Direct DNA Sequencing Using PCR OR Ligation Mediated PCR LMPCR, DNA, Cloned, DNA Polymerase, Taq Polymerase, Double Stranded, Single Stranded

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Home >> Isolation, Sequencing and Synthesis of Genes >>Direct DNA Sequencing Using PCR OR Ligation Mediated PCR - LMPCR

	Direct DNA Sequencing Using PCR OR Ligation Mediated PCR - LMPCR - Polymerase chain reaction (PCR) has also been used for DNA product. This method of DNA sequencing the sequencing is faster and more reliable and can utilize either the whole genomic DNA or cloned fragments for sequencing a particular DNA segment. The DNA sequencing using PCR involves two steps:
(i) generation of sequencing template, the structure of gene should be known. The structure of a gene earlier (double stranded or single stranded) using PCR and (ii) sequencing PCR products either with the thermolabile DNA polymerase or with the thermo stable Taq DNA Polymerase. Thus the DNA sequencing method using PCR eliminates the need of cloning the DNA in single stranded DNA phage vector i.e. M13. Although double stranded DNA product of PCR can be utilized for sequencing, this may cause difficulty due reassociation after denaturation thus preventing the sequencing primer from annealing to its complementary sequence to allow extension. To reduce this problem, either a variant of the standard method for sequencing double stranded DNA is employed or single stranded DNA templates are produced following asymmetric PCR earlier discussed in Chapter 4.
A number of thermolabile DNA polymerases have been used for sequencing of in vitro amplified DNA. Alternatively, Taq polymerase (thermostable enzyme) used for PCR, can also be used for sequencing reaction. In either case, Sanger's synthetic' method involving incorporation of dideoxynucleotide (ddNTP) for chain termination, is used for sequencing. However, Maxam and Gilbert's chemical degradation method can also be used. In Sanger's method, as usual, four mixtures are prepared, each using One of the four ddNTP. 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 stop solution in each tube and mixtures are run on Polyacrylamide sequencing gel to obtain the ladders, which can be read by computer or manually. This method using PCR helped in automation of DNA sequencing.

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