DNA Sequencing- Sanger’s dideoxy method or chain termination method

The chain terminator method is more efficient and uses fewer toxic chemicals and lower amount of radioactivity than the method of Maxam and Gilbert. Sanger method uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.

Chain termination method of DNA sequencing, is also referred to as dideoxynucleotide sequencing because of the use of the special types of ddNTPs. The ddNTPs are different from normal dNTPs. it possesses the hydrogen group instead of a hydroxyl group in the dNTPs.

Phosphodiester bond can’t form between two adjacent nucleotides due to the absence of 3'-hydroxyl group in ddNTPs. The nucleotide chain can’t synthesis further and hence it is known as the chain termination method.

The chain termination method requires a single-stranded DNA template,  DNA primer, a DNA polymerase, nucleotides radioactively or fluorescently labelled modified nucleotides that terminate DNA strand elongation.

The process of Sanger sequencing is broadly divided into 3 steps:

1. DNA extraction: using any of the DNA extraction protocols
2. PCR amplification: using the flanking primers, dNTPs,  ddNTPs, Taq DNA polymerase and PCR buffer. (ddNTPs  are fluorescent labelled, each with a characteristic dye, and appears in that colour during detection)
3. Identification of the amplified fragments: using autoradiography, PAGE, or capillary gel electrophoresis. 

PCR amplification is performed by designing four different reactions

Reaction	PCR reaction	modification
Reaction “A”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddATPs
Reaction “G”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddGTPs
Reaction “T”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddTTPs
Reaction “C”	Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers	Labeled ddCTPs

 

●      Each tube contains the same amount of PCR reagents but in each tube, extra ddNTPs are added as shown in the table.

●      The primer binds at the region of interest.

●      Now in the next step, the Taq DNA polymerase adds the dNTPs to the DNA strand.

●    Taq polymerase expands the growing DNA strand by the addition of the dNTPs. Once  it adds the ddNTP instead of dNTP, the chain expansion is stopped or terminated.

●      The termination process is complete in 4 different tubes for 4 different ddNTPs. For example, in the ddATP tube, terminates the chain at all the positions where the ddATPs are going to bind.

●      The amplified PCR products are loaded onto the polyacrylamide gel electrophoresis. The DNA fragments migrate into the gel based on the size of the fragments. The smaller fragments run faster towards the positive charge than the larger fragments.  

●      Depending upon the types of labeling the gel is then analyzed.

●      The DNA bands are visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image.

●    A dark band in a lane indicates a DNA fragment that is result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). If fluorescent labelled, the characteristic colour inidcates incorporation of that dieoxynucleotide.

 

Sangers chain termination method of DNA sequencing.

In electrophoresis, the smaller DNA fragments migrate faster than larger ones. So start reading the sequence from the smallest fragment on the positive side.

The first point in sequencing the DNA is to match the size of DNA. if the bands obtained in gel and the nucleotide sequence of DNA are similar, the reaction is completed properly.

For instance, if sequence length is 16, then 16 bands must be present in the gel. Arrange the sequences accordingly.

Sanger sequencing is the gold standard method for research as well as in the diagnosis because of its easy setup and high reproducibility. its automation had done easily.

 Traditionally, the results are interpreted on PAGE manually but now, the process is automated. A detector detects the fluorescence signals each time when the chain is terminated. The signals are recorded and analyzed computationally. The computational software generates various fluorescence peaks depending upon the amount of fluorescence emitted

Advantage

Chain termination methods have greatly simplified DNA sequencing.

Limitations

●       Non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence.

●       DNA secondary structures affecting the fidelity of the sequence.