Sunday, 29 September 2019

DNA Sequencing methods

DNA Sequencing methods

DNA Sequencing

DNA sequencing is the process of determining the precise order of nucleotides i.e., adenine (A), thymine (T), cytosine (C) and guanine (G) along a DNA strand. It helps in genome analysis and hence understanding the biological processes. Sanger’s classical method is an enzymatic chain termination method of sequencing developed by F. Sanger et al in 1977. This method is performed in various steps.

DNA Sequencing Steps

(1) DNA Isolation

Genomic DNA to be sequenced can be isolated from blood, saliva or tissue samples of an organism.

DNA Isolation

(2) DNA Fragmentation

The entire genomic DNA extracted is broken down into short fragments and amplified using PCR or cloning vector. Fragmented DNA is denatured to obtain single-stranded DNA (ssDNA), which is used as a template.

DNA Fragmentation

(3) DNA elongation

The fragmented sample DNA is divided into 4 separate reaction mixtures, each comprising of 4 normal deoxynucleotides (dNTPs) : dATP, dCTP, dGTP, dTTP, DNA polymerase enzyme, primer, single-stranded template DNA and any one of the four dideoxynucleotides (dd NTPs) in each of the flask Enzyme starts the synthesis of the complementary strand using normal dNTPs but the chain elongation ceases when a ddNTP gets incorporated into the synthesizing strand which prevents the addition of next nucleotide. Thus, a mixture of terminated fragments having different lengths is produced.

DNA elongation


(4) Each of the four reaction mixtures is run together on a polyacrylamide gel for electrophoresis. Separated fragments are visualized by autoradiography. From the position of the bands, the sequence of the original DNA template strand is read using sequencing software programs on the computer.

Dideoxynucleotides

Significance of Dideoxynucleotides NTPs

Dideoxynucleotides also are known as 2’, 3’ dideoxynucleotides are analogs to the normal dNTPs. The DNA polymerase cannot distinguish between them while catalyzing polymerization.

Dideoxynucleotides

The absence of 3’ OH in ddNTPs inhibits the chain elongation as the phosphodiester bond could not be formed with next nucleotide. Hence, terminates the DNA elongation.

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