Sanger method
(Chain Termination Method /Sanger Dideoxy Method)
The enzymatic method is called as Sanger Method. Sanger sequencing method was developed by
Frederick Sanger and his colleagues in 1977. The development of this technique won Sanger the
Nobel Prize in Chemistry in 1980.
Sanger sequencing dominated the DNA sequencing platform, bringing successful completion of the
Human Genome Project (HGP) in 2003. Although this technique has been replaced by next
generation sequencing methods, it is still used today for smaller-scale projects.
In order to perform the sequencing, one must first convert double stranded DNA into single
stranded DNA. This can be done by denaturing the double stranded DNA with NaOH.
A Sanger reaction consists of the following components:
Single stranded DNA fragment (ssDNA template): a DNA strand to be sequenced (one of the single
strands, which was denatured using NaOH).
All four deoxyribonucleotide triphosphates: i.e. dATP, dGTP, dTTP and dCTP
NOTE: At least one of the four deoxyribonucleotide triphosphates should be radioactive in each of
the four reaction mixture tubes in order to permit the autoradioautographic development of DNA
bands after the gel electrophoresis.
DNA polymerase: Each incubation tube will also carry DNA polymerase enzyme (Sequenase) in order
to copy the DNA template by adding nucleotides to the primer as the synthesis proceeds.
NOTE: Sequenase is an engineered E. Coli DNA polymerase I (known as ‘Klenow Fragment’), which is
used to copy the DNA template.
DNA primers: The enzyme Sequenase needs a primer to start end to end nucleotide synthesis.
Four reaction mixtures
In addition to radio-labelled primers and DNA polymerase (Sequenase), each incubation tube
(carrying a reaction mixture) should have all the four deoxyribonucleotides (dATP, dCTP, dGTP and
dTTP) and a particular dideoxyribonucleotide phosphate.
The four reaction mixtures should differ from each other in having a different dideoxyribonucleotide
phosphate analogue (ddNTP analogues: ddGTP, ddATP, ddTTP and ddCTP).
Example: Tube A will carry Sequenase, radio-labelled primers, all the four deoxyribonucleotides
(dATP, dCTP, dGTP and dTTP) and a particular dideoxyribonucleotide phosphate (ddATP in this case).
Dideoxynucleotides (ddNTPs) are chain-elongation inhibitors of DNA polymerase, used in the Sanger
method for DNA sequencing.
A dideoxynucleotide (ddNTP) is an artificial molecule that lacks a hydroxyl (OH) group at both the
2nd and 3rd carbons of the sugar moiety of DNA molecule. In contrast, a regular deoxynucleotide
triphosphate (dNTP) has the hydroxyl group on the 3rd carbon of the sugar.
The main purpose of the 3'-OH group is that it is used to form a phosphodiester bond between two
nucleotides - this allows a DNA strand to elongate.
During DNA replication, an incoming nucleoside triphosphate is linked by its 5' α-phosphate group
to the 3' hydroxyl group of the last nucleotide of the growing chain. With ddNTP, where there is no
3' - OH group, this reaction cannot take place, so elongation is terminated. Thus, each new strand
will stop randomly at positions where dNTP is replaced by ddNTP.
The concentration of ddNTP should be 1% of the concentration of dNTP. The logic behind this ratio is
that after DNA polymerase is added, the polymerization will take place and will terminate whenever
a ddATP is incorporated into the growing strand.
Thus, four sets of chain-termination fragments, corresponding to A, C, T and G, are produced in four
reaction mixtures.
Procedure
The single stranded DNA is mixed with primer and split into four reaction mixtures. Each reaction
mixture contains DNA polymerase, four deoxyribonucleotide phosphates (dNTPs) and a replication
terminator (ddNTP). Each reaction proceeds until a replication terminating nucleotide (ddNTP) is
added. The mixtures are loaded into four separate lanes of gel and the electrophoresis is used to
separate the DNA fragments.
STEPS:
1. Fragmentation and amplification: Fragment the DNA and clone the fragments into vectors.
2. Denaturation: Denature the double stranded DNA (by heat or NaOH) into single stranded DNA
fragments.
3. Attach the primer
4. Add 4 dNTPs + 1 ddNTP
5. Four different reaction vials are taken, each with the four standard dNTPs (dATP, dGTP, dCTP and
dTTP) and DNA polymerases. Difference among the vials is because of different type of ddNTP. Each
vial will have 1 ddNTP per 100 dNTPs.
6. After the occurrence of DNA synthesis, each reaction vial will have a unique set of single-stranded
DNA molecules of varying lengths. However, all DNA molecules will have the same primer sequence
at its 5' end.
7. Find the nucleotide sequence using gel electrophoresis: In the gel, we have varying sequences,
lined up according to size.
Autoradiography
In order to identify various terminated chains of DNA (the DNA strands) the ddNTPs would have to
be radioactively or fluorescently labelled beforehand. The DNA strands are then separated using gel
electrophoresis; then, the gel is read from top to bottom (3' to 5') to obtain the sequence.
Smaller strands migrate to the bottom, while larger strands stay up top (near the well). We can read
each molecule in order to find the DNA sequence
Merits of chain termination method
1. Not as toxic and less radioactivity than Maxam and Gilbert method.
2. Easier to automate - Leroy Hood and co-workers used fluorescently labelled ddNTPs and primers
for the first high-throughput DNA sequencing machine. This lowered the cost from $100 million to
$10,000 USD in 2011.
3. Highly accurate long sequence reads of about 700 base pairs.
4. Easier to get started. The kits that are commercially available contain reagents necessary for
sequencing - pre-aliquoted and ready to use.
Limitations
1.Non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence.
2.DNA secondary structures affecting the fidelity of the sequence.
Significance of DNA Sequencing
Information obtained by DNA sequencing makes it
possible to understand or alter the function of genes.
DNA sequence analysis demonstrates regulatory regions
that control gene expression and genetic “hot spots”
particularly susceptible to mutation.
Comparison of DNA sequences shows evolutionary
relationships that provide a framework for definite
classification of microorganisms including viruses.
Comparison of DNA sequences facilitates identification of
conserved regions, which are useful for development of
specific hybridization probes to detect microorganisms
including viruses in clinical samples.
DNA sequencing has become sufficiently fast and
inexpensive to allow laboratory determination of
microbial sequences for identification of microbes.
Sequencing of the 16S ribosomal subunit can be used to
identify specific bacteria. Sequencing of viruses can be
used to identify the virus and distinguish different strains.
DNA sequencing shows gene structure that helps
research workers to find out the structure of gene
products.
