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

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Abstract

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.

Introduction

Human DNA quantification has became an essential analysis to ensure the quality of PCR-based studies performed from low copy number and/or highly damaged DNA samples that are analyzed in a routine way by different scientific disciplines including ancient DNA studies [1], [2], molecular ecology [3], or forensic genetics [4]. Random allele dropout [5] is a very common PCR artifact when microsatellite typing is performed from low copy number samples like non-invasive shed hairs and fecal material [3], bones [4], or other forensic samples [6]. This could lead to false homozygous typing results. In vitro nucleotide missincorporation could also have a great impact in the accuracy of mitochondrial DNA (mtDNA) sequence analysis if PCR-amplifications start from very few copies or even single DNA molecules [1]. This scenario could be further complicated if samples are also subjected to degradation, to base pair modifications [7], or to different contamination levels. Therefore, the estimation of the number of human DNA template molecules is very useful and can be accomplished by high sensitive hybridization methods with primate specific alphoid DNA probes [8] or by competitive PCR [1] or, more recently, by using real-time quantitative PCR [3], [9], [10], [11], [12].

In this study we explore different designs to estimate both mitochondrial mtDNA and nuclear DNA content based on the detection of the 5′ nuclease activity of the Taq DNA polymerase [13] 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 (HVI) of the mtDNA control region [14]. The system used for the quantification of nuclear DNA was the PCR-amplification of a segment of the X–Y homologous amelogenin (AMG) gene [15], [16] 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 DNA quantitation but also sex determination. Both designs have been used to measure the DNA content from low copy number and/or highly damaged DNA samples retrieved from skeletal remains, trying to evaluate the potential utility of this technology to improve the quality of some PCR-based DNA studies (microsatellite typing and mtDNA sequencing) with interest for human identification purposes.

Section snippets

DNA samples

The following DNA samples were used as DNA standards:

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    Human genomic DNA 9947A at a 0.1 ng/μl concentration (Applied Biosystems, Foster City, CA, USA).

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    Human genomic DNA from the male cell line ATCC CCL-256.1 at a 2.5 ng/μl concentration (Reliagene Technologies, New Orleans, LA, USA).

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    Human amplicons (620 bp) obtained by PCR-amplification around the HV1 region, spanning nucleotides 15,800–16,420 [14], from donors of known mtDNA sequence. Amplicons were purified by filtration with centricon-100 devices

Human mtDNA quantification

The first limitation during the development of the mtDNA real-time PCR quantitative design was the trouble to purify enough human mtDNA (free from nuclear DNA) that can be quantified accurately by conventional spectrophotometric or fluorometric methods and therefore can be used to construct the standard curve. Trying to overcome this drawback we decided to PCR-synthesize, purify and quantify a 620 bp PCR-amplicon using DNA from different donors with known mtDNA sequences as DNA templates. This

Discussion

We have described two specific 5′ nuclease assays for target both mtDNA and nuclear human DNA. We have chosen two genetic markers very well established in the forensic and ancient DNA fields: the HV1 mtDNA region [14] and the X–Y homologous AMG gene [15]. This election greatly simplified the initial efforts of this study because we could choose previously reported primer sequences that have been validated for human identification purposes by several end-point PCR studies. These include

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