DNA Sequencing

Introduction

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule.

Sanger sequencing

Sanger sequencing, also called the chain termination method, is a way to find the DNA sequence. It was created by Frederick Sanger in 1977.
Instead of using lots of toxic chemicals and radioactivity like older methods, Sanger sequencing uses special molecules called dideoxynucleotide triphosphates (ddNTPs) to stop the DNA from growing.
To do Sanger sequencing, you need single-stranded DNA, a starting piece of DNA called a primer, a DNA builder called a polymerase and specially labelled molecules.
The DNA is split into four tubes, each getting one of the four regular building blocks (dATP, dGTP, dCTP, dTTP) and one of the special stopping blocks (ddATP, ddGTP, ddCTP, ddTTP).
After some building, the DNA pieces are separated by size on a gel. Then, using X-rays or UV light, the DNA sequence can be read by looking at where the bands stop.
This method can be even faster and cheaper if the primer is coloured with a special dye, making it easier to read.

In chain-termination PCR, a modified version of PCR, the DNA sequence is used as a template. Instead of regular nucleotides, modified ones called dideoxyribonucleotides (ddNTPs) are added. These ddNTPs stop the DNA from growing during the extension step.

In the second step, the chain-terminated pieces of DNA are sorted by size using gel electrophoresis. The DNA is loaded onto one end of a gel, and an electric current is applied. Smaller pieces move faster through the gel than larger ones, so they end up at the top, while larger pieces stay closer to the bottom.
In manual Sanger sequencing, the DNA from each of the four PCR reactions is put into separate lanes on the gel, allowing easy identification of which pieces correspond to each stopping molecule.
In automated Sanger sequencing, all the DNA pieces are put into one lane and sorted automatically inside the sequencing machine.

In the last step, we read the gel to decode the DNA sequence. Each band on the gel represents a nucleotide in the sequence.
In manual Sanger sequencing, the user reads the gel bands to determine the sequence.
In automated Sanger sequencing, a computer reads the bands using fluorescence and generates a chromatogram showing the sequence.

Maxam-Gilbert sequencing:

The Maxam-Gilbert sequencing procedure involves the following steps:

Radioactive Labeling: One end of the DNA fragment is labelled with a radioactive isotope, typically gamma-32P.
Chemical Treatment: The DNA fragment is treated with specific chemicals in separate tubes to generate breaks at specific nucleotide bases. Formic acid is used for purines (A+G), dimethyl sulfate for guanines (and some adenines), and hydrazine for pyrimidines (C+T). Sodium chloride (NaCl) is added to hydrazine for cytosines.
Fragmentation: The chemical treatment creates breaks in the DNA strand, resulting in a series of labelled fragments.
Electrophoresis: The labelled fragments from each chemical reaction are electrophoresed side by side on a gel to separate them by size.
Visualization: The gel is exposed to X-ray film for autoradiography, which reveals a series of dark bands corresponding to the location of each radiolabeled DNA fragment.
By analyzing the positions of the dark bands on the autoradiograph, the sequence of the DNA fragment can be determined based on the specific nucleotide bases that were cleaved by the chemical treatments.

Disadvantage of Maxam-Gilbert sequencing

Applications:

Let Me Explain