Strategy for rapid cloning of 5' cDNA ends and related genomic 5' regulatory DNA sequences using modified DLDA-PCR and Alu-PCR techniques

M. J. De Veer, R. T. Good, H. Sim, P. M. Smooker, R. J. Devenish, S. J. Ralph

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Many strategies for isolating novel genes produce only a segment of the target DNA sequences, usually located towards 3' ends of the cDNAs. Thus, polymerase chain reaction (PCR)-based techniques were developed to aid isolation of full-length cDNAs. However, these techniques are often difficult, with low rates of success. We describe improvements to DNA ligase- dependent amplification PCR applied using 3' sequence information derived from cloned differential gene-display PCR products. We have also modified Alu-PCR, using primers specific for mammalian interspersed Alu repetitive sequence, to permit rapid cloning of upstream regulatory DNA sequences from genomic DNA. In combination, the PCR techniques described provide a complete strategy for successful cloning of full-length cDNA and corresponding upstream regulatory DNA sequences.

Original languageEnglish
Pages (from-to)151-156
Number of pages6
JournalMethods in Molecular and Cellular Biology
Issue number2
Publication statusPublished - 1995

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