+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies



Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies



Forensic Science International 139(2-3): 141-149



We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 012485822

Download citation: RISBibTeXText

PMID: 15040907

DOI: 10.1016/j.forsciint.2003.10.008


Related references

Specific quantification of human genomes from low copy number DNA samples in forensic and ancient DNA studies. Croatian Medical Journal 44(3): 273-280, 2003

Copy number of bean mitochondrial genes estimated by real-time PCR does not correlate with the number of gene loci and transcript levels. Plant Molecular Biology 61(1-2): 1-12, 2006

Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results. Bmc Research Notes 10(1): 593, 2017

Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis. Biotechniques 33(2): 402, 2002

Differential nuclear and mitochondrial DNA preservation in post-mortem teeth with implications for forensic and ancient DNA studies. Plos one 10(5): E0126935, 2015

Preventing lactic acidosis in HIV HCV coinfected patients during interferon and ribavirin treatment Development of a real time PCR for mitochondrial DNA copy number in PBMCs. Hepatology 36(4 Part 2): 230A, 2002

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR. Nucleic Acids Research 29(3): E13, 2001

The Alterations in Mitochondrial DNA Copy Number and Nuclear-Encoded Mitochondrial Genes in Rat Brain Structures after Cocaine Self-Administration. Molecular Neurobiology 54(9): 7460-7470, 2017

fastMitoCalc: an ultra-fast program to estimate mitochondrial DNA copy number from whole-genome sequences. Bioinformatics, 2017

FastMitoCalc: an ultra-fast program to estimate mitochondrial DNA copy number from whole-genome sequences. Bioinformatics 33(9): 1399-1401, 2017

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples. Journal of Forensic Sciences 50(5): 1044-1060, 2005

Nuclear and mitochondrial DNAs microsatellite instability and mitochondrial DNA copy number in adenocarcinoma and squamous cell carcinoma of lung: a pilot study. Apmis 123(12): 1048-1054, 2015

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities. Scientific Reports 7: 14024, 2017

Two nuclear genes mrs3 and mrs4 which can suppress a mitochondrial splice defect when present on a high copy number plasmid belong to the mitochondrial carrier family. Yeast 6(SPEC ISSUE): S530, 1990

Reproducible quantitative PCR of mitochondrial and nuclear DNA copy number using the LightCycler. Methods in Molecular Biology 197: 129-137, 2002