An allelic ladder is an artificial mixture of the common alleles present in the human population for a particular STR marker (Sajantila et al. 1992). They are generated with the same primers as tested samples and thus provide a reference DNA size for each allele included in the ladder. Allelic ladders have been shown to be important for accurate genotype determinations (Smith 1995). These allelic ladders serve as a standard like a measuring stick for each STR locus. They are necessary to adjust for different sizing measurements obtained from different instruments and conditions used by various laboratories (see Chapters 14 and 15).
Allelic ladders are constructed by combining genomic DNA or locus-specific PCR products from multiple individuals in a population, which possess alleles that are representative of the variation for the particular STR marker (Sajantila et al. 1992, Baechtel et al. 1993). The samples are then co-amplified to produce an artificial sample containing the common alleles for the STR marker (Figure 5.3). Allele quantities are balanced by adjusting the input amount of each component so that the alleles are fairly equally represented in the ladder.
Principle of allelic ladder formation. STR alleles from a number of samples are separated on a polyacrylamide gel and compared to one another. Samples representing the common alleles for the locus are combined and re-amplified to generate an allelic ladder. Each allele in the allelic ladder is sequenced since it serves as the reference material for STR genotyping. Allelic ladders are included in commercially available STR kits.
For example, to produce a ladder containing five alleles with 6, 7, 8, 9, and 10 repeats, individual samples with genotypes of (6,8), (7,10), and (9,9) could be combined. Alternatively, the combination of genotypes could be (6,9), (7,8), and (10,10) or (6,6), (7,7), (8,8), (9,9), and (10,10).
Additional quantities of the same allelic ladder (second- and third-generation ladders) may be produced by simply diluting the original ladder 1/1000-1/1 000 000 parts with deionized water and then re-amplifying it using the same PCR primers (Baechtel et al. 1993). It is imperative that allelic ladders be generated with the same PCR primers as used to amplify unknown samples so that the allele 'rungs' on the ladder will accurately line up with that of the repeat number of the unknown sample when the unknown is compared to the ladder. As will be seen in the next section, commercial manufacturers now provide allelic ladders in their STR typing kits so that individual laboratories do not have to produce their own allelic ladders.
CHOICE OF MARKERS USED BY THE FORENSIC DNA TYPING COMMUNITY
For DNA typing markers to be effective across a wide number of jurisdictions, a common set of standardized markers must be used. The STR loci that are commonly used today were initially characterized and developed either in the laboratory of Dr. Thomas Caskey at the Baylor College of Medicine (Edwards et al. 1991, Hammond et al. 1994) or at the Forensic Science Service in England (Kimpton et al. 1993, Urquhart et al. 1994). The Promega Corporation (Madison, Wisconsin) initially commercialized many of the Caskey markers while Applied Biosystems (Foster City, California) picked up on the Forensic Science Service (FSS) STR loci as well as developing some new markers.
Today both Applied Biosystems and the Promega Corporation have STR kits that address the needs of the DNA typing community and cover a common set of STR loci. The availability of STR kits that permit robust multiplex amplification of eight or more STR markers has truly revolutionized forensic DNA. Matching probabilities that exceed one in a billion are possible in a single amplification with 1 ng (or less) of DNA sample. Just as impressive is the fact that results can be obtained today in only a few hours compared to the weeks that restriction fragment length polymorphism (RFLP) methods took just a few years ago.
One of the first STR multiplexes to be developed was a quadruplex created by the Forensic Science Service that comprised the four loci TH01, FES/FPS, VWA, and F13A1 (Kimpton et al. 1994). This so-called 'first-generation multiplex' had a matching probability of approximately 1 in 10 000. The FSS followed with a second-generation multiplex (SGM) made up of six polymorphic STRs and a gender identification marker (Gill et al. 1996, Sparkes et al. 1996). The six STRs in SGM are TH01, VWA, FGA, D8S1179, D18S51, and D21S11 and provide a matching probability of approximately 1 in 50 million. The gender identification marker amelogenin will be described in more detail later in this chapter.
The first commercial STR kit capable of multiplex amplification became available from the Promega Corporation in 1994 for silver stain analysis. This kit consisted of the STR loci CSF1PO, TPOX, and TH01 and is often referred to as the 'CTT' triplex using the first letter in each locus. The CTT triplex only had a matching probability of ~1 in 500 but was still widely used in the United States in the mid-1990s as it was the first available STR multiplex kit and could be performed with a fairly low start-up cost.
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