Amplifying DNA with arbitrary oligonucleotide primers.

G e n e t i c molecular markers are DNA segments that behave as landmarks for genome analysis. These segments usually represent variant or polymorphic sites that can be identified using general strategies such as molecular hybridization or enzymatic amplification of DNA. For years, DNA-based diagnostic markers have been used in general organismal identification and in the construction of genetic linkage maps. (1) The widely used restriction fragment length polymorphisms (RFLPs), for example, are molecular markers identified by endonuclease restriction and blot hybridization of DNA. (z) DNA amplification using PCR (3'4) has also been used extensively in many applications to study polymorphic loci, like hypervariable minisatellites (s) or microsatellites harboring simple sequence repeats, (6-8) and to generate sequence-tagged sites (STSs) (9) for genetic and physical mapping. Several strategies involving DNA replication, DNA ligation, or RNA transcription have been used for in vitro amplification of nucleic acids. (1~ However, PCR remains the most widely accepted amplification tool. Primer-directed amplification of DNA, at first used in PCR to amplify cognate regions present at very low levels in the genome, has extended DNA analysis to regions adjacent to sequenced DNA segments, to unknown DNA, and even to the study of RNA-expressed sequences. (~s-~7) Amplification strategies can be grouped according to the mechanism of the amplification process (Table 1). Amplification with specific primers in the PCR, for example, is a determinate process that requires prior knowledge of the template sequence and targets usually one defined amplification site. Similarly, amplification of interspersed repetitive sequences (IRS), like Alu-PCR (18) or REP-PCR, (19) are determinate processes that target multiple sites of defined sequence in both DNA strands. In contrast, amplification with degenerate primers in random indeterminate amplification processes, like random primed amplification (RPA), (2~ primer-extension preamplification (PEP), (21) and random PCR (rPCR), (22) take advantage of stochastic annealing events that randomly amplify nucleic acid stretches or even whole genomes by targeting sites of a noncognate nature. These random DNA amplification procedures are generally used for radioactive or fluorescent labeling of nucleic acids, to increase the amount of DNA in the construction of representative cDNA libraries, or for PCR typing of single haploid cells. Other kinds of amplification processes, although still determinate, do not require prior knowledge of template sequence and can target single or multiple sites in a genome or template molecule. These strategies can use either one arbitrary primer in conjunction with a specific primer (23) or one or more arbitrary primers (24-26) to study single or multiple amplicons, respectively, and can even be extended to the analysis of RNA populations. (27-28) In particular, strategies like gene-walking PCR (23) and differential cDNA PCR (27) study specific template regions arbitrary in length but juxtaposed to a known DNA segment defining a specific primer. Like anchored PCR, (29) these hemispecific reactions allow analysis of unknown genomic regions corresponding to mRNA sequences, or adjacent to STSs or sequenced stretches of DNA.