Amplification of human genomic DNA sequences with polymerase chain reaction using a single oligonucleotide primer.
We present two examples of exponential nucleic acid amplification with the polymerase chain reaction (PCR) in the presence of only one amplification primer. Cloning and sequencing of the PCR products generated by amplification of human genomic DNA revealed that the amplified sequence contained only one primer and its complement, at the two ends of the PCR product. Although these experiments were performed with primers derived from the sequence of the prostate specific antigen (PSA) gene and the normal epithelial cell-specific 1 gene (NES1), the amplified sequences were novel and had no homology with either PSA or NES1 DNA. While both PSA and NES1 genes reside on chromosome 19q13.3-q13.4, the amplified sequences were found by mapping to reside on chromosome 5q12 and 5p15.1-p15.3, respectively. When we examined the mechanism of amplification by PCR using one primer in these two cases, we found that there was a high homology between the PSA primer or the NES1 primer and the two regions flanking the amplified sequence of chromosome 5q12 or 5p15. This indicated that the single PSA or NES1 primer could anneal on both strands of the DNA of that region, and mediate the exponential amplification. Since this phenomenon occurred to us twice with a limited number of different PCR reactions performed in our laboratory (< 20), we believe that it may represent a common artifact of PCR. Moreover, it appears that the palindromic primer binding sites can anneal to each other forming DNA cruciforms.
Luo
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Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
Diamandis
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D001483:Base Sequence; D002874:Chromosome Mapping; D002888:Chromosomes, Human, Pair 19; D002895:Chromosomes, Human, Pair 5; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D006801:Humans; D007610:Kallikreins; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000235:genetics; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D009363:Neoplasm Proteins; D004247:DNA; C100936:KLK10 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen