RecA-mediated multistrand formation for cloning PCR products into vectors: simplified process for 5'-rapid amplification of cDNA ends.
I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.
Shigemori
Yasushi
Y
Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. syasushi@kazusa.or.jp
D020137:Base Pair Mismatch; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005822:Genetic Vectors; D010957:Plasmids; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases