search.noResults

search.searching

dataCollection.invalidEmail
note.createNoteMessage

search.noResults

search.searching

orderForm.title

orderForm.productCode
orderForm.description
orderForm.quantity
orderForm.itemPrice
orderForm.price
orderForm.totalPrice
orderForm.deliveryDetails.billingAddress
orderForm.deliveryDetails.deliveryAddress
orderForm.noItems
CONTINUING EDUCATION :: MOLECULAR DIAGNOSTICS


15-20 different loci in a single reaction. (Technically, challenges in making so many competing reactions amplify with near equal efficiency may make it sim- pler to limit individual PCR reactions to four to six loci per multiplex set, and then combine products of several such reactions for the detection step.) Considering for a moment just one of our loci, if we PCR amplify it in a diploid organism such as a human, we expect products from two alleles—one on each host chromosome pair. For each locus we expect to get either no amplicon (in the event the locus is deleted), or a product of a size which is determined by the number of repeat elements present in the micro- satellite. If we take a triploid repeat as an example and assume some space for flanking sequences where our PCR primers are located, we might come across a pop- ulation sample at this locus see amplicon sizes of 112 to 130 nucleotides in length at 3 nt intervals. Each of these discrete sizes will occur at some frequency in the population. If we extend this to the diploid situation, we expect to see no amplicon (homozygous locus deleted, both alleles); one size product (where both alleles happen to be homozygous for the same repeat number); or two products (where the alleles are heterozygous for repeat number). In any case you can either score no, one, or two products and the prod- ucts if present will be at known, predetermined sizes. A common nuance to the methodology is the addition of a final longer PCR extension step which allows for uniform monoadenylation of all products (the DNA polymerase commonly used in this applica- tion has a tendency to add a single “A” to 3’ ends of amplicons). If this is not allowed to go to completion, the end product is some mix of blunt and monoad- enlyated products, giving rise to two product bands one bp apart. The longer final extension encour- ages all products to be monoadenylated and thus generate a single uniform peak—a nice thought but one which in reality is also influenced by adjacent sequences, meaning individual VNTR loci tend to have distinctive “personalities” as evidenced by char- acteristic ratios of these two peaks, or sometimes even a descending series of “stutter” products—evidence of DNA slippage at the locus during PCR. From an instrumentation perspective, we can use capillary electrophoresis (conveniently available in the form of trusty old Sanger sequencing machines) to reliably separate these products by size and color, with sizing accuracy assisted by inclusion on a dedi- cated dye channel of a size standard in each reaction. Now if we extend this to a number of such loci and can identify the product(s) from each by some combi- nation of dye label and/or size, we can generate a vast number of possible combinations of product size and color patterns—each reproducibly generated by one individual’s template DNA. With enough such loci combined, not only are the results reproducible per individual, but also unique (or approaching unique, to some astronomically high probability) to that individual. With the loci commonly used for human VNTR typing, only some 12-15 markers are needed to reach acceptable levels of “uniqueness.”


10 MAY 2019 MLO-ONLINE.COM


Interpretation


At least two stages of interpretation of the data occur. The first is in directly examining the capillary elec- trophoresis data for expected markers, and “binning” the data. As we now understand, each product can occur in multiple pre-set sizes based on the repeat element length; we’ve also an inkling that not all loci are well behaved and may have split peak (incom- plete monoadenylation) or stutter issues. While this may sound complex, in reality it becomes fairly straightforward to interpret results and call each locus as either null (homozygous deleted), hetero- zygous with two size values, or homozygous with a single size value. (Some among you may note there’s another possibility, hemizygous deletion. This will also show as a single peak for the one present allele size, and while the peak area may be less than would be expected for a homozygote of that size, we gen- erally can’t detect or call this with certainty and could erroneously call this homozygous. Loci are chosen such that deletions are very rare and so this condition isn’t expected to be observed.) Note that because we’re looking for peaks at known, expected sizes—and these differ by at least two and more commonly three, four, or five nucleotides— minor variations in migration due to e.g. differing ionic strength between samples are not significant. Sizes as reported by the instrument are “binned” to the closest matching size; an apparent 161.7 bp prod- uct when there’s known 161 and 166 alleles can be comfortably called as 161. By carrying out this scoring/binning on all marker sets, an ordered series of values is generated. While the exact number depends on the set of markers chosen, as an example one common commercial iteration of this method uses 15 classical VNTR loci (yielding 30 values) and one additional loci, which while not strictly a classical VNTR can be treated as one and yields two more values (which indicate whether the source material was male or female) for a total of 32 values. This set of values is the sample “fingerprint.”


Applications


Where do we find VNTR typing used in clinical (non- forensic) settings today? They can be applied any time we wish to link two samples, with two of the


most common settings being: t Paternity testing. Barring any de novo changes in repeat copy number, a child should inherit one half of their VNTR markers from each parent. The appearance of even a single locus in the offspring bearing a size not rep- resented in the putative parents, means either that a de novo size change has occurred (pos- sible, but rare) or much more likely, that one (or both) parents isn’t as thought. Given the total number of loci examined and the popu- lation diversity at each locus, it would be vanishingly rare for someone to not show at least some diversity from a nonrelated puta- tive father. Of course, it goes without saying


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52