Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content.

Development of PCR for the amplification of nucleic acids has revolutionalized several fields of molecular biology. Since the introduction of this method several parameters of the system have been modified to amplify longer nucleic acid fragments (Foord and Rose 1994, and references within). A variety of DNA polymerases have been used for PCR because of their improved thermal stability (Lawyer et al. 1993) and editing functions (Lundberg et al. 1991; Mattila et al. 1991). Use of a two-enzyme approach (Klentaq I and Pfi~ DNA polymerase) has enabled the amplification of fragments up to 35 kb in length (Barnes 1994; Cheng et al. 1994). Several reagents have been shown to facilitate DNA strand separation either because they disrupt base pair ing [e.g., d imethylsulfoxide (DMSO), formamide] or isostabilize DNA [e.g., te traalkylammonium (TAA) salts such as tetramethylammonium chloride (TMAC), tetraethyla m m o n i u m chloride, and N,N,N-trimethylglycine (betaine)] (Melchior and yon Hippel 1973; Rees et al. 1993). Some of these reagents have been included in PCR reactions to amplify GCrich templates. Recently, it has been shown that at lower concentrations TMAC can enhance the amplification of AT-rich DNA fragments, and at