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Sanger sequencing

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13 views

Sanger sequencing

Uploaded by

lucylit0666
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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*Sanger

sequenci
ng
Submitted by
Banazir M
* Introduction
DNA sequencing
The process of determining the order of the
nucleotide bases in a DNA molecule.
Sanger sequencing (dideoxy chain
termination method)
• It is a widely used technique for determining
the nucleotide sequence of DNA.
• It was developed by Frederick Sanger in
1977.
• Remains in use today for smaller-scale
projects due to its high accuracy.
*Principle
The method relies on the incorporation of modified
nucleotides called dideoxynucleotides
(ddNTPs), which terminate DNA synthesis
because they lack the 3'-OH group for
phosphodiester bond formation required for chain
elongation. The fragments of different lengths are
then run on a gel to determine the sequence.
DNA Template: The DNA fragment to be
sequenced.
Primer: A short single-stranded DNA molecule
that is complementary to the region at the
start of the DNA to be sequenced. It binds to
the template and provides a starting point for
DNA synthesis.
DNA Polymerase: An enzyme that
synthesizes new DNA strands by adding
nucleotides to the growing chain.
Deoxynucleotides (dNTPs): The building
blocks of DNA (adenine (A), guanine (G),
cytosine (C), and thymine (T)).
Dideoxynucleotides (ddNTPs): Modified
nucleotides that terminate DNA strand
elongation. Each ddNTP is fluorescently labeled
with a different color corresponding to each of
the four bases (ddATP-b, ddGTP-y, ddCTP-g,
The DNA polymerase extends the primer
by adding dNTPs to the growing DNA
strand. Occasionally, a ddNTP is
incorporated instead of a dNTP, causing
the chain to terminate.

This process results in a collection of DNA


fragments of varying lengths, each ending
with a ddNTP.
Steps of Sanger Sequencing
1. Preparation of DNA Template:
The DNA sequence to be determined is
denatured into single strands.
A short primer is annealed to a
complementary region on one strand of the
DNA template.
2. Reaction Setup:
Four separate reaction mixtures are prepared,
each containing:
• DNA template
• DNA polymerase
• A primer
• Normal nucleotides (dNTPs: dATP, dTTP,
dGTP, dCTP)
• One type of fluorescently
labeled dideoxynucleotide
(ddNTP) (ddATP, ddTTP, ddGTP, or
DNA Synthesis and Termination:
DNA polymerase extends the primer by
adding nucleotides complementary to the
template strand.
When a ddNTP is incorporated into the
growing chain, elongation stops because
ddNTPs lack a 3'-OH group.
Separation of Fragments:
The resulting DNA fragments of varying
lengths are separated by size
using capillary electrophoresis or gel
electrophoresis.
Each fragment ends with a ddNTP, allowing
the terminal nucleotide to be identified.
the oligonucleotides will be arranged from
smallest to largest, reading the gel from
In manual Sanger sequencing, the user
reads all four lanes of the gel at once,
moving bottom to top, using the lane to
determine the identity of the terminal
ddNTP for each band. For example, if the
bottom band is found in the column
corresponding to ddGTP, then the smallest
PCR fragment terminates with ddGTP, and
the first nucleotide from the 5’ end of the
original sequence has a guanine (G) base.
Detection and Analysis:
In automated Sanger sequencing, the
fluorescent labels on the ddNTPs are
detected using a laser during
electrophoresis.
The sequence of the DNA is deduced
based on the order of the fluorescent
signals.

https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/
genomics/sequencing/sanger-sequencing
Output
The result is an electropherogram, a
graph displaying colored peaks
corresponding to the DNA sequence.
The colors represent the different
nucleotides, and their order reveals the
DNA sequence.
APPLICATIONS
•Gene Mutation Analysis
•Clinical diagnostics for genetic disorders (e.g., cystic fibrosis, sickle cell
anemia).
•Cancer mutation detection (oncogenes, tumor suppressor genes).
•Species identification for biodiversity studies and forensic investigations.

•DNA Barcoding
•Species identification for biodiversity studies and forensic
investigations.

• Validation of NGS Results


Quality control and confirmation of novel variants or
discrepancies in NGS data.

• Microbial Identification and Phylogeny


• Pathogen identification (e.g., bacteria, viruses).
• Phylogenetic analysis of organisms.

• Forensic Applications
DNA profiling for identification in forensic science.
*Advantages
1.High accuracy (up to ~99.99% for
small sequences).
2.Ideal for sequencing small regions
of DNA.
3.Low Error Rates: Low error rates
compared to some high-throughput
sequencing methods.
4.Read Lengths: Longer read lengths
compared to most NGS methods,
making it useful for resolving
repetitive regions and complex
sequences.
*Disadvantages
1.Limited throughput (not suitable
for sequencing entire genomes
quickly).
2.Expensive
3. Time-consuming compared to
modern techniques like next-
generation sequencing (NGS).

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