Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

We describe here a simple, rapid, and efficient cDNA cloning strategy that is based on the DNA polymerase chain reaction technique developed by Saiki et al

M. A. Frohman; M. K. Dush; G. R. Martin

2006

Scholarcy highlights

  • We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of lowabundance mRNAs
  • We describe here a simple, rapid, and efficient cDNA cloning strategy that is based on the DNA polymerase chain reaction technique developed by Saiki et al
  • Full-length cDNA clones can be reconstructed from separate but overlapping 3' and 5' rapid amplification of cDNA ends" products, or they can be synthesized by using primers whose sequences were obtained by analysis of the extreme 5' and 3' ends of the RACE products for PCR amplification of reverse-transcribed mRNA
  • In some circumstances other reaction products were visible after ethidium bromide staining, but they did not hybridize to the int-2 probe; we found that such nonspecific PCR products could be mini
  • The RACE protocol described here provides an alternative to other cDNA cloning methods that is advantageous in many respects
  • Using the RACE protocol, we found that the length of the poly(A) tail on the cDNA products did not exceed 34 nucleotides, even though no precautions were taken to limit tail length
  • It should be possible to use a modified rapid amplification of cDNA ends" protocol to construct general cDNA libraries: one could reverse transcribe using17-adaptor primer, tail the strand products with G or C residues, generate a strand with a different adaptor on its 5' end, and amplify the pooled cDNAs. Since the RACE protocol as presently described requires less than 1 ng of mRNA and this amount could be reduced considerably, it is conceivable that the construction of such general cDNA libraries could be carried out with the RNA from a single cell

Need more features? Save interactive summary cards to your Scholarcy Library.