2Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
Analysis of mitochondrial DNA in forensic samples is routinely carried out by direct sequencing of hypervariable regions within the non-coding displacement loop. Although the accuracy and sensitivity of this method cannot be questioned, it is both time-consuming and labor intensive. Finding a way to rapidly pre-screen forensic samples—prior to sequencing, to reduce the number of samples that need to be sequenced—would greatly benefit forensic laboratories. Herein, we describe an assay for discrimination of DNA from different individuals based on high-resolution melting analysis of the two hypervariable regions HVI and HVII of the mitochondrial genome. By clearly distinguishing the DNA melting curves of six different individuals, we show that this assay has the potential to function as a rapid and inexpensive pre-screening method for forensic samples prior to DNA sequencing.
The identification of forensic material is routinely carried out by analysis of short tandem repeats (STRs) within nuclear DNA (1). However, in cases where the DNA is degraded or only available in scarce amounts, analysis of mitochondrial DNA (mtDNA) is often useful due to its high copy number per cell (2). Most of the mtDNA sequence variation is condensed within the hypervariable regions I and II (HVI and HVII) (3) of the non-coding displacement loop (D-loop). Forensic mtDNA identification is carried out by Sanger sequencing of HVI and HVII allowing detection of the single nucleotide polymorphism (SNP) variation occurring within these regions (4). Although useful and reliable, this method is laborious, time-consuming, and expensive. A simple method to rapidly pre-screen crime scene DNA (to exclude suspects and reduce the number of evidence samples that need to be sequenced) would facilitate the process of forensic mtDNA analysis. Although methods for rapidly genotyping mitochondrial polymorphisms have been reported, these techniques are either destructive and open-tube (5), or require the use of costly allele-specific probes (6) or expensive mass spectrometers (7).
High-resolution melting (HRM) analysis is a novel, closed-tube method for rapid analysis of genetic variation within PCR amplicons (8). A wide range of HRM applications have been reported, such as SNP genotyping, mutation discovery, and DNA methylation analysis (9,10,11). The HRM technique was initially thought to require the use of a new generation of saturating DNA dyes (12); however, it was recently shown to work equally well with SYBR Green I, a non-saturating dye (13,14,15). In HRM-designated instruments, the decrease in fluorescence caused by the transition of dsDNA to ssDNA with increasing temperature is carefully monitored and, with the aid of tailor made analysis software, different genetic variants can be discriminated by their characteristic melting curves.
SuperConvection is a novel technology to minimize thermal heterogeneity within samples by inducing enhanced mass-transport in the reaction mixture (U.S. Patent no. 6783993) (16). We have implemented this technology into a new real-time PCR instrument, QuanTyper-48 (AlphaHelix Molecular Diagnostics AB, Uppsala, Sweden), along with a sophisticated in-tube temperature measurement system, which enables an exact control of sample temperature to further increase the sensitivity and accuracy of HRM.
Herein, we present superconvective HRM as a method for screening mtDNA variation in forensic samples prior to sequencing. Our objective was to develop an assay that allows for the exclusion of non-matching forensic material, thereby reducing time and cost of mtDNA sequencing, by discriminating between different individuals based on their characteristic high-resolution melting curves. This is a proof-of-concept study and more work will be done to further test the limits of the procedure.Materials and methods DNA samples
Genomic DNA. Genomic DNA from six anonymous Swedish blood donors—denoted B14, B15, B18, B20, C2, and C5—was used in the study. Blood samples were collected after informed consent. The DNA was isolated from whole blood using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). DNA concentration was measured using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and the samples were diluted to a final concentration of 10 ng/µL. The DNA samples were sequenced with respect to HVI and HVII using the Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3100 genetic analyzer (Applied Biosystems).
A/T SNP template DNA. Two oligonucleotide pairs, corresponding to the Y-chromosomal region NCBI36:Y:20327149–20327198, were synthesized to represent an A/T and a T/A genotype and were used as templates in the A/T SNP PCR.Primer design
A/T SNP PCR. Primers were designed to flank the Y-chromosomal SNP position NCBI36:Y:20327175 and amplify a 50-bp amplicon from the A/T SNP template DNA. The primer sequences are depicted in Table 1.
HVI/HVII PCR. HVI and HVII primer design is detailed elsewhere (17) and the primer sequences are summarized in Table 1. Briefly, primers were designed to flank highly polymorphic regions of HVI and HVII. The HVI amplicon is 243 base pairs in length and spans from position 16105 to 16348 of the mtDNA genome (18), while the HVII amplicon is 242 base pairs long and spans from position 45 to 287. HVII and HVI amplicon SNP sequences for each of the six individuals are summarized in Tables 2 and 3.