Disruption of phase during PCR amplification and cloning of heterozygous target sequences.
PCR amplification of genomic DNA or cDNA has become a standard tool for identification of mutations underlying genetic disease. There are inherent limitations in the application of this method in compound heterozygotes. One problem which is encountered is the disruption of phase (linkage) between heterozygous polymorphisms represented on heterologous alleles. A test system was used to demonstrate and quantitate the disruption of phase between two polymorphic restriction sites. Phase is disrupted in approximately 1% of the PCR amplified material, possibly due to incomplete chain elongations and subsequent priming on the heterologous allele. Phase is disrupted in approximately 1/4 of cloned PCR fragments, possibly due to excision repair of heteroduplexes during cloning. The implications of these disruptions for the use of PCR in identifying mutations are discussed.
Jansen
R
R
Howard Hughes Medical Institute, Department of Cell Biology and Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Ledley
F D
FD
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005784:Gene Amplification; D008040:Genetic Linkage; D006579:Heterozygote; D006801:Humans; D007535:Isomerases / Q000235:genetics*; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics*; D008780:Methyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D007535:Isomerases; D008765:Methylmalonyl-CoA Mutase