PRINCIPLES OF DNA

SEQUENCING AND
GENESCAN
G.SANKARI DEVI
DEPARTMENT OF HAEMATOLOGY, CMC,
VELLORE
Introduction

The process of determining the

order of nucleotide bases along
a DNA strand is called
sequencing.
It was first developed in 1977
Chain termination method
Chemical degradation method
Autoradiography method
 The Two Rapid Sequencing
Techniques:
- Enzymatic Method of Sanger et al.,
(1977)
- Chemical Degradation Method of
Maxam and Gilbert ((1977)
 Principle
Both methods generate separate population of
labelled oligonucleotides that begin from a fixed point
and terminate randomly at a fixed residue.
These population of oligonucleotides are then
resolved by electrophoresis under conditions that can
discriminate between individual DNAs that differ in
length by as little as one nucleotide.
When the populations are loaded in adjacent lanes of
a sequencing gel, the order of oligonucleotides along
the DNA can be read.
Sanger method of di-deoxy mediated
chain termination:
Sanger synthesised chemical analogues of the dNTPs that
lacked the OH at the 3 position of the ribose ring referred
to as di-deoxy NTPs, or ddNTPs.

The 3 OH is essential for chain extension, without it, chain

extension terminates.

To achieve chain termination at all positions

for a given nucleotide, both the dNTP and the ddNTP
are present in the sequencing reaction. If dNTP is
incorporated, synthesis proceeds, if ddNTP is
incorporated, synthesis is terminated.
Principle of sequencing
Chain termination sequencing involves the synthesis
of new strands of DNA complementary to a single
stranded Template.
The template DNA is supplied with a mixture of four
dNTPS, four ddNTPS each labeled with a different
color fluorescent tag, and DNA polymerase.
All four dNTPS are present, chain elongation
proceeds until, by chance, DNA polymerase inserts a
ddNTPs. It results in new set of DNA chains all of
different lengths.
ddATP PCR
ddGTP DNA dNTPS
ddCTP
+ Polymerase + + Product
ddTTP

TGTGGGAATTTTGA

ACACCCTTAAAACT
A CA CC C T T AA

T
G
G
T
G
G
G
A
A
T
T
 Method

PCR for the specific template

check amplification.
Pre sequencing wash is
performed on the remaining
PCR product as follows.
 Pre sequencing sample processing:

After checking the amplification add sterile

water to the remaining PCR product make up to
100l mix it several times
Transfer this to Polystyrene Filter Plates to purify
PCR products by Vacuum filtration.
Fix this plate on a multi screen vacuum manifold
with under drain support grid
Apply Vacuum at 20 Hg for 10 minutes or until
the wells dried completely.
Add 100 l of sterile water again and repeat the
steps.
Add 25 l of sterile water into each well and
vigorously mix before sample retrieval with a
pipette
 Load 5 l of the retrieved product
in 2% agarose gel in 0.5X TBE
check the amplification of the
purified product.
Depending upon the intensity of
product take1or 2l of purified
PCR product for the sequencing
reaction
 Sequencing Reaction Set-up:
Set up a 10 l sequencing reaction for each sample as follows,

1. Big dye terminator ready reaction mix (RR mix) 1 l

2. 5X Big dye Buffer 1 l

3. Primer (1 Pico mole/ l ) 1.6 l

4. Sterile Water 5.4

l
5. PCR Product 1 l

Thermal cycling Program For Sequencing PCR:

960C for 15 seconds
500C for 20 seconds
600C for 4 minutes
Go to 1, 25 times
150C for ever.
 Post sequencing Reaction Purification:

The presence of unincorporated dye labeled ddNTP and

salts in the sample can have a significant negative effect
on the quality of sequencing.

Procedure for Post sequencing reaction cleanup :

Add 30 l of injection solution to a Sequencing PCR

product
Mix gently by pipetting up and down several times.
Transfer diluted products into the post sequencing plate.
Place the SEQ96 plate on the vacuum manifold.
Set the vacuum to 20 Hg.
Apply vacuum until the solution has been completely
dried in the wells.
Remove the SEQ96 plate from the manifold add 30 l of
injection solution to the same wells and repeat the above
step.
For elution, add 25uL of injection solution into
the bottom of each well and retreive the
sequencing product by pipetting up and down
20 times.
Alternatively, the DNA can be resuspended
by shaking for 10 minutes on a micro plate
shaker.
Transfer the purified sequencing product into
0.5ml tube and close tube with septa.
Label appropriately and hand it over to the
person in charge to load the sample into the
machine.
Gene scan
PRINCIPLE:
The purpose of the procedure is to describe how to
use the ABI310 sequencer to perform the bone
marrow chimerism analysis.
This is a capillary based sequencer, which when
used in concert with the Genescan software package
is able to identify and quantify fragment peaks as they
pass through by a capillary gel matrix and are
fluoresced by a laser. The genescan software is able
to determine when the peak passed by the laser and
what the amplitude and area of the fragment curve is.
Using the peak height information along with the
known patterns of the pre-recipient and donor
samples,it is mathematically possible to calculate the
percent of engraftment in the post-recipient sample.
The following table to perform
the analysis with ABI 310 genetic
analyzer
Step 1

In a genetic analyzer sample tube add

Deionized Formamide 14
Gene scan Rox 500 size standard 0.5
PCR product 1
Total volume 15.5
 Close each tube.
Mix the products and spin the
mixture.
Denature the sample in the heat block
for 5minutes at 95o C
Chill on ice and spin briefly in a micro
centrifuge.
Load the sample in an ABI 310
genetic analyzer.