Low Copy Number DNA Samples Research Articles

Challenges with co-amplification of microbial DNA in interpretation of STR profiles obtained from human skeletal remains

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.

Subduing the influence of PCR inhibitors on amplifying aged, degraded, and low copy number DNA: PCR enhancer cocktail-p and rescue PCR.

PCR inhibitors are a formidable problem to the study of aged, degraded, and/or low copy number DNA. As a result, there is a need to find alternate methods that ameliorate the efficacy of PCR. In this study, we attempted to use genetic methods to identify the species of salmonid (Oncorhynchus spp.) remains recovered from archaeological sites along the Feather River located in northern California, United States. In the process of doing so, we compared the efficacy of a PCR enhancer cocktail called “PEC-P” and a reagent rich PCR recipe called “rescue PCR” over standard PCR. Across all treatments (full concentration and 1:10 dilute eluates subjected to standard PCR, PEC-P, and rescue PCR) species identification was possible for 74 of 93 archaeological fish specimens (79.6%). Overall, six of the 93 samples (6.5%) consistently yielded species identification across all treatments. The species of ten specimens (10.8%) were uniquely identified from amplicons produced with either PEC-P or rescue PCR or both. Notably, the species of seven samples (7.5%) were uniquely identified with standard PCR over the alternative treatments. Considering both full concentration and 1:10 dilute eluates (N = 186), standard PCR performed as well as PEC-P (p = 0.1451) and rescue (p = 0.6753). Yet, considering results from full concentration eluates alone (N = 93), PEC-P (60.2%) outperformed both standard PCR (44.1%; p = 0.0277) and rescue PCR (40.9%; p = 0.0046). Stochasticity observed in our study cautions us against choosing a “best” performing method of those explored here and suggests their respective potentials to improve success may be sample dependent. When working with samples compromised by PCR inhibitors, it is useful to have alternative methodologies for subduing the problem. Both PEC-P and rescue PCR represent useful alternative methods for the study of aged, degraded, and/or low copy number DNA samples compromised by PCR inhibitors.

Estimation of the number of contributors of theoretical mixture profiles based on allele counting: Does increasing the number of loci increase success rate of estimates?

DNA mixtures are more frequently encountered in casework due to increased kit sensitivity, protocols with increased cycle number, and requests for low copy number DNA samples to be tested. Generally, the first step in mixture interpretation is determining the number of contributors, with the most common approach of maximum allele count. Although there are previous studies regarding the accuracy of this approach, none have evaluated the accuracy with the newly expanded U.S. core STR loci. In this work, 4,976,355 theoretical mixture combinations were generated with the PowerPlex® Fusion 6C system which includes 23 autosomal STR loci and three Y-STR loci. The number of contributors could be correctly assumed for 100% two-person and 99.99% three-person mixtures, whereas, four-, five-, and six-person mixtures were correctly assumed in 89.7%, 57.3%, and 7.8% of mixtures, respectively. Y-STR analysis showed the 3 Y-STR markers are only accurate for two-person male mixtures (96.7%). This work demonstrates that maximum allele count using the expanded U.S. core loci is not much improved from previous smaller panels, reiterating that this method is not as accurate beyond three contributors.

Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products.

AimTo show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods.MethodsThe DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands were captured on streptavidin-coated plates, where everything but the dye-labeled target sequence was washed, eliminating all the background noise, released, and run on a genotyping instrument in a single-strand configuration.ResultsThe IPCRp-based multiplex kit (6 loci) revealed equine DNA full genotype profiles, ie, appearance of all six loci, when sampled from fresh feces in 87% of the samples and partial genotype profile (appearance of one to five loci) in 13% of the samples, for a total of 100% genotyping success rate.ConclusionThese results indicate that the IPCRp amplification method, coupled with the Qiagen DNA Stool Mini Kit extraction can maximize the likelihood of obtaining horse DNA genotypes from fecal samples.

Reduction of stutter ratios in short tandem repeat loci typing of low copy number DNA samples

Increased height of stutter peaks is a phenomenon with low copy number (LCN) short tandem repeat (STR) typing that can impact interpretation. An alternative strategy of lowering the annealing/extension temperature (LT) at 56 °C was designed to attempt to decrease the heights of stutter peaks. STR typing results were generated in terms of stutter ratios using LT-PCR conditions and compared with data obtained using standard (STD) PCR conditions. DNA samples ranging from 100 to 25 pg were amplified using reagents contained in the AmpFℓSTR Identifiler PCR Amplification or AmpFℓSTR Identifiler Plus PCR Amplification kits with 32 or 34 PCR cycles. Stutter ratios decreased by an average of 14.7%, 14.9% and 18.1% at 100, 50 and 25 pg of template DNA under LT conditions compared with those of STD conditions in the Identifiler Kit amplified samples. The LT conditions also decreased average stutter ratios by 13.3% compared with those of STD conditions in the Identifiler Plus Kit amplified samples. The overall PCR efficiency obtained with STD and LT conditions with the two STR kits was comparable in terms of the number of detected alleles, peak heights and peak height ratios. These results support the hypothesis that a lower temperature annealing/extension step reduces the likelihood of slippage during PCR by enhancing the stability of the DNA polymerase/template DNA complex or the stability of the generated duplex than the conditions of the standard extension step. This stability in turn would result in lower stutter ratios.

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR® Identifiler® Plus for 28 cycles. Following amplification, samples were denatured at 99.9°C for 5min and incubated with one unit of DSN at 62°C in a 28μl volume for 1min. Nuclease activity was terminated through the addition of equal volume of 10mM EDTA and 95°C incubation for 2min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.

DcDegenerate Oligonucleotide Primed-PCR for Multilocus, Genome-wide Analysis From Limited Quantities of DNA

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples (<or= 0.100 ng). Experiments involved altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases. Increasing the degeneracy of the primer and the number of cycles that use a low annealing temperature should improve the nonspecific amplification of the DOP-PCR reaction. The addition of proofreading enzymes should allow for longer amplification products, increasing the genome coverage of the reaction. Low-input DNA quantities were examined for the primer and the cycle number studies using standard DOP-PCR parameters. The optimized DOP-PCR technique was then implemented for the polymerase study. All DOP-PCR products were amplified by using a multiplex microsatellite amplification kit to evaluate products from multiple chromosomes, followed by separation and detection by capillary electrophoresis. The 10 N primer, 12 nonspecific cycles, and the addition of the DeepVent proofreading enzyme all significantly increased the number of short tandem repeat alleles successfully amplified. All modifications also lowered the rate of allele drop-in, or sporadic additional allele occurrence, when compared with DOP-PCR results published earlier. Further, an average of > 0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results show that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples.

Introduction of the DNase in forensic analysis

Laboratory DNA contamination is a frequent problem in forensic genetics, especially when extra PCR cycles are used to obtain acceptable amplified products from low copy number DNA samples. We have evaluated the use of the DNase I for forensic genetics studies. Initially we introduced known genomic DNA in the PCR reaction mix in order to simulate DNA contamination. Different conditions of action an inactivation of the DNase I were tested. Finally, we performed amplifications of low copy number samples employing homemade and commercially available STR amplification systems; in both cases decontamination was successful. We demonstrate that DNase I may be useful to reduce laboratory contamination in forensic genetics labs. Our protocol may be easily introduced in standard PCR protocols of commercial kits in order to be used routinely in low copy number DNA amplification.

Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA

Evidentiary items sometimes contain an insufficient quantity of DNA for routine forensic genetic analysis. These so-called low copy number DNA samples (<100 pg of genomic DNA) often fall below the sensitivity limitations of routine DNA analysis methods. Theoretically, one way of making such intractable samples amenable to analysis would be to increase the number of starting genomes available for subsequent STR (short tandem repeat) analysis by a whole genome amplification strategy (WGA). Although numerous studies employing WGA have focused primarily on clinical applications, few in-depth studies have been conducted to evaluate the potential usefulness of these methods in forensic casework. After an initial evaluation of existing methods, a modified WGA strategy was developed that appears to have utility for low copy number forensic casework specimens. The method employs a slight, but important, modification of the “improved primer extension preamplification PCR” method (I-PEP-PCR), which we term mIPEP (modified-I-PEP-PCR). Complete autosomal STR and Y-STR (Y chromosome short tandem repeat) profiles were routinely obtained with 5 pg of template DNA, which is equivalent to 1–2 diploid cells. Remarkably, partial Y- and autosomal STR profiles were obtained from mIPEP-treated DNA recovered from bloodstains exposed to the outside environment for 1 year whereas non-mIPEP-treated samples did not produce profiles. STR profiles were obtained from contact DNA from single dermal ridge fingerprints when the DNA was subjected to prior mIPEP amplification but not when the mIPEP step was omitted.

Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci

In this validation study, we have evaluated the efficacy and the validity of the SGM Plus test using an amplification regime of 34 cycles. We obtained valid DNA typing results from pristine extracts with an extremely low DNA content. In this context, the aspects of single cell PCR typing were also evaluated. In these experiments, the allele dropout phenomenon was clearly demonstrated. From actual casework samples, we obtained conclusive DNA profiles from highly purified extracts of bone and teeth that failed to demonstrate typing results using the standard PCR protocol of 28 cycles. Moreover, low copy number (LCN) DNA typing offered us the possibility to reanalyse crime samples that failed to produce a conclusive profile after 28 cycles. Unfortunately, several complications accompany ultrasensitive PCR amplification. During our validation studies, we have observed increased risk of contamination, allelic dropout, locus dropout and heightened stutters. Analyses of heterozygote balance, between-loci balance and stutter heights, show that the 34-cycle PCR has its own characteristic features. We finally show that reamplification of SGM Plus PCR products by an extra 6 PCR cycles offers a promising new alternative if too little of the original sample extract is left for a complete reanalysis.