The below mentioned article provides a study note on gene sequencing.
When a particular gene of interest or a DNA fragment is isolated, the sequencing of that piece of DNA becomes essential. The sequence of DNA refers to the order of nucleotide bases along its sugar phosphate backbone.
No technique can determine the sequence of bases in an entire gene in a single experiment, so it is necessary to cut the whole gene into fragments of manageable size (few hundred base pair long) and purify each fragment. There are two different methods which are now routinely used for determination of DNA sequences.
(a) Maxam & Gilbert’s Chemical Degradation Method:
In this method the following steps are involved (Fig. 22.17):
1. The 3’ ends of DNA fragments are labelled.
2. The labelled strands are then separated, of which both strands are labelled at 3’ ends.
3. 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.
4. Fragments of different sizes having 32P are produced.
5. Electrophoresis is done using each of the four samples in four different lanes of the gel.
6. Autoradiograph of the gel helps to determine the sequence from the position of bands in four lanes.
(b) Sanger and Coulson’s Dideoxynucleotide Synthetic Method:
This method is based on utilization of DNA polymerase, 2′-3′ dideoxynucleotides, 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:
1. Four reaction tubes are set up each containing single stranded DNA sample to be sequenced, all four dNTPs (radioactively labelled) and an enzyme for DNA synthesis (DNA polymerase I).
2. 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).
3. 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.
4. 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.
(c) 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:
1. Generation of sequencing templates (double stranded or single stranded using PCR).
2. 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 formamide and mixtures are allowed to run polyacrylamide sequencing gel, which can be read by computer or manually.