TY - JOUR
T1 - Quantification of heteroplasmic mitochondrial DNA mutations for DNA samples in the low picogram range by nested real-time ARMS-qPCR
AU - Biffi, Stefania
AU - Bortot, Barbara
AU - Carrozzi, Marco
AU - Severini, Giovanni Maria
PY - 2011/6
Y1 - 2011/6
N2 - In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples.
AB - In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples.
KW - ARMS-PCR
KW - heteroplasmy
KW - mitochondrial DNA
KW - nested real-time PCR
KW - single-fiber PCR
KW - SYBR Green
UR - http://www.scopus.com/inward/record.url?scp=79958041835&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79958041835&partnerID=8YFLogxK
U2 - 10.1097/PDM.0b013e3181efe2c6
DO - 10.1097/PDM.0b013e3181efe2c6
M3 - Article
C2 - 21532488
AN - SCOPUS:79958041835
VL - 20
SP - 117
EP - 122
JO - Diagnostic Molecular Pathology
JF - Diagnostic Molecular Pathology
SN - 1052-9551
IS - 2
ER -