Sanger’s method of DNA
sequencing
14 -11-2022
14-08-2023
Frederick Sanger
English biochemist
• The Nobel Prize in Chemistry 1958 - for his
work on the structure of proteins,
especially that of insulin
• The Nobel Prize in Chemistry 1980 - for
their contributions concerning the
determination of base sequences in
nucleic acids
What is DNA sequencing?
• DNA sequencing refers to the general laboratory technique for
determining a DNA molecule's exact sequence of nucleotides, or
bases.
• The sequence of the bases (often referred to by the first letters of
their chemical names: A, T, C, and G) encodes the biological
information that cells use to develop and operate.
This method of sequencing is also called as
Enzymatic method or Chain termination method
• The key ingrediants of Sanger’s DNA sequencing are
1. A DNA polymerase enzyme – helps in elongating the DNA fragment
2. A primer, which is a short piece of single-stranded DNA that binds
to the template DNA and acts as a "starter" for the polymerase
3. The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
4. The template DNA to be sequenced
5. Dideoxy versions of all four nucleotides (ddATP, ddTTP, ddCTP,
ddGTP), each labeled with a different color of dye. These are the
chain-terminating nucleotides.
In 1977, Fredrick Sanger, had an idea. If the 3’OH is absent there would be no
reaction with the incoming nucleotide and the reaction would stop. This formed the
basis of the classical Sanger’s sequencing.
• io
All the dideoxy nucleotides are added at a very
low concentration and they are radiolabeled.
• A) direct labeling (B) indirect labeling via a prostetic group and (C) indirect labeling via
complexation. The different methods allow incorporating various types of radiolabels into a
protein: (A) iodine (B) fluorine and (C) metallic radionuclides.
• Polyacrylamide gel electrophoresis
separates the DNA fragments
based on size.
• The smallest-sized fragment travels
to the base of the gel.
• The largest-sized fragment travels
slowly and is at the top of the gel.
• The DNA travel in a buffered
system under electric charge.
Modern Sanger Sequencing method
• The ddNTPs are labeled with fluorescent material.
• Fluorescent dyes, or fluorophores, are compounds that absorb
light at a given wavelength and emit light at a higher wavelength,
producing fluorescence in various colors.
• These dyes can be grouped into categories such as
• organic dyes (e.g., fluorescein, rhodamine),
• biological fluorophores (e.g., green fluorescent protein, phycoerythrin,
allophycocyanin)
• The ratio (10: 1) is optimal for determining sequences from the
primer up to =400 nucleotides.
• For determination of longer sequences either the dNTP concentration
in the labeling reaction should be increased (e.g., 5-fold) or the
concentration of ddNTP in the termination reaction should be
decreased (e.g., dNTP/ddNTP ratio of 30:1).
Uses and limitations
1. Sanger sequencing gives high-quality sequence for relatively long
stretches of DNA (up to about 900 base pairs).
2. It's typically used to sequence individual pieces of DNA, such
as bacterial plasmids or DNA copied in PCR.
3. It is expensive and inefficient for larger-scale projects, such as the
sequencing of an entire genome or metagenome (the “collective
genome” of a microbial community)
Applications
1. Comparing DNA sequences will help to understand the role of
inheritance in susceptibility to disease. - an increased likelihood of
developing a particular disease based on a person's genetic makeup.
Ex: In AIDS, the role of variation in the CCR5 gene in resistance to
infection and slower disease progression.
The presence of an intronic SNP (RANTES gene) is associated with
increased disease progression to AIDS in both European and African
Americans.
2. Physicians are using sequence data to identify the particular type of
cancer a patient has - for better choices of treatments.
Ex: Breast, soft tissue sarcoma, bone, leukemia, brain, and
adrenocortical carcinoma are caused by Li-Fraumeni Syndrome.
A gene called TP53 (codes for a protein p53). A mutation in this gene
causes the gene to lose its ability to function correctly.
Approximately 70% of families with LFS will have a mutation in the
TP53 gene.
3. Undiagnosed Diseases program (NHGRI supported)
uses DNA sequencing to identify the genetic causes of
rare diseases.
Ex: Charcot-Marie-Tooth disease (OMIM 601472)
variants in GARS gene, results in extreme growth
retardation.
Saul-Wilson syndrome (OMIM 618150), is a rare skeletal
dysplasia. Genomic sequencing analysis identified a de
novo variant in COG4.
Saul-Wilson syndrome
• sparse eyebrows, and thin
vermillion of the upper lip,
prominent forehead and
veins
4. The Cancer Genome Altas project (supported by NHGRI and the
National Cancer Institute) uses DNA sequencing to find the genomic
details of about 30 cancer types.
Acute myeloid leukemia, Breast Ductal Carcinoma, Bladder Urothelial
carcinoma
5. Understand gene activity in different tissues and the role of gene
regulation in disease.
6. By comparing genome sequences of different types of animals and
organisms such as chimpanzees and yeast, provides an insight into the
biology of development and evolution.
Review of the steps in Sanger sequencing
1. The ssDNA with a short complementary primer molecule is the beginning
step of the sequencing.
2. The 3’ hydroxyl end of the primer is binds to the DNA to be sequenced.
3. The Klenow fragment of DNA polymerase I starts at the primer and
synthesis a complementary copy of the particular DNA region.
4. DNA sequencing employs four different reaction mixtures, each contains
all four deoxyribose nucleoside triphosphates.
5. In addition to dNTPs, each of the four mixtures contains one of the four
dideoxyribonucleoside triphosphates (ddNTPs) , which lacks both 2’ and
3’ hydroxyl group.
6. As the 3’ hydroxyl group is required for the formation of phosphodiester
bond, the presence of ddNTPs causes chain termination.
References
• DNA Sequencing Core Facility
https://biology.unt.edu/~jajohnson/Chromatogram_Interpretation
• https://agctsequencing.wordpress.com/tag/troubleshooting-
sequencing/
