Enhanced PCR amplification of VNTR locus D1S80 using peptide nucleic acid (PNA).
Use of the polymerase chain reaction (PCR) to amplify variable numbers of tandem repeat (VNTR) loci has become widely used in genetic typing. Unfortunately, preferential amplification of small allelic products relative to large allelic products may result in incorrect or ambiguous typing in a heterozygous sample. The mechanism for preferential amplification has not been elucidated. Recently, PNA oligomers (peptide nucleic acids) have been used to detect single base mutations through PCR clamping. PNA is a DNA mimic that exhibits several unique hybridization characteristics. In this report we present a new application of PNA which exploits its unique properties to provide enhanced amplification. Rather than clamping the PCR, PNA is used to block the template making it unavailable for interstrand and intrastrand interactions while allowing polymerase to displace the PNA molecules and extend the primer to completion. Preferential amplification is reduced and overall efficiency is enhanced.
Demers
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DB
Fairfax Identity Laboratories, Genetics & IVF Institute, VA 22031, USA.
Curry
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ET
Egholm
M
M
Sozer
A C
AC
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D006580:Genetic Carrier Screening / Q000379:methods*; D005838:Genotype; D006801:Humans; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D009695:Nucleic Acid Renaturation; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D010455:Peptides; D004247:DNA