Get Complete Information on DNA Sequencing

1. Objective :

To perform DNA sequencing and determine the DNA sequence by reading an autoradiogram obtained from a sequencing gel.

2. Introduction :

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There are several methods for DNA sequencing but the most popular one is the dideoxynucleotide triphosphate (ddNTP) method or chain termination method proposed by F. Sanger’s group in 1977. The basic steps are followed in this method to get an autoradiogram.

Step I:

Two complementary strands are separated yielding two single strand by denaturing the DNA fragment to be sequenced with alkali. Each of the two strands is separately sequenced.

Step II:

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The sequencing reaction mixture includes a DNA single strand (which is to be sequenced) suitable primer radio-labelled, NTPs and DNA polymerase. This mixture is distributed into 4 tubes, labeled A, T, G and C. Small amount of ddATP, ddTTP, ddGTP and ddCTP are added in A, T, G, C tubes, respectively.

Step III:

Then, the reaction mixture from each tube is run on a polyacrylamide gel in four separate lanes labeled A, T, G and C. In each lane, the DNA strands are separated (according to their size) wherein the DNA fragments differing even in one nucleotide in length can be resolved.

Step IV:

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By aligning the gel tightly with an X-ray film followed by developing the film in suitable developer, an autoradiogram is prepared. The DNA bands appear as dark bands on the autoradiogram.

Step V:

Reading of the autoradiogram.

3. Principle :

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DNA polymerase makes copies of the DNA single strand which is to be sequenced that extends a primer annealed to the 3′ end of the DNA strand.

The extension terminates and a shorter fragment is generated when occasionally a dideoxy derivative is added in each of the four tubes. For example, in a tube where ddCTP is present, fragments of different lengths ending in residue C are obtained which correspond to various positions of G in the DNA strand which is being sequenced (C is complementary to G).

In the same way in the ddNTP-labelled tubes, fragments of various lengths are obtained which corresponds to the position of the complementary base in the DNA strand being sequenced. Using the radioactive primers the position of a fragment can be viewed using an autoradiogram (X- ray) film.

4. Procedure

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(i) Procure an autoradiogram; its bands are read as A, T, G or C depending on the lane they appear and in the increasing order of their distance from the lower (anodic) end.

(ii) Synthesis of the new complementary strand occurs in a 5′-8′ direction which is terminated at various points due to the incorporation of ddNTPs.

5. Result :

The sequence of the original DNA strand is -5’TAATGATCGC3′ -. For detail go through the text.