in a particular sample would suggest incomplete digestion of the PCR product.
Allele-specific amplification has been used in DNA diagnosis of genetic disease. Diagnosis can be based solely on absence of an amplification product, so it is a difficult technique to control adequately. Absence of a fragment can indicate either failure of the amplification procedure or absence of the allele in question from the test sample. If the former applies, a typing error would result. For this reason, the committee recommends that this method not be used for forensic analysis.
DNA sequence analysis of PCR products is commonly carried out either manually or with automation. Some ambiguity of nucleotide sequence at one or more positions is common and can signal DNA contamination or a technical problem in the analysis. Another important problem occurs when the DNA sequence of a fragment demonstrates variation at more than one position in the nucleotide sequence. Because both alleles at a locus are sequenced in the procedure, it is difficult to determine what fraction of the variation is contributed by each allele. For example, if heterozygosity is observed at two positions in a sequence, one cannot know, without further experimentation, whether one allele contains both variants or each allele contains one.
New methods of detection of PCR products will surely be devised. Well-controlled, extensive studies of the methods will be required before their use in forensic science, and the quality-assurance procedures described in Chapter 4 will be important to ensure their rigorous testing and reliability.
One commercial kit for forensic PCR analysis has been marketed. Other such kits will probably be ready for commercial markets soon. The committee sees a potential for introduction of unreliable kits and the misuse of kits. The existence of a kit suggests ease of use and low chance of technical error. The committee believes that nonexpert laboratories will run a significant chance of error in using kits. We therefore recommend that a standing committee (discussed later in this chapter) consider the issue of regulatory approval of kits for commercial use in forensic DNA analysis. Even though no precedent exists for regulation of tests in forensic applications, we believe that it might be necessary for a government agency to test and approve kits for DNA analysis before their actual forensic use.
PCR analysis has a number of desirable features for forensic applications. It requires very little DNA (less than for a Southern blot by a factor of 100-150) in the evidence sample. It is thus feasible to amplify dozens of