Tandemly repeated sequences are powerful tools for genotyping and linkage analysis. The sequences that are the core of the repeated units represent a relatively wide diversity of size (2–80 bp), sequence, and genome distribution. They are extremely valuable as genetic markers because of their highly polymorphic and abundant presence in the genomes of higher organisms. Tandemly repeated sequences can be broadly categorized into two groups— VNTRs and STRs—based primarily on the size range of the core sequence that is repeated.

Figure 6.6 VNTR analysis of human blood specimens for YNZ 22 alleles performed on GeneGel Excel 12.5/24.

Figure 6.7 Separation of amplified microsatellite sequences from different cancer tissues performed on GeneGel Clean 15/24.

Variable-number tandem repeats (VNTRs) describe allelic variants of tandemly repeated core sequences. VNTRs represent simple head-to-tail repeated sequences that can typically range in length from 10 to 80 bps and are highly polymorphic as to the number of repeats at a given locus. They occur fairly frequently in the genome, but there are relatively few different types. Not surprisingly, longer VNTRs tend to show greater polymorphism and thus greater utility for genotyping. Occurring every few kilobases on average, they are not evenly dispersed throughout the genome, tending to cluster toward the telomeric ends of chromosomes.

A working VNTR fragment size range is 50 to 1,500 base pairs. These sequences are typically identified using a strategy similar to that of RFLP analysis, where genomic DNA is digested with an endonuclease and separated by electrophoresis. The gels are then analyzed by southern blotting, using sequence-specific probes to the repeated core sequence.

Short tandem repeat (STR) sequences, or microsatellites, consist of much shorter (2–10 bp) core sequences whose allelic variants are tandemly repeated as many as hundreds of times at different genetic loci. They are typically more evenly dispersed throughout the genome as compared with the larger VNTRs, and they represent ideal tools as genetic markers because of their rich diversity, wide distribution, and polymorphism. Common repeats used for typing and linkage analysis are "CA" or "ACTT" sequences.

Specifically designed as amplification-based detection methods, STR and microsatellite-based DNA typing offer some practical advantages over typing methods based on larger repeat sequences. For example, PCR amplification using primers targeted to a specific STR sequence typically generates 50-to-500-bp-sized fragments without compromising allelic diversity. This allows for easier sizing of a wider range of alleles on a single electrophoretic separation, as compared with larger tandem repeat sequences that typically produce an order-of-magnitude greater range in fragment size diversity. In addition, the smaller average size of STR and microsatellite alleles allows the technique to exhibit a greater tolerance for crude or partially degraded genomic DNA samples, because the need for longer intact sequence domains is reduced. As a corollary, these techniques also show a greater tolerance for rapid and simplified sample preparation methods that make analysis faster and easier.

References

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