Question: When amplifying DNA fragments that contain STR repeat regions, it is possible to have a phenomenon known as allele dropout. Sequence polymorphisms are known to occur within or around STR repeat regions. These variations can occur in three locations (relative to the primer binding sites): within the repeat region, in the fl anking region, or in the primer binding region. If a base pair change occurs in the DNA template at the PCR primer binding region, the hybridization of the primer can be disrupted, resulting in a failure to amplify and, therefore, failure to detect an allele that exists in the template DNA. More simply, the DNA template exists for a particular allele but fails to amplify during PCR due to primer hybridization problems. This phenomenon results in what is known as a null allele . Fortunately null alleles are rather rare because the flanking sequence around STR repeats is fairly stable and consistent between samples.
No primer set is completely immune to the phenomenon of null alleles. However, when identical primer sets are used to amplify evidence samples and suspect reference samples, full concordance is expected from biological materials originating from a common source. If the DNA templates and PCR conditions are identical between two samples from the same individual, then identical DNA profiles should result regardless of how well or poorly the PCR primers amplify the DNA template. The potential of null alleles is not a problem within a laboratory that uses the same primer set to amplify a particular STR marker. However, with the emergence of national and international DNA databases, which store only the genotype information for a sample, allele dropout could potentially result in a false negative or incorrect exclusion of two samples that come from a common source. To overcome this potential problem, the matching criteria in database searches can be made less stringent when searching a crime stain sample against the DNA database of convicted offender profi les (see Chapter 12). That is, the database search might be programmed to return any profiles with a match at 25 out of 26 alleles instead of 26 out of 26. Whenprimers are selected for amplification of STR loci, candidate primers are evaluated carefully to avoid primer binding site mutations. Sequence analysis of multiple alleles is performed, family inheritance studies are conducted, withinlocus peak signal ratios for heterozygous samples are examined, apparent homozygous samples are reamplified with lower annealing temperatures, and statistical analysis of observed versus expected homozygosity is performed on.