Noninvasive genotyping of 9 Y‐chromosome specific STR loci using circulatory fetal DNA in maternal plasma by multiplex PCR

Deng, Zhihui; Wu, Guo-Guang; Li, Qian; Zhang, Xuan; Li, DaCheng; Gao, Su-qing; Lan, Y.‐X

doi:10.1002/pd.1422

Cited by 31 publications

(21 citation statements)

References 19 publications

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“…[1][2][3][4] Prenatal genetic diagnosis using maternal plasma or serum has been developed recently. 5,6 However, when these techniques are applied to blood samples, it must be considered that PCR inhibitors in the specimens may lead to possible false-negative or reduced sensitivity despite advanced DNA purification methods. For example, even after DNA purification steps are used before PCR, a 14% false-negative rate has been observed for hepatitis B virus detection, most probably due to incomplete removal of PCR inhibitors.…”

mentioning

confidence: 99%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

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PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition , enhance PCR amplification , and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent , L-carnitine , D-(؉)-trehalose , and heparin. These cocktails , in combination with two inhibitor-resistant Taq mutants , OmniTaq and Omni Klentaq , enabled efficient amplification of exogenous , endogenous , and high-GC content DNA targets directly from crude samples containing human plasma , serum , and whole blood without DNA purification. In the presence of these enhancer cocktails , the mutant enzymes were able to tolerate at least 25% plasma , serum , or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples , both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial

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mentioning

confidence: 99%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

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“…Scheffer et al combined both PCR results for the Y-linked sequences, SRY and DYS14, for fetal gender determination and concluded that the DYS14 assay targeted a multi-copy sequence and therefore had a higher sensitivity than SRY (30). Deng et al sought nine Y-STR loci of fetal DNA in maternal blood and found that the numbers of Y-STR loci, where the maximum allelic size was less than 100, 137 and 180 bp, observed in Chinese individuals, were one, five, and nine loci, respectively (31). The present study applied the conventional PCR method specialized for amplifying polymorphic X and Y miniSTRs, and utilized an algorithmic based genotyping approach to find fragmented fetal DNA in maternal plasma with high sensitivity and specificity.…”

Section: Discussionmentioning

confidence: 99%

Algorithm-Based Fetal Gender Determination Using X and Y Mini-Short Tandem Repeats at Early Gestational Ages

Aghanoori

Shahriary

Khorrami

et al. 2016

Jentashapir J Health Res

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Background: Detection of fetal DNA in maternal blood has been examined by many research groups for a few years; thereby, scientists have a shorter way to take to approach prenatal diagnosis of abnormal pregnancies. The Y chromosome sequences have recently become the most common applicable indices for fetal sex determination. Objectives: We conducted an algorithmic X and Y mini-Short Tandem Repeats (STRs) genotyping method that could solve the problem of false negative (no detection of Y sequences) results of previous methods. Patients and Methods: Blood samples were obtained from 106 pregnant women and their spouses. Conventional PCR amplified 19 mini-Short Tandem Repeats (STRs) and three non-STR markers, which were subsequently genotyped by the means of Polyacrylamide gel electrophoresis (PAGE). Results: Sensitivity and specificity of the mini-STR genotyping method for fetal DNA detection were calculated (95.9% and 98%, respectively) with a confidence interval of 95%. In addition, sensitivity and informativeness were computed for each of the single mini-STR markers in our conventional PCR method. We also introduced the minimum number of mini-STRs needed to reach maximum validity for fetal gender determination in clinical settings. Conclusions: Algorithm-based mini-STR genotyping method significantly increases the reliability (sensitivity and specificity) of gender determination using free fetal DNA and could be applied in prenatal clinical testing.

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“…Because foetal cfDNA sequences are fragmented and generally present in sizes of <150 bp (with few longer than 250 bp) [17], it is difficult to obtain sufficient effective loci using a commercial STR typing kit, especially for a female foetus [9,10]. Although mini-STR analysis has been implemented as an alternative [11], SNP analysis provides a greater guarantee of genotyping fragmented DNA because the variations of interest are much shorter.…”

Section: Introductionmentioning

confidence: 99%

“…In forensic science, recent research has sought to develop a universal approach to non-invasive paternity analysis by genotyping the paternally inherited alleles in the maternal plasma DNA using STR and single-nucleotide polymorphism (SNP) markers [9][10][11][12][13][14][15][16]. Because foetal cfDNA sequences are fragmented and generally present in sizes of <150 bp (with few longer than 250 bp) [17], it is difficult to obtain sufficient effective loci using a commercial STR typing kit, especially for a female foetus [9,10].…”

Section: Introductionmentioning

confidence: 99%

An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM

et al. 2017

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Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.

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Noninvasive genotyping of 9 Y‐chromosome specific STR loci using circulatory fetal DNA in maternal plasma by multiplex PCR

Abstract: This assay provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Cited by 31 publications

References 19 publications

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Algorithm-Based Fetal Gender Determination Using X and Y Mini-Short Tandem Repeats at Early Gestational Ages

An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM

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