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CONTINUING EDUCATION :: MOLECULAR DIAGNOSTICS


Biology If there’s enough of them to see as distinct bands on CsCl gradients, it should come as no surprise that there are many microsatellites in the human genome. Overall, they comprise about three percent of the total human nuclear genome, dispersed more or less uniformly across all chromosomes. Ones based on odd number elements (three or five nucleotides) are about half as common as ones with even number ele- ments (two, four, or six nucleotides) and for unknown reason—or if there even is a reason—triplet SSRs are most common in association with coding regions. It’s unclear if microsatellites have a consistent useful biological function, in most cases either appearing to do nothing or else being pathogenic. Pathogenic examples related to trinucleotide repeats have their own collective name as trinucleotide repeat disorders. One of the best-known examples of these is Hunting- ton’s disease. In this case, the repeat element CAG occurs within an exonic (protein coding) portion of the HTT gene with each repeat coding for an addi- tional glutamine residue in series in the mature Hun- tingtin protein. At n< 26, protein behavior is normal and there is little risk to offspring, but as n increases (and thus the number of glutamine residues present in a row in the protein) there is a progressive loss of proper gene function evidenced by increased risk of Huntington’s disease in offspring, risk of Huntington’s disease in the proband, and then increasing sever- ity of disease and decrease in age of symptom onset. At approximately n>40, full penetrance (worst case presentation) is observed and a 50 percent risk of disease transmission to offspring.


For our current consideration,


what’s of key importance here is that the repeat numbers (n) for a single microsatellite locus can sometimes change between


A very common method for DNA profiling is known as


Variable Nucleotide Tandem Repeat (VNTR) typing.


generations. One mechanism by which this occurs is known as polymerase slippage. Essentially, this happens during DNA replication if polymerase activ- ity pauses and the nascent strand briefly detaches from template before reannealing; it may anneal in register with regard to the repeat element either upstream or downstream of where it detached. As polymerase activity now resumes the progeny strand has either gained or lost copy numbers of the repeat, respectively. It is observed that gain of repeats occurs much more frequently than loss. Considering this mechanism, it further stands to reason that the lon- ger a microsatellite is, the greater the chances for a slippage event to occur during replication. Overall there is thus a trend toward getting longer over time, and the degree of instability increasing as they get longer, in effect creating a positive feedback process. This does not on evolutionary timescales become a runaway process and must be constrained by loss of organism fitness as more and more chromosomal regions become endless short repeats.


Another mechanism which can lead to changes in mini and microsatellite element repeat number is unequal crossing over. Readers are reminded that during meiosis, breakage and rejoining of homolo- gous chromosome pairs allows for the recombina- tion of alleles. These breakages and joins occur along homologous sequence elements on the two chromo- somes, but as in the case of polymerase slippage it is possible for a shift in register by some multiple of the repeat element size to occur during strand align- ment. The resulting recombined chromosomes will have one which has gained repeats while the other has lost an equal number. In common with poly- merase slippage, this type of instability is increas- ingly likely to occur as the length of the region—that is, the number of repeats n—increases. It’s important that there are mechanisms for change in repeat copy number (this creates differen- tiable markers over time) and that such changes are individually quite rare, occurring on an evolutionary timescale (meaning that we can expect in almost all cases, alleles sizes remain fixed between generations). Overall, we are presented with a large and disperse set of VNTRs across the genome, and we should expect them to behave as fixed in size when going from parent to progeny. This randomness of dispersion across the genome, relatively high population diversity per locus, and low frequency of further variation between organism generations underlies their utility as genetic markers.


Technique


In practice, VNTR loci for evalu- ation are selected based on hav- ing highly conserved flanking sequences; a large number of repeat number sizes seen across the population of interest (in


other words, high allelic diversity); and good ampli- fication behavior (reliable amplification of true loci across a wide range of input concentrations, few or no spurious amplification products). For ease of use and convenience in assay performance it’s further desirable to multiplex loci into single amplification reactions, so loci with dissimilar product sizes (that is, non-overlapping products) and similar amplifica- tion kinetics and common reaction requirements are selected to group together. For each individual VNTR locus, PCR primers are selected which meet these criteria, and one PCR primer of the pair for each locus is fluorescently labelled. As the end detection method will support five separable dye channels, a total of four target dyes are available, meaning that the requirement above for amplicons of dissimilar size is not absolute, when amplicons of two loci may be of overlapping size, they can be differen- tially dye labeled. By a combination of differentiable expected product sizes and use of different dyes where sizes might overlap, it is possible to multiplex


MLO-ONLINE.COM MAY 2019 9


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