A system for shotgun DNA sequencing
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Implication
Experimental study demonstrates rapid shotgun DNA sequencing using a modified M13mp7 phage vector, highlighting streamlined enzymatic cloning and dideoxy sequencing.
Key Points
- To engineer an enhanced M13 phage vector with a multipurpose cloning site for rapid and streamlined chain-terminator DNA sequencing.
- Inserted a synthetic multipurpose cloning site adding 14 codons into the beta-galactosidase gene of bacteriophage M13mp2.
- Eliminated two endogenous restriction endonuclease sites in viral gene II through site-directed single base-pair mutations to construct M13mp7.
- Performed direct cloning of restriction-digested DNA fragments coupled with chain-terminator sequencing using a 15-base synthetic primer.
- Retained functional intracistronic complementation of the lac gene product despite the 14-codon synthetic insertion.
- Enabled direct and versatile cloning of DNA fragments generated by a wide variety of restriction endonucleases.
- Achieved rapid enzymatic dideoxy nucleotide sequencing across cloned inserts using a universal 15-base primer.
Cite This Study
Messing et al. (1981) studied this question.