Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection.
A novel method for detection of any mutation located within a PCR-amplified DNA sequence was demonstrated. The method is based on the inhibition of spontaneous DNA branch migration. Partial duplexes produced by PCR amplification of a test and a reference genomic DNA sample anneal to form four-stranded cruciform structures. Spontaneous DNA branch migration results in dissociation of these structures when the test and reference sequences are identical. Any base substitution, deletion or insertion inhibits branch migration and produces stable cruciform structures. When suitable ligands are attached to the PCR primers, the cruciform structures can be detected by standard immunochemical methods. This approach was tested using several commonly occurring mutations within the human cystic fibrosis gene. New methods for increasing the specificity of PCR amplifications are described that were used for successful mutation analysis.
Lishanski
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A
Advanced Diagnostics Division, Dade Behring Inc., San Jose, CA 95135, USA. alla_lishanski@dadebehring.com
Kurn
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N
Ullman
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EF
D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004261:DNA Replication; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons; D006801:Humans; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA