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DNA Sequencing -
Determination of nucleotide or base sequence of a DNA molecule/fragment is known as DNA sequencing. At present, DNA sequencing is possible for only 700-800 bp long DNA fragments. DNA sequencing has become feasible as a result of the following important developments:
(1) the availability of restriction endonucleases,
(2) the development of highly sensitive gel electrophoretic techniques, which can separate DNA fragments differing by one nucleotide only,
(3) the gene cloning and PCR techniques making available large quantities of individual DNA fragments, and
(4) the development of two reliable, relatively easy and rapid DNA sequencing procedures, called (a) Maxam and Gilbert procedure, and (b) the enzymatic procedure.
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Maxam and Gilbert Procedure
In the first DNA sequencing technique, called Maxam and Gilbert procedure, the DNA fragment to be sequenced is end labelled by the addition of 32p-dATP either at the 5-ends (by the enzyme polynucleotide kinase) or at the 3-ends (by the enzyme deoxynucleotidyl transferase) of its two strands. The end-labelled fragment is now
(i) digested with a restriction endonuclease, which cleaves it into only two fragments of unequal lengths. As a result, only one end of each of the two fragments thus produced will be labelled. The two unequal fragments are separated through gel electrophoresis and they are sequenced separately.
Alternatively, (ii) the end labelled fragment is denatured and its two complementary strands are separated through gel electrophoresis. For some
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unknown reason, the two complementary strands of a DNA molecule generally show different mobilities during gel electrophoresis. The samples of complementary strands thus separated are sequenced separately. It may be noted that each strand will be labelled at one end (either 5' or 3') only.
The single end labelled double or single stranded DNA samples thus produced are subjected to base specific chemical cleavage so that in a reaction mixture cleavage occurs only at one of the following four sites: sites having G, C, G + A or C + T. Each DNA sample is partially digested in four separate reaction mixtures (one each for specific cleavage at the sites of G, C, G + A or C + T).
In these reaction mixtures, each DNA fragment/strand is expected to be cleaved, on an average only once at anyone of the sites having the particular base for which the reaction mixture is specific, each such site in the DNA fragment/strand being equally likely to be cleaved.
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The base specific cleavage of DNA fragments involves the following steps:
(1) modification of the concerned base,
(2) removal of the modified base from the DNA strand, and
(3) induction of strand break (break in the sugar phosphate back-bone) in the position from which the modified base has been removed. Such a cleavage generates a mixture of end-labelled DNA fragments of variable lengths.
Digests of double stranded DNA are denatured before they are subjected to electrophoresis. The digests from the four reaction mixtures are then subjected to gel electrophoresis in separate lanes of the same gel to separate the fragments according to their lengths. The base sequence is determined by sequential reading of the bands developed in the four lanes of the gel through autoradiography.
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The Enzymatic Procedure
The second technique is an enzymatic procedure, commonly referred to Sanger Coulson method, developed by F. Sanger and coworkers. In this technique, the DNA fragment to be sequenced is denatured and the complementary strands are separated through electrophoresis.
One of the two complementary strands (or often both the strands, but in separate experiments) is used as template for DNA replication catalysed by the Klenow fragment (E.coli DNA polymerase I minus the first of its 323 amino acids, i.e the sequence having 5' --> 3' - exonuclease activity). Single-stranded samples of DNA
fragments may also be obtained by cloning DNA fragments in a single stranded DNA virus, e.g., M13, vector. In the reaction system for DNA replication, at least one of the four deoxyribonucleotides is radioactive in order to permit the autoradiographic development of bands after gel electrophoresis.
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A small primer sequence with a free 3'-OH group must be provided with the template strand for DNA replication to proceed, since a free 3'-OH is absolutely essential for DNA polymerase I to catalyse DNA replication.
Four different reaction mixtures are prepared for the replication of each DNA strand to be sequenced. In one of the reaction systems, 2', 3'-dideoxycytidine triphosphate (ddCTP) is added in a concentration about 1/100th of the normal deoxycytidine triphosphate present in the system. ddCTP acts as a terminator of the polynucleotide chain being newly synthesized on the template strand.
Chain termination by ddCTP is achieved due to the fact that it (and the other 2'-3'-dideoxynucleotides) does not have a free 3'-OH group as a result of which further nucleotides cannot be added to the new chain. At the concentration used here, ddCTP would terminate the newly synthesized polynucleotide chains at anyone of all the possible sites where cytosine is to be incorporated in the new chain.
In one each of the three other reaction mixtures using the same DNA fragment as template, 2',3'-dideoxythymidine triphosphate (ddTTP), 2',3'-dideoxyadenpsine triphosphate (ddATP), or 2',3'-dideoxyguanosine triphosphate (ddGTP) is used as chain terminator to terminate the polynucleotide chains at anyone of all the positions where T, A or G, respectively, are to be incorporated in the new chain (each 2',3' -dideoxynucleotide is used in a separate reaction mixture).
The partially synthesized (due to chain termination) DNA chains from each of the above four reaction mixtures are separated from the template strand by denaturation. The four single stranded samples are now separately subjected to gel electrophoresis (in separate lanes of a gel) in order to separate the strands according to their size.
The bands in the gels are developed onto an X-ray film through autoradiography. The fastest moving fragment will be the smallest one, and each subsequent band will be one nucleotide longer than the previous one.
Therefore, by comparing the bands of the four gel lanes thus obtained, nucleotide sequence of the DNA fragment can be determined. The position of a band in the gel from a reaction mixture will indicate the position of the base of which 2',3'-dideoxynucleotide triphosphate was used as chain terminator in that mixture.
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