Part of the Methods in Molecular Biology™ book series (MIMB, volume 65)
Rapid Sequencing of cDNA Clones Direct Sequencing Using Sequential Linear/Asymmetric PCR
Following the isolation of a clonal recombinant phage or plasmid after screening a cDNA library, the first analyses that are routinely performed are the determination of the insert size and the sequence of the 3′- and 5′-ends of the cloned fragment. Prior to the use of PCR, this was a laborious process, especially when the cDNA library had been constructed using a vector derivative of bacteriophage λ. I recently described a PCR-based protocol that rapidly generated this information from single-phage plaques and, additionally, produced material suitable for subcloning into plasmid vectors (1). The procedure, which is outlined in Fig. 1, involves an initial PCR reaction using an oligonucleotide primer pair which flank the cDNA insertion site to produce sufficient quantities of double-stranded DNA for sequencing. An aliquot of this reaction is then subjected to further PCR, but with only one of the primers. Any residual second primer is rapidly depleted during this second reaction and large quantities of single-stranded DNA are generated. This material is then used as a template for sequencing using the second oligonucleotide to prime the reaction.
KeywordsMicrofuge Tube cDNA Insert Oligonucleotide Prime Pair SeaPlaque Agarose Fresh Microfuge Tube
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- 2.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
© Humana Press Inc., Totowa, NJ 1996