Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies.
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.
Alonso
Antonio
A
Instituto Nacional de Toxicología, Servicio de Biología, Luis Cabrera 9, 28002 Madrid, Spain. a.alonso@mju.es
Martín
Pablo
P
Albarrán
Cristina
C
García
Pilar
P
García
Oscar
O
de Simón
Lourdes Fernández
LF
García-Hirschfeld
Julia
J
Sancho
Manuel
M
de La Rúa
Concepción
C
Fernández-Piqueras
Jose
J
D053523:Amelogenin; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D004247:DNA / Q000032:analysis*; D016172:DNA Fingerprinting / Q000379:methods*; D003746:Dental Enamel Proteins / Q000235:genetics; D005260:Female; D018732:Forensic Anthropology / Q000379:methods; D018628:Gene Dosage; D015186:Hominidae / Q000235:genetics; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012732:Sex Determination Analysis; D020080:Tandem Repeat Sequences; D014083:Tooth Germ
C507199:AMELX protein, human; D053523:Amelogenin; D003746:Dental Enamel Proteins; D004247:DNA