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      Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

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          Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

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          Most cited references 32

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          Analysis of protein-coding genetic variation in 60,706 humans

          Summary Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. We describe the aggregation and analysis of high-quality exome (protein-coding region) sequence data for 60,706 individuals of diverse ethnicities generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of truncating variants with 72% having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human “knockout” variants in protein-coding genes.
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            Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

            A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
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              Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis.

               D Sen,  W Gilbert (1988)
              We have discovered that single-stranded DNA containing short guanine-rich motifs will self-associate at physiological salt concentrations to make four-stranded structures in which the strands run in parallel fashion. We believe these complexes are held together by guanines bonded to each other by Hoogsteen pairing. Such guanine-rich sequences occur in immunoglobulin switch regions, in gene promoters, and in chromosomal telomeres. We speculate that this self-recognition of guanine-rich motifs of DNA serves to bring together, and to zipper up in register, the four homologous chromatids during meiosis.

                Author and article information

                G3 (Bethesda)
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                30 January 2017
                March 2017
                : 7
                : 3
                : 1019-1025
                [* ]Department of Pathology, University of Otago, Christchurch 8140, New Zealand
                []Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand
                Author notes
                [1 ]Corresponding author: Department of Pathology, University of Otago, Christchurch, P.O. Box 4345, Christchurch, 8140 New Zealand. E-mail martin.kennedy@
                Copyright © 2017 Stevens et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 33, Pages: 7


                polymerase chain reaction, methylation, allelic dropout, pcr, g-quadruplex


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