Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Anjum, Ghulam Murtza; Du, Wei‐Dong; Klein, Rachel Herndon; Amara, Umme; Huber‐Lang, Markus; Schneider, E. Marion; Wiegand, Peter

doi:10.1002/elps.200900325

Cited by 6 publications

(5 citation statements)

References 38 publications

(38 reference statements)

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“…A histogram for homoplasmic 3460G > A detection of pyrosequencing assay was shown as a reference (D). entire mitochondrial genome was only possible using various individual pyrosequencing reactions on a number of PCR fragments [22]. In this study, we carried out 4 individual PCR amplifications and 4 pyrosequencing reactions (40µl of PCR solution) for each sample to achieve the 4 mutation analyses (11778G > A, 14484T > C, 3460G > A and 13708G > A).…”

Section: Comparison Of Three Snp Typing Methodologies In Detecting Lhmentioning

confidence: 99%

A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON)

Chen

Cao

et al. 2011

Disease Markers

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Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

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Section: Comparison Of Three Snp Typing Methodologies In Detecting Lhmentioning

confidence: 99%

A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON)

Chen

Cao

et al. 2011

Disease Markers

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“…MDA-based WGA has been frequently used in DNA sample preparation for genotyping and sequencing in recent years [18-21]. High call rates (97.5%) and excellent concordance rates were achieved from WGA samples using high-density SNP arrays [19,20].…”

Section: Introductionmentioning

confidence: 99%

Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development

et al. 2012

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BackgroundGenotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized.ResultsTo assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA.ConclusionThe relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.

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“…In these circumstances DNA typing of mitochondrial displacement loop (D-loop) region is usually the method of choice for investigators since most of the variations in mtDNA among individuals are found within the D-loop and can be useful for discriminating among unrelated individuals. 80 The HV I/HV II mtDNA linear array assay, decreases sequencing efforts substantially and thereby reducing the cost and time in forensic analysis. 81 …”

Section: Methods Of Mtdna Analysismentioning

confidence: 99%

DNA Profiling and forensic dentistry – A review of the recent concepts and trends

Manjunath

Chandrashekar

Mahesh³

et al. 2011

Journal of Forensic and Legal Medicine

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Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Cited by 6 publications

References 38 publications

A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON)

A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON)

Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development

DNA Profiling and forensic dentistry – A review of the recent concepts and trends

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