RPCR: a powerful tool for random amplification of whole RNA sequences.

Abstract

T h e polymerase chain reaction (PCR), at first used to amplify known DNA sequences present at very low levels in genomic DNA samples, ~1-3) was rapidly extended to amplif icat ion of RNA sequences by the use of cDNA. ~4's) The PCR method, which requires mult iple heat-denaturation steps and was originally established with the Klenow fragment of Escherichia coli DNA polymerase I, now employs thermostable forms of DNA polymerase and two gene-specific primers (GSP) that flank the DNA segment to be amplified. Although PCR is the most widely utilized amplif icat ion protocol, known RNA sequences can be amplified alternatively with a RNA transcription-based amplif icat ion system (TAS), ~6'7) changing the final product from double-stranded DNA to singlestranded RNA. The TAS reaction needs two GSP, with the downstream GSP linked to a RNA polymerase (T7, T3, or SP6) b inding site, and is carried out by using sequential cDNA synthesis and RNA transcription, which are displayed as seven steps, including three heat-denaturation steps. Since the original description of the TAS protocol, the reaction conditions have been modif ied to achieve more productive and quantitative amplification. Thus, the self-sustained sequence replication (3SR) ~8,9) reaction was modeled after the general scheme employed during retroviral replication and does not require heat-denaturation; 108 copies of each target molecule can be produced in less than 1 hr with all three enzyme activities from avian myeloblastosis virus reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase operating in an efficient and concerted manner . Other protocols such as rapid amplification of cDNA ends (RACE) ~°-12) use only one GSP and permit amplif icat ion of a region between a single known short sequence in a cDNA molecule and its unknown 3' or 5' end. Also described as one-sided PCR ~13) or anchored PCR, ~4) RACE 3'-end and 5'-end procedures are performed by using a homopolymer ic nucleotide containing an adapter primer that anneals to the cDNA's 3' or 5' termin i and one GSP. RACE using a randomly primed first-strand cDNA has also been reported recently, ~1s,16) with the 3' extension technique assuming the amplification of DNA fragments localized both in large 3' unsequenced regions and downstream to a 5' known sequence. Last, an elegant method that employs head-to-tail ligation of specifically primed, single-stranded, firststrand cDNAs with RNA ligase followed by PCR amplification ~17) was developed to yield 5' double-stranded cDNA copies in sufficient abundance for cloning; although there is actually no available data about the efficiency of this technique, it has the potential advantage of c ircumventing the usual problems associated with the homopolymeric tailing.