Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA.
Errors introduced during PCR amplification set a selectivity limit for microsatellite analysis and molecular mutation detection methods since polymerase misincorporations invariably get confused with genuine mutations. Here we present hairpin-PCR, a new form of PCR that completely separates genuine mutations from polymerase misincorporations. Hairpin-PCR operates by converting a DNA sequence to a hairpin following ligation of oligonucleotide caps to DNA ends. We developed conditions that allow a DNA hairpin to be efficiently PCR-amplified so that, during DNA synthesis, the polymerase copies both DNA strands in a single pass. Consequently, when a misincorporation occurs it forms a mismatch following DNA amplification, and is distinguished from genuine mutations that remain fully matched. Error-free DNA can subsequently be isolated using one of many approaches, such as dHPLC or enzymatic depletion. We present feasibility for the main technical steps involved in this new strategy, conversion of a sequence to a hairpin that can be PCR-amplified from human genomic DNA, exponential amplification from picogram amounts, conversion of misincorporations to mismatches and separation of homoduplex from heteroduplex hairpins using dHPLC. The present hairpin-PCR opens up the possibility for a radical elimination of PCR errors from amplified DNA and a major improvement in mutation detection.
Kaur
Manjit
M
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Makrigiorgos
G Mike
GM
D016477:Artifacts; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards
D009692:Nucleic Acid Heteroduplexes; D004247:DNA