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

Stevens, Aaron J.; Taylor, Millie G; Pearce, F. Grant; Kennedy, Martin A.

doi:10.1534/g3.116.038687

Cited by 19 publications

(23 citation statements)

References 33 publications

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“…2b and 2f). The failure of amplification of target region of the allele harboring deletion or substitution mutations could also be attributed to allelic drop out during PCR and also the fact that extension of the annealing failure is allele and mismatch position-dependent [23][24][25].…”

Section: Discussionmentioning

confidence: 99%

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

Ijaz

Nakazato

Setou

et al. 2022

Preprint

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The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3` ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We demonstrate six examples of CRISPR/Cas9-engineered knockout cells, to screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR coupled with TBE-High-Resolution PAGE is a simple method for genotyping of the CRISPR/Cas9-engineered cell lines, which can be performed without special equipment and techniques.

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Section: Discussionmentioning

confidence: 99%

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

Ijaz

Nakazato

Setou

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…Most ADO mechanisms are determined by the presence of a single-nucleotide variant (SNV) situated inside the primer-binding sequences on the targeted DNA, the SNV causing the failure of the primer-template annealing and the consequent amplification error. Concomitant presence of a differential allelic methylation and G-quadruplex motifs in some regions of the genome, DNA degradation leading to PCR refractory breaks, imperfect PCR conditions preventing DNA template accessibility, and presence of both homopolymer tracts and pseudogenes were also described as potential determinants of ADO or preferential amplification ( Piyamongkol et al, 2003 ; Stevens et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Sanger Validation of High-Throughput Sequencing in Genetic Diagnosis: Still the Best Practice?

et al. 2020

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Next-generation sequencing (NGS)’s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq® and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% “high quality” NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be a priori assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.

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“…The new primer has a forward position at exon 15 and reverse position at partial exon 16 based on Insee et al (2014) research. The specific CHD1-W gene site position has one deletion base to amplify the W chromosome that would likely impact mismatch to create two "alleles" if heterozygous, which could be distinguished by Sanger sequencing (Simsek and Adnan 2000;Stevens et al 2017). We chose that site with considering requires good primer characteristics (Wang 2016;Bustin et al 2020) also, indel polymorphisms are less frequent in exonic regions than in intronic regions so the primer designed to hybridize DNA to exonic regions would be more likely to amplify readable sequences (Tezuka et al 2012).…”

Section: Primer Designmentioning

confidence: 99%

Short Communication: A new primer set in CHD1 gene for bird sex identification

et al. 2021

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Abstract. Vera F, Wajjwalku W, Yuda P, Daryono BS. 2021. Short Communication: A new primer set in CHD1 gene for bird sex identification. Biodiversitas 22: 4977-4982. Determine sex is difficult for many bird species that are sexually monomorphic or only dimorphic in the adult stage. Many molecular markers have been developed for DNA sexing, which were mostly based on identifying Z and W chromosomes of avians. The sex determination of birds was mostly applied universal primer set such as P2/P8 or 2550F/2718R. However, those universal primers that were designed sometimes could not good result consistency in non-ratite birds or ratite birds. Therefore, we improved a specific primer design with deletion site in W chromosome to amplify the female-specific segments on Chromodomain-helicase DNA binding protein-1 (CHD1) gene from alignment sequences CHD1-Z and CHD1-W of Macrocephalon maleo S. Müller, 1846. This study aims to design a new pair of primer targeting a section of the CHD1 gene that can be amplified in non-ratite birds, the name of a new primer is In-Sex F/In-Sex R. A new primer set amplified DNA fragments in around 650 or 550 base pairs of CHD1-Z and also, 350 base pairs of CHD1-W. The result has successfully amplified the sex of multiple species on orders Galliformes, Passeriformes, Accipitriformes, and Strigiformes. This analysis can be helpful to the effort of in-situ or ex-situ management and conservation programs e.g the mating system in birds. And also, the analysis can be helpful for sexing data in the case of captive birds before releasing in natural habitat or reintroduction programs.

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

Cited by 19 publications

References 33 publications

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

Sanger Validation of High-Throughput Sequencing in Genetic Diagnosis: Still the Best Practice?

Short Communication: A new primer set in CHD1 gene for bird sex identification

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