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SPECIAL FEATURE :: PCR


PCR: a versatile tool—from detecting genetic diseases and cancers to monitoring stability after bone marrow transplants


By Teresa Snyder-Leiby, PhD P


olymerase chain reaction (PCR) is the foundation for hundreds of molecular DNA tests for the detection and monitoring of a wide range of genetic diseases. Molecular testing methods and examples of the diseases


detected include:1-12 t Qualitative fluorescence PCR (QF-PCR): Detection of aneuploidy (such as Down syndrome) and repeat expansion


diseases (such as Fragile X and Huntington’s disease). t Multiplex Ligation-dependent Probe Amplification MLPA®


and Methylation Specific (MS-MLPA® ) (MRC-


Holland reference): Detection of diseases from insertions/ deletions/point mutations; including predisposition to cancer (BRCA 1), neuromuscular disorders (Duchenne


muscular dystrophy), and aneuploidy. t Microsatellite instability: Detecting cells deficient in DNA repair capabilities, microsatellite instability-high (MSI-


H), or mismatch repair deficient (dMMR) in solid tumors. t QF-PCR using human identity chemistries is also an essential tool in monitoring engraftment post-transplant.


QF-PCR - aneuploidy


Aneuploidy (an abnormal number of chromosomes) is the most frequent genetic disorder observed in live births and miscarriages, with trisomies being the most prevalent, accounting for approximately 53 percent of all chromo- some abnormalities. QF-PCR is quantitative, and the labo- ratory and data analysis steps can be partially automated for accurate results of more samples in less time than the traditional karyotype method. QF-PCR uses chromosome- specific primers to amplify DNA fragments for each of the chromosomes of interest. DNA fragments are separated with capillary electrophoresis and analyzed by fragment size and number of fragments. The peak height or area, is used to quantify the amount of DNA amplified, determining if the expected pair of each chromosome or aneuploidy is present. Three DNA fragments—visualized as peaks of approxi- mately the same height in an electropherogram, or peak ratios of 2:1 and 1:2—are indications that the sample is in the trisomic range. Custom chemistries or commercial PCR kits provide primer sets that will detect chromosomes 13, 18, 21, X, and Y. In many cases these multiplexes are sufficient for conclusively detecting and quantifying aneuploidy. The peak pattern shown in Figure 1 is an example of a sample that is in the trisomic range for Down syndrome. Peak ratios at loci D21S11 (1:1:1), D21S1437, and IFNAR (1:2) are typical indicators of trisomy 21. Loci D13S634 and D21S1311 contain only single peaks (non- informative). The remaining loci have high hetero- zygous balance, which is typical of two copies of the homologous chromosomes.


Repeat expansion diseases


Expansions of simple sequence repeats, mainly but not limited to tri-nucleotide repeats, are responsible for over


20 JUNE 2019 MLO-ONLINE.COM


40 human diseases. In general, an increasing number of repeats results in more severe phenotypes and the number of repeats increase (expand) as the disease gene is inherited. PCR primers are used to amplify the triplet or hexanucleo- tide repeats. These fragments are separated by capillary elec- trophoresis. Large repeats are in the high molecular weight range. Molecular weight and number of repeats do not have a strictly linear relationship for larger fragments. Reporting requires a correction for the non-linear relationship. DNA control(s) with fragments of a known number of repeats are included in the PCR and capillary electrophoresis. The size and known number of repeats of the control DNA are used to calculate correction and mobility factors for converting the sample peak size (molecular weight) into a number of repeats.


Figure 2 is a report containing the electropherogram of a Huntington’s control sample used to calculate the correc- tion and mobility factors. Figure 3 is a report of a sample with a single fragment in the normal range and detection of a full expansion of 176 CAG repeats.


MLPA®


Multiplex ligation-dependent probe amplification (MLPA®


) is a PCR-based technique developed by MRC- Holland. Since its introduction in 2002, it has become a widely-used and effective technique for detecting copy number variations associated with many common diseases and cancers. MLPA is simpler, more sensitive, and less time intensive when compared to other tradi- tional techniques for detecting copy number variations, including FISH, and Southern blotting. The MLPA reaction is composed of five steps:


1. DNA denaturation; 2. Hybridization of MLPA probes; 3. Ligation reaction of the two probes hybridized to their target sequence; 4. PCR reaction; and 5. Separation of resulting DNA fragments by capillary elec- trophoresis. Peak height or area of test samples is compared to control samples with normal copy number for each probe.


Figure 4 is an example of a sample with no duplica- tions or deletions for a spinal muscular atrophy (SMA) test. The red trace in the electropherogram is the normal sample. The blue trace is the test sample. Figure 5 illustrates heterozygous deletions at the two SMN_2 probes, as the test sample peak heights are half the height of the normal sample.


Microsatellite instability


Microsatellites are stretches of DNA where a 1-5 base pair sequence is repeated several times. The most common microsatellite in humans is a dinucleotide repeat of CA which occurs tens of thousands of times across the genome.


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