Novel method for detection of small amounts of RNA based on the semi-nested polymerase chain reaction.

Abstract

A technique for the amplification of very small quantities of cDNA based on a semi-nested polymerase chain reaction system has been developed. The technique uses an additional blocking oligonucleotide "primer" in the second round nest which is complementary to one of the first round primers. This prevents amplification of first round product or of any other unwanted products dependent on that primer, but allows amplification of second round product, using first round product as the template. The second round primers are added through the oil layer, eliminating the possibility of introducing first round product aerosol into the atmosphere, as would be the case in a fully nested system. Contamination is therefore minimised, while amplification of the desired product is maximised.