DNA amplification in vitro using T4 DNA polymerase.
We have evaluated in vitro DNA amplification by polymerase chain reaction using either T4 DNA polymerase or Klenow fragment of Escherichia coli DNA polymerase I. Both polymerases under optimal salt conditions permit efficient amplification of exon 3 of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene from human genomic DNA and from plasmid containing the HPRT cDNA. DNA sequences amplified from human genomic DNA, using two 20-nucleotide primers flanking the ends of the exon, showed a marked difference between the two polymerases. T4 DNA polymerase yielded only the expected amplified DNA fragment, whereas Klenow fragment produced many lower-molecular-weight bands in addition to the expected DNA fragment. On the basis of the reported fidelity of in vitro DNA synthesis using Klenow fragment and T4 DNA polymerase, it is expected that the latter will create substantially fewer errors during the amplification process. For these reasons, T4 DNA polymerase should be particularly valuable for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected.
Keohavong
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Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge 02139.
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Cariello
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Thilly
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D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005091:Exons; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D013604:T-Phages / Q000201:enzymology
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004259:DNA-Directed DNA Polymerase