Comprehensive insight into sanger sequencing

1. Sequencing
• Sanger sequencing
• Chain termination sequencing
Sunil Bhandari
Balkumari College

2. Sequencing:
• The fundamental way of analyzing the structure of DNA/protein,
whether it is a recombinant plasmid, a natural gene, or a whole
genome or synthesized proteins, is to determine the sequence of
bases of which it is composed.
• Sequencing is the process of determining the actual sequence of
nucleotides (As, Ts, Cs, and Gs present in the DNA) or determining the
actual amino acid sequence present in the protein.
• Today, with the right equipment and materials, sequencing a short
piece of DNA/protein is relatively straightforward.

3. • Sequencing is the primary way of characterizing a
macromolecules, whether it be determining the order
of amino acids in a protein or of bases in a nucleic acid
• Protein sequencing was a very important tool before
genes could be cloned and sequenced.
• However, with the advent of recombinant DNA
technology, sequencing of encoded protein being
deduced from the sequence of the gene.
• In Feb 2001, Human Genome Project and Celera
published the human genome sequence of no less than
three billion base pairs. (30,000 genes, 3 billions bases).

5. Principle of Sangar Sequencing
Principle
“The dideoxy chain termination DNA sequencing technology (Sanger Sequencing)
utilizes the fact that DNA polymerases will incorporate a chain terminating 2’, 3’
dideoxynucleotide triphosphate ddNTP at the appropriate complementary
position but synthesis will be stopped by the incorporation of the ddNTPs at
the 3 end because the next nucleotide to be added lacks the required 3 hydroxyl
group for dNTP phosphodiester bond formation. (Sanger et al 1977)

6. What happens when
dNTP’S are changed
into ddNTP’s?
• ddNTP’s do not have any
hydroxyl group for
further phosphodiester
bond formation, thus no
further base is added
afterwards. This is
called chain termination.

7. Sangar sequencing/
chain termination reaction
• is a process of PCR with a few more bases on it
so,
• Part-I of process is: PCR
• Part-II of process is: Chain detection

8. Process
of
sanger
sequen-
cing

12. 5’-TAGCTGATACGAG-3’
OUTCOME OF GEL RADIOGRAPHY IS
THE COMPLIMETORY STRAND
COMPLIMENTOTY OF COMPLIMENT
STRAND IS TEMPLATE
THUS
OUR TEMPLATE:
3’-ATCGACTATGCTC-5’

15. Automation:
Sequencing Generation

16. 1st generation sequencing
• Protocol is much more similar to sanger sequencing.
• Sanger sequencing had been automated and also known as first generation
sequencing
• It utilizes all same protocol up to template replication (PCR) and
final imaging step was changed
• The complicated (plus time consuming) and hazardous X ray
exposure of polyacrylamide gel ( had been replaced by the
addition of detectors that detects the fluorescently labelled
ddNTPs bands when they escape out of gel during
electrophoresis
• Then detectors detection was recorded on computers and the
whole sequence is retrieved directly from computers

18. High throughput sequencing: NGS
High throughput sequencing methods- fast and cheap
ways to sequencig and easy to analyze large genomes.
A variety of different approaches are being used.
• They generally involve the amplification of DNA templates by the
polymerase chain reaction, and the physical binding of template
DNA to a solid surface or to tiny beads called microbeads
• These techniques are often referred to as massively parallel DNA
sequencing, because thousands or millions of sequencing
reactions are run at once to greatly speed up the process

19. NGS Example: Real time sequencing
 The method of real time sequencing involves imaging the
continuous incorporation of dye labelled nucleotides during DNA
synthesis.
Pacific Biosciences platform:
• Single DNA polymerase molecules are attached to the bottom surface
of individual zero mode waveguide detectors ( Zmw detectors).
• These Zmw detectors can obtain sequence information while
phospholinked nucleotides are being incorporated into the growing
primer strand.
• Zmw detectotr sends signals to the computers.
• DNA sequence easily determined.

20. Fig: Pacific Biosciences platform: (Zmwdetectors)