Simple & Rapid Generation of Complex DNA Profiles for the Undergraduate Laboratory

DNA profiles can be generated by a variety of techniques incorporating different types of DNA markers. Simple methods are commonly utilized in the undergraduate laboratory, but with certain drawbacks. Here, I present an advancement of the Alu dimorphism technique involving two tetraplex PCR analyses that yield 6,561 possible genotypes. This technique is simple to perform in an undergraduate laboratory. DNA markers are segments of nucleotides with known locations in the genome that can be informative in DNA profiling and genetic mapping studies if variations exist. Mini-satellites, a.k. a. variable number tandem repeats (VNTRs), consist of variants that are the result of 15-100 base pair (bp) repeated DNA fragments, and microsatellites are variants resulting from 2-7 bp repeated DNA fragments. VNTRs are commonly analyzed using the Southern blot procedure in order to detect numerous loci simultaneously, thereby generating a highly informative DNA profile. This is accomplished using a radioactive probe that consists of a short stretch of DNA nucleotides that are shared among the VNTR loci. Microsatellites require the use of high-resolution techniques such as standard acrylamide gel electrophoresis or the use of an automated DNA sequencer. Several microsatellite loci can be analyzed simultaneously by the multiplex polymerase chain reaction (PCR) technique. This methodology involves using primer pairs from several loci in a single reaction. Although there are commercial suppliers that offer kits for this technique, including the primers and markers for various alleles, cost becomes a limiting factor in undergraduate teaching laboratories. Based on cost, practicality, equipment availability, and isotope use, undergraduate laboratories are restricted in the types of analyses that can be performed. The two techniques typically used in the undergraduate laboratory are either the isolation of the VNTR locus referred to as D1S80, or an Alu dimorphism such as TPA-25 (Bloom et al., 1996), and are available as kits through commercial suppliers. Both simply involve PCR and analysis by electrophoresis on an agarose gel. D1S80 is a choice marker since it contains 29 different alleles yielding a possible 435 different genotypes [n (n+1)/2]. The primary drawback is the resolution limitation of agarose gels, creating difficulty in distinguishing individual alleles. Alu dimorphisms are variants that differ by the presence or absence of an Alu element. Alu elements are 300 bp retroposons (RNA-mediated transposable elements) that are amplified in the primate genome by the process of retrotransposition (retroposition) (Figure 1). The more recent integrations are not fixed in the human genome and therefore yield this type of variation (Roy-Engel et al., 2001), which has been useful in human population studies (Batzer et al., 1994; Roy-Engel et al., 2001) supporting the African origins of humans. The advantages of using the Alu dimorphism include its ease of use and simplicity of identification of the allelic variants. The drawback is the limited information attainable with only three possible genotypes (Figure 2). I previously developed a system to assay four Alu-containing loci using multiplex PCR (Kass, 2003). This is a simple, rapid technique for analyzing 81 ([3.sup.4]) possible genotypes. However, when using this for a simple forensic-type study in an undergraduate genomics laboratory, two individuals had the same genotype; and when incorporating this for a paternity test illustration, it was difficult to rule out one potential father. Therefore, I developed a second tetraplex reaction presented here for the first time. The two tetraplex reactions yield 6,561 combinations, dramatically increasing forensic and paternity capabilities of this tool. Additionally, the individual Alu variants that were chosen are referred to as "intermediate frequency (IF) polymorphisms", i.e., both the presence and absence forms are commonly found among populations (Roy-Engel et al. …