Single-molecule dilution and multiple displacement amplification for molecular haplotyping.
Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.
Paul
Philip
P
Allogen Laboratories, The Cleveland Clinic, Cleveland, OH 44195, USA. pp@tt.ccf.org
Apgar
Josh
J
D000483:Alleles; D000941:Antigens / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D006239:Haplotypes; D006579:Heterozygote; D006358:Hot Temperature; D006801:Humans; D007201:Indicator Dilution Techniques; D007962:Leukocytes / Q000276:immunology; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D013997:Time Factors
D000941:Antigens; D004247:DNA; D004259:DNA-Directed DNA Polymerase