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

Abstract

A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaql and P fu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.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 pairing [e.g., dimethylsulfoxide (DMSO), formamide] or isostabilize DNA [e.g., tetraalkylammonium (TAA) salts such as tetramethylammonium chloride (TMAC), tetraethylammonium 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 higher concentrations it can inhibit enzyme amplification (Chevet et al. 1995). However, templates with high GC contents are variably amplifiable or completely unamplifiable even with the addition of DMSO, formamide, glycerol, TMAC, or 7-deazaguanosine triphosphate (7cdGTP), which lowers the melting temperature of the substituted DNA (McConlogue et al. 1988;Bookstein et al. 1990;Hung et al. 1990;Sarkar et al. 1990;Pomp and Medrano 1991). In spite of all the advances described above there is no single method that has been developed that could amplify to a similar extent all DNA templates in a heterogeneous mixture.There are a number of PCR applications (e.g., construction of cDNA library from a small number of cells, representational differential analysis of two different genomic DNA, or cDNAs from two different cell types) for which it would be highly desirable to have a method whereby a mixture of DNA fragments of similar length (0.5-10 kb) can be amplified with equal efficiency regardless of base composition. Therefore, we explored amplification by using isostabilizing quarternary amines that did not inhibit the DNA polymerases. Betaine is a natural amino acid analog and has many useful properties such as osmoprotection of Escherichia coli cells, stabilization of proteins against thermal denaturation, and isostabilization of DNA (Csonka 1989;Santoro et al. 1992; Rees et al. 1993). However, use of betaine as a reagent for amplification of GC-rich templates has not been explored.In this report we studied the efficiency of ...

The twist gene is a common target of retroviral integration and transcriptional deregulation in experimental nephroblastoma

“…As a result of an extremely high GC content, this region could not be correctly amplified and sequenced by conventional PCRbased procedures. Using the modified PCR buffer containing 1.3 m betain and 1.3% DMSO (Baskaran et al, 1996), we confirmed the presence of additional 50 nucleotides (nucleotides À22 through À71 in Figure 4, GenBank accession number AY126449) in the chicken genome.…”

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

“…To minimize random point mutations, Pfu DNA polymerase was used in the PCR. Addition of betaine (1 M) in the PCR mixture was used to improve the amplification of DNA by reducing the formation of secondary structure in the GC-rich region when necessary (45). In the first round of saturation mutagenesis, pBS(Kan)EH was used as the template, and mutagenesis was performed individually at sites Phe-108, Ile-111, Ile-219, and Cys-248.…”

Generation of T-cell receptor retrogenic mice

“…This system provides two different buffers: an Advantage 2 buffer and a high-fidelity (HF) buffer. The latter is generally used for most reactions; however, longer fragments or more difficult templates may require manipulation of buffer volumes as per manufacturer's instructions, or the addition of 5% (vol/vol) DMSO and/or 1 M betaine [19][20][21][22] .…”