Dual asymmetric PCR: one-step construction of synthetic genes.
We have developed a one-step process for constructing synthetic genes. Four adjacent oligonucleotides 17-100 bases in length having short overlaps of 15-17 bases are used as primers in a PCR mixture. The quantity of the two internal primers is highly limited, and the resultant reaction causes an asymmetric single-stranded amplification of the two halves of the total sequence due to an excess of the two flanking primers. In subsequent PCR cycles, these dual asymmetrically amplified fragments, which overlap each other, yield a double-stranded, full-length product.
Sandhu
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GS
Dept. of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905.
Aleff
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RA
Kline
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BC
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005813:Genes, Synthetic; D006629:Hirudins / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D006629:Hirudins; D009838:Oligodeoxyribonucleotides; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase