Top 3 Methods used in Gene Sequencing

The following methods highlight the top three methods used in gene sequencing. The methods are: 1. Maxam & Gilbert’s Chemical Degrada­tion Method 2. Sanger and Coulson’s Dideoxynucleotide Synthetic Method 3. Direct DNA Sequencing using PCR.

1. Maxam & Gilbert’s Chemical Degrada­tion Method:

In this method the following steps are involved (Fig. 18.17)

i. The 3′ ends of DNA fragments are labelled.

ii. The labelled strands are then separated, of which both strands are labelled at 3′ ends.

iii. The mixture is divided into four sam­ples, each treated with a different reagent having the property of destroy­ing either only G or only C or A and G, T and C; the concentration is adjusted in such a way that 50% of target base is destroyed.

iv. Fragments of different sizes having ³²P are produced.

v. Electrophoresis is done using each of the four samples in four different lanes of the gel.

vi. Autoradiograph of the gel helps to deter­mine the sequence from the position of bands in four lanes.

2. Sanger and Coulson’s Dideoxynucleotide Synthetic Method:

This method is based on utilization of DNA polymerase, 2′-3′ dideoxy- nucleotides, and different dNTPs. Here the di-deoxynucleotide terminates the chain reaction of DNA synthesis which produces the different length of chains.

The method involves, the following steps:

i. Four reaction tubes are set up each con­taining single stranded DNA sample to be sequenced, ail four dNTPs (radioactively labelled) and an enzyme for DNA synthesis (DNA polymerase I).

ii. Each tube also contains a small amount of (much smaller amount relative to four dNTPs) one of the ddNTP, so that four tubes have each different ddNTP, bring­ing about termination at a specific base – Adenine (A), Cytosine (C), Thymine (T), and Guanine (G).

iii. The fragments generated by random incorporation of ddNTP leads to termi­nation of reaction and so the different fragments are produced which can be separated by high resolution polyacrylamide gel, four adjoining lanes are loaded by four different samples.

iv. The gel is then auto-radiographed, the position of different bands in each lane can be visualized, and based on the position of the bands, the DNA sequence can be read out very easily.

3. Direct DNA Sequencing using PCR:

PCR is also used for sequencing the amplified DNA product. This method is more reliable, less time consuming and can utilize either the whole genomic DNA or cloned fragments for sequen­cing a particular DNA segment.

The sequencing in this method involves only two steps:

i. Generation of sequencing templates (double stranded or single stranded using PCR).

ii. Sequencing of PCR products either with the thermolabile DNA polymerase or with the thermo-stable Taq DNA poly­merase.

So, here the DNA sequencing does not require any cloning vector. In both the cases of enzyme, Sanger’s dideoxy method or Maxam- Gilbert’s chemical method can be used. In Sanger’s method, as usual, the sequencing primer is labelled with ³²P and the mixtures with amplified DNA, Taq polymerase and appropriate buffer are incubated at 70°C for 5 min.

The reac­tion is stopped by addition of form-amide and mixtures are allowed to run polyacrylamide sequencing gel, which can be read by computer or manually.

Genome Projects:

The technology for large- scale DNA sequencing has enable scientists to undertake genome sequencing project in a realistic time scale. Since the time of first ‘large’ genome sequencing in bacteriophage X in 1983, the projects on different groups have been com­pleted.

Some notable examples include the bac­terium Escherichia coli, the yeast Saccharomyces cerevisae, the weed Arabidopsis thaliana, the rice Oryza sativa, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, the mouse Mus musculus, the primate chimpanzee Pan troglodytes, the human Homo sapiens sapiens.