For human identification purposes it is important to have DNA markers that exhibit the highest possible variation or a number of less polymorphic markers that can be combined in order to obtain the ability to discriminate between samples.
As will be discussed further in Chapter 7, forensic specimens are often challenging to PCR amplify because the DNA in the samples may be severely degraded (i.e., broken up into small pieces). Mixtures are prevalent as well in some forensic samples, such as those obtained from sexual assault cases containing biological material from both the perpetrator and victim.
The small size of STR alleles (~100-400bp) compared to minisatellite VNTR alleles (~400-1000 bp) make the STR markers better candidates for use in forensic applications where degraded DNA is common. PCR amplification of degraded DNA samples can be better accomplished with smaller product sizes (see Chapter 7). Allelic dropout of larger alleles in minisatellite markers caused by preferential amplification of the smaller allele is also a significant problem with minisatellites (Tully et al. 1993). Furthermore, single base resolution of DNA fragments can be obtained more easily with sizes below 500 bp using denaturing polyacrylamide gel electrophoresis (see Chapter 12). Thus, for both biology and technology reasons the smaller STRs are advantageous compared to the larger minisatellite VNTRs.
Among the various types of STR systems, tetranucleotide repeats have become more popular than di- or trinucleotides. Penta- and hexanucleotide repeats are less common in the human genome but are being examined by some laboratories (Bacher et al. 1999). As will be discussed in Chapter 6, a biological phenomenon known as 'stutter' results when STR alleles are PCR amplified. Stutter products are amplicons that are typically one or more repeat units less in size than the true allele and arise during PCR because of strand slippage (Walsh et al. 1996). Depending on the STR locus, stutter products can be as large as 15% or more of the allele product quantity with tetranucleotide repeats. With di- and trinucleotides, the stutter percentage can be much higher (30% or more) making it difficult to interpret sample mixtures (see Chapter 7). In addition, the four base spread in alleles with tetranucleotides makes closely spaced heterozygotes easier to resolve with size-based electrophoretic separations (see Chapter 12) compared to alleles that could be two or three bases different in size with dinucleotides and trinucleotide markers, respectively.
Thus, to summarize, the advantages of using tetranucleotide STR loci in forensic DNA typing over VNTR minisatellites or di- and trinucleotide repeat STRs include:
■ A narrow allele size range that permits multiplexing;
■ A narrow allele size range that reduces allelic dropout from preferential amplification of smaller alleles;
■ The capability of generating small PCR product sizes that benefit recovery of information from degraded DNA specimens; and
■ Reduced stutter product formation compared to dinucleotide repeats that benefit the interpretation of sample mixtures.
In the past decade, a number of tetranucleotide STRs have been explored for application to human identification. The types of STR markers that have been sought out have included short STRs for typing degraded DNA materials, STRs with low stuttering characteristics for analyzing mixtures, and male-specific Y chromosome STRs for analyzing male-female mixtures from sexual crimes (Carracedo and Lareu 1998). The selection criteria for candidate STR loci in human identification applications include the following characteristics (Gill et al. 1996, Carracedo and Lareu 1998):
■ High discriminating power, usually > 0.9, with observed heterozygosity > 70%;
■ Separate chromosomal locations to ensure that closely linked loci are not chosen;
■ Robustness and reproducibility of results when multiplexed with other markers;
■ Low stutter characteristics;
■ Predicted length of alleles that fall in the range of 90-500 bp with smaller sizes better suited for analysis of degraded DNA samples.
In order to take advantage of the product rule, STR markers used in forensic DNA typing are typically chosen from separate chromosomes to avoid any problems with linkage between the markers (see Chapter 20).
Was this article helpful?
Are You Suffering From Social Withdrawal? Do People Shun Or Ostracize You Because You Have A Hard Time Getting Some Of Your Words Out? Or Does Your Child Get Teased At School Because They Stutter And Cant Speak Like Everyone Else? If you have answered yes to any of the above, then you are in the tiny percentage of people that stutter.