Minisatellite variable repeat MVRPCR

Halki Diabetes Remedy

Most Effective Diabetes Treatment

Get Instant Access

The majority of minisatellite loci consist of heterogeneous arrays of two or more repeat unit types (MVRs) that differ slightly. The human hypervariable minisatellites characterized to date vary not only in the number of repeat copies (allele length) but also in the interspersion pattern of variant repeat units within the array. This internal variation provides a highly informative approach to the study of allelic variation and the processes of mutation. Interspersion patterns can be determined by MVR mapping and this reveals far more variability than allele length analysis.

The first MVR mapping was developed at locus D1S8 (minisatellite MS32) (Wong et al., 1987). This particular minisatellite consists of a 29 bp repeat unit showing two classes of MVR that differ by a single base substitution, resulting in the presence or absence of an HaelII restriction site (Jeffreys et al., 1990). Extremely high levels of variation in the interspersion patterns of two types of repeat within the alleles have been revealed by HaelII digestion of PCR-ampli-fied alleles. Subsequently, a technically simpler version of a PCR-based mapping system (MVR-PCR) was invented (Jeffreys et al., 1991b) (see also a detailed review by Jeffreys et al., 1993). In addition to a primer at a fixed site in the DNA flanking the minisatellite, this assay reveals the internal variation of repeats by using an MVR primer specific to one or more types of variant repeat. The MVR-specific primers at low concentration will bind to just one of their complementary repeat units in the PCR, thus MVR locations will be represented as an array of sequentially sized, amplified DNA fragments. To analyse two different types of repeat, MVR-PCR uses a single flanking primer plus two different MVR primers to generate two complementary ladders of amplified products corresponding to the length between the flanking primer and the location of one or other of the repeat types within the minisatellite repeat array. In order to prevent the progressive shortening of PCR products by internal priming of the MVR-specific primers, 'tagged' amplification that uncouples MVR detection from subsequent amplification is used.

The MVR-PCR products are separated by electrophoresis on an agarose gel and detected by Southern blotting and hybridization to an isotope-labelled minisatellite probe. The PCR reaction for each of the two MVR-specific primers is carried out in a separate tube, and products are loaded in adjacent lanes in an agarose gel. Some work has been done that involves the labelling of variant primers with different-coloured fluorescent tags and amplifying both sets of MVR products in a single reaction, to facilitate comparison of the complementary MVR profiles (Rodriguez-Calvo et al., 1996; Hau and Watson, 2000). The MVR-PCR technique has revealed enormous levels of allelic variation at several human hypervariable minisatellites: MS32 (D1S8) (Jeffreys et al., 1991b; Tamaki et al., 1992a, 1992b, 1993), MS31A (D7S21) (Neil and Jeffreys, 1993; Huang et al., 1996), MS205 (D16S309) (Armour et al., 1993; May et al., 1996), CEBl (D2S90) (Buard and Vergnaud, 1994), g3 (D7S22) (Andreassen and Olaisen,

1998), YNH24 (D2S44) (Holmlund and Lindblom, 1998), B6.7 (Tamaki et al, 1999; Mizukoshi et al., 2002) and the insulin minisatellite (Stead and Jeffreys, 2000).

At a minority of loci such as MS32, where almost all repeat units are of the same length, a diploid MVR map of the interspersion patterns of repeats from two alleles superimposed can be generated from total genomic DNA and encoded as a digital diploid code (Jeffreys et al., 1991b). Since different length repeats will cause the MVR maps of each allele to drift out of register, most minisatellites are not amenable to this diploid coding, and instead only single allele coding is possible. Both single allele and diploid codes are highly suitable for computer databasing and analysis.

Allele-specific MVR-PCR methods (Monckton et al., 1993) have been developed to map single alleles from total genomic DNA using allele-specific PCR primers directed to polymorphic SNP sites in the DNA flanking the minisatellite (Figure 5.4). These SNP primer pairs are identical, with the exception of a 3' terminal mismatch that corresponds to the variant flanking base. This method is extremely expedient since it does not involve the time-consuming separation of the alleles by agarose gel electrophoresis. Another added advantage of allele-

Figure 5.4 Allele-specific MVR-PCR of MS32 (DIS8). Alleles from the genomic DNA of three individuals were directly mapped utilizing an SNP (Hf) flanking the minisatellites (allele-specific MVR-PCR). The numbers to both sides of the figure indicate the code position, e.g. in sample 1 the Hf+ allele can be read as xatataaaaat . . .'

Figure 5.4 Allele-specific MVR-PCR of MS32 (DIS8). Alleles from the genomic DNA of three individuals were directly mapped utilizing an SNP (Hf) flanking the minisatellites (allele-specific MVR-PCR). The numbers to both sides of the figure indicate the code position, e.g. in sample 1 the Hf+ allele can be read as xatataaaaat . . .'

specific MVR-PCR is its ability to recover individual-specific typing data from DNA mixtures (Tamaki et al., 1995a).

The MVR-PCR technique is the best approach for exploiting the potential of hypervariable minisatellite loci because of the unambiguous nature of MVR mapping and the generation of digital MVR codes suitable for computer analysis. Code generation does not require standardization of electrophoretic systems, is immune to gel distortions and band shifts, does not involve error-prone DNA fragment length measurement and does not require side-by-side comparisons of DNA samples on the same gel.

For these reasons, MVR-PCR is well-suited for use in forensic analysis. The potential for forensic applications have been demonstrated by obtaining authentic diploid MVR coding ladders from only 1 ng of genomic DNA from bloodstains, saliva stains, seminal stains and plucked hair roots (Yamamoto et al., 1994a). This is done by determining the source of saliva on a used postage stamp (Hopkins et al., 1994), by making MVR coding ladders quickly without any need for blotting and hybridization (Yamamoto et al., 1994b) and by maternal identification from remains of an infant and placenta (Tamaki et al., 1995a). While forensic samples in general contain partially degraded DNA, MVR-PCR does not require intact minisatellite alleles. Such DNA samples yield truncated codes due to the disappearance of longer PCR products, but these codes are still compatible with the original allele information. Although replicate runs on the same sample and reading consensus codes are required, reliable codes can be obtained from as low as 100 pg of genomic DNA by MVR-PCR at MS32 (Jeffreys et al., 1991b).

The MVR-PCR method at MS32 and at minisatellite MS31A has also been applied to paternity testing. The potential for establishing paternity in cases lacking a mother was demonstrated by a major contribution to the paternity index made possible by the extremely rare paternal alleles at these two loci (Huang et al., 1999). Similarly, these rare alleles proved vital in confirming the relationship between a boy and his alleged grandparents despite an inconsistency (i.e. mutant allele) between the father and grandparents at one of the STR loci (Yamamoto et al., 2001). Significant germline mutation rates to new length alleles have been observed at some hypervariable loci (Jeffreys et al., 1988), which will generate false paternal exclusions in about 1.8% of paternity cases. In such cases, allele length measurements do not allow the distinction of nonpaternity from mutation. In contrast, detailed knowledge of the mutation processes, coupled with MVR analysis of allele structures, can help distinguish mutations from non-paternity. This theory was tested at MS32 using both real and simulated allele data (Tamaki et al., 2000). Since MVR-PCR allows information to be recovered from at least 40 repeat units, a mutant paternal allele will be identical to the progenitor paternal allele - with the exception of the first few repeats. Most germline mutation events altering repeat array structures are targeted to this region, most likely due to its proximity to a flanking recombination hotspot that appears to drive repeat instability (reviewed in Jeffreys et al., 1999). Thus, a mutant paternal allele in a child will tend to resemble one of the father's alleles more than most other alleles in the population. This approach is unlikely to work at extremely hypervariable minisatellite loci such as CEB1 and B6.7, given their very high rate of germline instability coupled with complex germline mutation events that can radically alter allele structure in a single mutation event (Buard et al., 1998; Tamaki et al., 1999).

The MVR-PCR technique reveals enormous levels of variation, unmatched by any other single-locus typing system. At MS32, for example, almost all alleles in several ethnic populations surveyed were different. However, different alleles can show significant similarities in repeat organization (Jeffreys et al., 1991b). Heuristic dot-matrix algorithms have been developed to identify significant allele alignments and have shown that approximately three-quarters of alleles mapped to date can be grouped into over 100 sets of alignable alleles, indicating relatively ancient groups of related alleles present in diverse populations (Tamaki et al., 1995b). Some small groups of alleles can display a strong tendency to be population-specific, consistent with recent divergence from a common ancestral allele (Figure 5.5). In most groups, the 5' ends of the aligned MVR maps show most variability due to the existence of the flanking recombination hotspot, therefore MVR allele analysis at MS32 can serve not only as a tool for individual identification but also for giving clues regarding the ethnic background of an individual (Tamaki et al., 1995b). Another locus that provided a clear and detailed view of allelic divergence between African and non-African populations is MS205 (Armour et al., 1996). A restricted set of allele families was found in non-African populations and formed a subset of the much greater diversity seen in Africans, which supports arguments for a recent African origin for modern human diversity at this locus. Very similar findings emerged from MVR analyses of the insulin minisatellite, again pointing to a major bottleneck in the 'Out of Africa' founding of non-African populations (Stead and Jeffreys, 2002).

Finally, MVR-PCR has been developed at the Y-chromosome-specific variable minisatellite DYF155S1 (MSY1), with potential for extracting male-specific information from mixed male/female samples (Jobling et al., 1998). This marker is also useful for paternity exclusion and, if adequate population data are available and the allele is rare, can be used in individual identification and paternity inclusion. The now-extensive use of Y-chromosomal microsatellites in forensic applications is discussed in Chapter 9.

Unfortunately, MVR-PCR has rarely been used in forensic analysis despite its simplicity, considerable discriminatory power and its ability to reveal enormous levels of variation.

Was this article helpful?

0 0
Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Post a comment