Genotyping Errors with the Polymerase Chain Reaction

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Abstract
We wish to describe a situation in which use of the polymerase chain reaction (PCR) can lead to genotyping errors. We have previously reported conditions for the analysis of a restriction-fragment–length polymorphism (RFLP) at the D7S8 locus on chromosome 7 using amplification of DNA followed by digestion with the restriction enzyme PstI.* While using this method for linkage diagnosis of cystic fibrosis, we encountered a patient who appeared to be homozygous for the absence of the restriction site according to the PCR; the patient was heterozygous, however, because Southern blot analysis of DNA revealed two bands, one indicating the presence of the restriction site. The substitution of one of the two oligonucleotide primers for the PCR with another oligonucleotide that binds to a site a few hundred base pairs away yielded a heterozygous result consistent with the Southern blot result. Thus, PCR amplification failed because of variation of the primer binding region of a chromosome having the polymorphic restriction enzyme site for PstI; the result was that the original PCR generated no DNA from that chromosome. Family studies indicated the presence of the same atypical result in one of three offspring of the patient and in the patient's mother. The results are consistent with the presence of a relatively rare genetic variation (only if the less common sequence occurs in at least 1 percent of the chromosomes in the population should the variation be called a polymorphism). Subsequently, we found similar results in two other families. We estimate that these instances of this variation occurred among 200 to 300 independent chromosomes.

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