Electrophoresis in DNA sequencing

1. ELECTROPHORESIS IN DNA
SEQUENCING
BY
S.SANDHIYA

2. INTRODUCTION
DNA is a long rod-like molecule which moves
through polyacrylamide gels with limited mobility.
Moreover, DNA has a uniform charge-distribution
which means that this mobility is directly
proportional to the size of the molecule.
For these reasons, electrophoresis of DNA in
polyacrylamide gels allows separation of molecules
differing by as little as a single nucleotide

3. SANGER DIDEOXYNUCLEOTIDE SEQUENCING OF
DNA
SEQUENCING OF DNA
FOOTPRINTING OF DNA
SINGLE STRAND CONFORMATION POLYMORPHISM
ANALYSIS OF DNA
SEQUENCING STRATEGIES
FOR THE STUDY OF DNA

4. SANGER DIDEOXYNUCLEOTIDE SEQUENCING OF
DNA
In Sanger dideoxynucleotide sequencing, the
sequence of a single DNA strand is determined
In order to obtain large amounts of single stranded
DNA the M13 bacteriophage system is used.

5.  DNA to be sequenced is cloned into the
RF form of M13.
 This is transformed into competent lac-
E. coli and replicated first into several
copies of RF M13 and then into many
copies of single stranded Ml3.
 Coat proteins surround this DNA to form
the mature bacteriophage which is
released into the medium.
 Single stranded DNA is collected by
phenol-chloroform extraction and
ethanol precipitation.
 This is used as template for DNA

6.  A small amount of competitive
inhibitors of DNA polymerase,
dideoxyadenine, dideoxyguanine,
dideoxythymidine or
dideoxycytosine (ddNTPs) is
included in each of four separate
reactions, together with
substrates for this enzyme; dATP,
dGTP, dCTP and dTTP
 The experiment therefore
results in four sets of DNA
daughter-strands which are
truncated fragments
complementary to the DNA to
be sequenced.

7. SEQUENCING OF DNA
The four mixtures are then electrophoresed in an
unusually long (up to 48 cm) and thin (0.4 mm)
continuous polyacrylamide (4–6%) gels in the
presence of urea (8 M) and at approx. 30–40 ◦C
A shark’s tooth comb is used to create small
reservoirs or wells at the top of the gel.
These form between the teeth of the comb and the
gel surface

8.  Truncated fragments for each of
four chain termination reactions
are separated in polyacrylamide
gels containing 8 M urea.
 Smaller fragments electrophorese
furthest into the gel (for clarity,
only those for ddG are shown in
the illustration but bands in the C,
A and T lanes arise from
corresponding fragment sets for
ddC, ddA and ddT).
 Bands are visualised by
autoradiography on X-ray film.
 The sequence obtained is
complementary to the sequence
AUTORADIOGRAM OF DNA SEQUENCING GEL

9.  Primer walking approach for sequencing 1 kb DNA
fragment. New oligonucleotide primers are designed
from 3 ends of previously sequenced region. In this
way sequencing of a template can be continued until
completed.

10.  Automated DNA sequencing.
Instead of a radiolabel, primer
strands are labeled with a
different fluor or each ddNTP.
 These have distinct excitation
(λ1) and emission (λ2)
wavelengths and can
therefore be easily
distinguished by the detector.
 A single track can be used for
separation of all four ddNTP
nested fragments.
AUTOMATED DNA
SEQUENCING.

11. FOOTPRINTING OF DNA
In eucaryotes, most DNA sequences are noncoding,
that is they do not code for protein
Examples include satellite DNA, intervening
sequences within genes (introns) and regulatory
sequences.
A widely-used experiment allowing the identification
of regulatory DNA sequences is DNA footprinting

12. This experiment involves mixing genomic DNA (or
smaller fragments derived from genomic DNA) with an
extract containing the protein factor under
investigation followed by digestion with a small
amount of DNA-ase I.

13. An adaptation of DNA footprinting allows simultaneous
identification and sequencing of the regulatory site

14. SINGLE STRAND CONFORMATION
POLYMORPHISM ANALYSIS OF DNA
 widely-used screening method for the identification of
mutations in DNA called single strand conformation
polymorphism (SSCP) analysis
 The procedure involves heating DNA fragments (200– 400 bp)
of a single gene from a range of samples (e.g. different
human individuals) in either NaOH or formamide to denature
double-stranded DNA.
 This technique has come to be widely-used in the screening
of human DNA for mutations responsible for genetically-
based diseases.

15. (a)DNA is denatured to make it single-
stranded. When placed in non-
denaturing conditions, intrachain
hydrogen bonding can occur.
Mutations in single-stranded DNA
may alter the intrachain hydrogen
bonding pattern giving a
conformational polymorphism
(b) Wild-type and mutant DNA have
altered mobilities in fragments
containing mutations. These are
visualised as band-shifts in
polyacrylamide non-denaturing gels.
Bands can either be shifted up (sample
2) or down (sample 3).