Written by Sanger sequencing which is also known as chain termination is a method used to determine DNA nucleotide sequence. This method was formulated in the year 1977 by Laureate Frederick Sanger together with his colleagues and that's why through his last name, the medical term Sanger Sequence started. This method became the most popular and widely used sequencing method, especially for the validation of deep sequencing results and smaller-scale projects.
Its 2 Specific Purposes
sanger sequencing maintains an important place in clinical genomics for at least 2 particular purposes. First, this sequencing serves as an orthogonal procedure for verifying sequence variants specified by NGS or next-generation sequencing. Second, this method delivers a means of patching the coverage of regions that are inadequately covered by NGS.
How Does Sanger Sequencing Work?
Sanger sequencing can be performed in an automated fashion which is the most common one via a sequencing machine or it can be done manually. Each of the methods used follows 3 basic steps.
Sanger Sequencing Steps
There are 3 fundamental steps in Sanger sequencing.
1. DNA Sequence For Chain Termination PCR
The DNA sequence of interest is utilized as a template for a particular type of PCR or Polymerase Chain Reaction which is called chain-termination PCR.
2. Size Separation by Gel Electrophoresis
In the 2nd step, the chain-terminated oligonucleotides are segregated by size using gel electrophoresis.
3. Gel Analysis & Determination of DNA Sequence
This last step involves reading the gel in determining the input of the DNA sequence.
Manual vs Automated Sanger Sequencing
In manual Sanger sequencing, the oligonucleotides from each of the 4 PCR reactions are run in 4 different lanes of a gel. This enables the user to know which oligonucleotides are appropriate or correspond to each ddNTP.
In an automated one, all oligonucleotides are run in sole capillary gel electrophoresis with the use of a sequencing machine.
