M13 Phage as Vectors for DNA Sequencing, Sanger's Methods, Cloning Vectors, Restriction Sites, Commercial Primer

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	M13 Phage as Vectors for DNA Sequencing - M13 is a filamentous bacteriophage of E. coli and contains a 7.2 kb long single stranded circular DNA. M13 phage has been variously modified to give rise to a M13 mp series of cloning vectors which can be used for cloning of a wide variety of DNA fragments particularly for the purpose of sequencing through Sanger's method of dideoxy chain termination. Cloning vectors of M13 mp series have a lac Z gene that complements gal host (e.g. JM 103 or JM 104) giving blue colonies. But if a foreign DNA segment is inserted at one of the polylinker sites associated with lac Z gene, it inactivates lac Z gene and no complementation is possible.
Thus on transformation, only white or clear plaques are obtained thus permitting selection of recombinant M13mp plaques. Once the foreign DNA is cloned in M13 vector, commercially available oligonucleotide primers are used for copying the insert in presence of dideoxynucleotides, so that fragments of different sizes with known termini are produced, permitting the determination of the sequence. Reversed order of the restriction sites in polylinker is present in a pair of vectors like M13mp8 and M13mp9 permitting sequencing from both the ends of double stranded DNA molecule. The extension of the commercial primer or cloned fragment in Mil vector in the presence of radioactively labelled nucleotides will also allow generation of radioactive single stranded probes