoresis

Agarose is a linear polysaccharide made up of

the basic repeat unit agarobiose, which
comprises alternating units of galactose and
3,6-anhydrogalactose.
The resolution of an agarose gel is inferior to
that obtained by PAGE.
The bands formed in an agarose gel are
relatively fuzzy because the pore size cannot
be accurately controlled.

Reptation

Topology determines mobility

The charge per unit length of DNA is constant, and in

a gel you would expect friction to increase in direct
proportion to the length of DNA a 2000 bp fragment
should experience twice as much friction as a 1000
bp fragment.
Therefore there should be a direct , inverse
relationship between the mass of DNA and its
migration rate through a gel.
Migration distance is however inversely proportional
to the log of size, that is large fragments > 30 Kb are
not well resolved.

Pulse Field Gel Electrophoresis

Is a method to resolve large DNA fragments( 10,000 kb)

Instead of a constant electric field, the direction of the
electric field is constantly changed. The time of the
pulse determines the separation of fragments.
Shorter fragments resolve with a short pulse and
longer fragments with a longer pulse
Modifications of PFGE are
1. Field inversion gel electrophoresis (FIGE), where the
polarity of the electrodes is periodically inverted the
during electrophoresis.
&
2. Contour-clamped homogeneous electric field (CHEF)
Here, electrophoresis reorients the DNA at smaller
oblique angle, generally between 96 and 120.

http://www.bio.davidson.edu/Courses/genomics/method/pulse_field.html

http://www.bio.davidson.edu/Courses/genomics/method/pulse_field.html

+
Field inversion gel electrophoresis
(FIGE)
Fragments under 2000kb

Contour-clamped homogeneous electric field

(CHEF)

Extraction of DNA fragments from Gel --- When is this of use??

PCR

Insert from
vector

PCR

Insert from
vector

500bp

500bp

Excise fragment

500bp

Agarase

DNA eluted
Purification

Detection of DNA by Southern Blotting

The original method of blotting was developed by Ed Southern in 1975.
This method detects DNA fragments in an agarose gel that are complementary to a
given nucleic acid sequence (Southern, 1975).
The basic steps are:
Running the DNA gel.
Denaturation using alkali
Transfer and immobilization on a suitable membrane (either electrophoretically or
through capillary action)
The DNA is fixed on the membrane by baking at 80 C or UV cross-linking.
Hybridization with complementary radiolabelled probe either single stranded DNA/ RNA.

Binding occurs by non-covalent hydrogen bonding.

The membrane is then washed extensively to remove non-specifically bound probe.
The specific interactions are detected by exposing the membrane to X-ray film

Detection of complemenatry Nucleic Acid

The original method of blotting was developed by Ed Southern in 1975.
Southern Blotting to detect the presence of complementary /related sequences

Separation of
fragments on gel
Denaturation with
alkali

Transfer
Complementary sequences
Immobilization to membrane
can be detected on X-ray film
by UV crosslinking.
Incubation with labeled single
stranded DNA or RNA as probe
Washing to remove nonspecificically bound probe

Colony hybridization : Screening of libraries

Library

Replica plating

Transfer to NC

Probe hybridization

Variants of Southern Blotting

Northern Blotting: Detection of RNA sequences using a similar methodology

Western Blotting: Detection of Proteins
South Western Blotting: Detection of DNA binding proteins
Proteins are separated as above
Incubated with radiolabeled DNA fragments
Detection of the label will indicate that the particular protein interacts with DNA

Western Blotting / Immunoblotting

A technique to detect specific proteins based on
antibody detection (Towbin et al, 1979)
Gel NC
-ve

+ve

Separation on Gel

Proteins Transferred to NC
Blotting
sheets

Filter Pads
Transfer

Detection with enzyme conjugated antibodies

V
V

V
Direct

Indirect

Insoluble coloured product indicates the presence of the protein

Antibody based screening of expression libraries

V
Library

Replica plating

Transfer to NC

Antibody based detection

DNA is commonly cloned in plasmid vectors

The basic steps involved in making a recombinant molecule are

Cutting the vector and DNA as required

Pasting them together
Amplifying the rDNA
The cutting and pasting is done using a variety of enzymes.
These vectors carry drug resistance markers that can be used to select
recombinants

1. DNA is extracted
from blood/tissue

2. PCR/RE digestion of DNA & vector

3. Ligation in the vector

How is DNA cloned ?

6. Expression in a
suitable host

5. Screening
for positives

4. Transformation into a host

Common Terminology of Cloning Enzymes

1. Kinases
2. Phosphatases
3. Reverse transcriptase

4. Endonucleases
5. Exonucleases
6. Polymerase

Phosphatases & Kinases

Phosphatases remove the phosphate group from nucleic acids or proteins.
These enzymes are most active at alkaline pH - hence the name.

In molecular biology phosphatases are used to remove the 5 PO4 from a DNA strand
The commonly used phosphatases are
Bacterial Alkaline phosphatase (BAP)
Calf Intestinal alkaline phosphatase (CIAP)
Shrimp Alkaline phosphatase

5
5

Bacterial alkaline Phosphatase (BAP) is the most active enzyme but also
the most difficult to destroy after the dephosphorylation reaction

CIAP is the most commonly used in Molecular Biology, though less active
than BAP, it is easily destroyed by heating at 75C in the presence of
EDTA.
The amount of CIAP/ug DNA as well as the time has to be carefully
selected.
Shrimp Alkaline Phosphatase is derived from a cold-water shrimp and is
easily destroyed by heat (65 C for 15 minutes).

What is the use of Phosphatase in Cloning?

Ligation
P

-OH
P

-OH

Ligation

Amp r+

-OH
P

-OH

=
Ligation

Vector self ligation

High background
More screening

What is the use of Phosphatase in Cloning?

-OH
P

+
P

-OH
P

Colonies contain
insert

-OH

=
Ligation

+
Alkaline
phosphatase
Less
background

Ligation

-OH

-OH

No self ligation of vector

Kinase
Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate
from ATP to the 5' end of either DNA or RNA.
It is a product of the T4 bacteriophage, and commercial preparations are usually
products of the cloned phage gene expressed in E. coli.

The enzymatic activity of PNK is utilized in two types of reactions:

The "forward reaction" :
PNK transfers the phosphate from ATP to the 5' end of a polynucleotide (DNA / RNA).
The target nucleotide is lacking a 5' phosphate either because it has been dephosphorylated
or has been synthesized chemically.

5 HO-

-OH 3
ATP

PNK
ADP

5 PO4

-OH 3

The "exchange reaction:

Target DNA or RNA that has a 5' phosphate is incubated with an excess of ADP

PNK will first transfer the phosphate from the nucleic acid onto an ADP,
This forms ATP and leaves a dephosphorylated target.
PNK will then perform a forward reaction and transfer a phosphate from ATP onto the target

nucleic acid.
5PO4

-OH 3

ADP

PNK

ATP

5 HO-

-OH 3

ATP
ADP
5PO4

PNK
-OH 3

What is the use of Kinase in Cloning?

Phosphorylating linker and adapters, or fragments of DNA as a prelude to ligation, which
requires a 5' phosphate.
This includes PCR products of, which are typically generated using non-phosphorylated
primers.

+ PCR products
lack a 5-P

+
PCR products
lack a 5-P

+
Ligation

+
Ligation

 Separation of
fragments on gel
Denaturation
with alkali

Transfer
Immobilization on membrane
Incubation with labeled single
stranded DNA or RNA as probe

Complementary
sequences
detected on X-ray film

In combination Phosphatase and Kinase may be used for Radiolabeling probes

Phosphatase :
Removing 5' phosphates from fragments of DNA prior to labeling with radioactive
phosphate

5 PO4

5 PO4

Kinase:
Radiolabeling oligonucleotides, usually with 32P, for use as hybridization probes.

5 HO-

-OH 3
ATP32

PNK
ADP

P32

-OH 3