A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis.

DNA STR profiles have been generated from 1 ng and low copy number (LCN) templates using 28 and 34 cycles of amplification, respectively. Characteristics which facilitate the interpretation of profiles, such as heterozygous balance, allelic dropout and stutter proportions have been quantified. We demonstrate that a reduction in DNA template coupled with an increase in amplification cycle number produces an increased rate of allelic dropout out which can be correlated to the peak areas of those alleles observed. In addition, the LCN conditions increase the degree of peak area asymmetry observed from heterozygotes and the size range of stutters. Analysis of the data allows us to develop sets of guidelines appropriate for interpreting both single and mixed DNA profiles.     

Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle “low copy number” (LCN) method for analysis of trace forensic DNA samples

The investigation of samples with low amounts of template DNA remains at the forefront of forensic DNA research and technology as it becomes increasingly important to gain DNA profile information from exceedingly trace levels of DNA. Previous studies have demonstrated that it is possible to obtain short tandem repeat (STR) profiles from <100 pg of template DNA by increasing the number of amplification cycles from 28 to 34, a modification often referred to as “low copy number” or LCN analysis. In this study, we have optimised post-PCR purification techniques applied after only 28 cycles of PCR, as well as using modified capillary electrophoresis injection conditions and have investigated the progressive application of these enhanced approaches. This paper reviews the characteristics of the profiles obtained by these methods compared with those obtained on the same samples after 34-cycle PCR. We observed comparable sensitivity to 34-cycle PCR in terms of the number of profiles with evidence of DNA and the number of allelic peaks per profile and we noted improved peak height and area magnitude with some sample types. Certain parameters reported to be adversely affected in 34-cycle LCN investigations, such as non-donor allele peaks and increased stutter peak ratio, were reduced by this approach. There are a number of advantages for trace samples in progressing from the standard 28-cycle process to the post-PCR processing method as compared to 34-cycle PCR method, including reduced sample consumption, reduced number of PCR amplifications required, and a staged approach to sample processing and profile interpretation.     

An Investigation of PCR Inhibition Using Plexor®‐Based Quantitative PCR and Short Tandem Repeat Amplification

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor^® real‐time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and C_T takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex^® 16 HS System. The overall results demonstrate that real‐time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.     

A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications

In forensic DNA profiling use is made of the well-known technique of PCR. When the amount of DNA is high, generally unambiguous profiles can be obtained, but for low copy number DNA stochastic effects can play a major role. In order to shed light on these stochastic effects, we present a simple model for the amplification process. According to the model, three possible things can happen to an individual single DNA strand in each complete cycle: successful amplification, no amplification, or amplification with the introduction of stutter. The model is developed in mathematical terms using a recursive approach: given the numbers of chains at a given cycle, the numbers in the next can be described using a multinomial probability distribution. A full set of recursive relations is derived for the expectations and (co)variances of the number of amplicon chains with no, 1 or 2 stutters. The exact mathematical solutions of this set are given, revealing the development of the expectations and (co)variances as function of the cycle number. The equations reveal that the expected number of amplicon chains without stutter grows exponentially with the cycle number, but for the chains with stutter the relation is more complex. The relative standard deviation on the numbers of chains (coefficient of variation) is inversely proportional to the square root of the expected number of DNA strands entering the amplification. As such, for high copy number DNA the stochastic effects can be ignored, but they play an important role at low concentrations. For the allelic peak, the coefficient of variation rapidly stabilizes after a few cycles, but for the chains with stutter the decrease is more slowly. Further, the ratio of the expected intensity of the stutter peak over that of the allelic peak increases linearly with the number of cycles. Stochastic models, like the one developed in the current paper, can be important in further developing interpretation rules in a Bayesian context.     

二组分混合DNA样品STR图谱解释

对混合样品STR图谱的结果进行解释。实验模拟二组分DNA混合样品 ,复合扩增荧光检测 10个基因座 ,比较混合样品谱带 ,计算等位基因峰面积比。结果发现 :二组分DNA混合样品的等位基因数增加 ,样品的混合比例不同就出现峰面积的不平衡。在等位基因峰面积比值与样品组分混合比例接近时 ,可由峰面积比值推断混合样品的混合比例。在混合比例为 1∶2 0时 ,基本上检测不到来自少量混合成分的等位基因 ,表现为单一组分图谱 ;在混合比例为 1∶10时 ,含量低的组分的等位基因峰面积接近与主要组分的“Stutter”峰面积 ,与来自主要组分的等位基因峰面积差异很明显。能检出混合样品中少量成分等位基因的最高混合比例为 1∶10     

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

PCR扩增循环数与低拷贝模板DNA的STR分型

目的探讨PCR扩增循环数对低拷贝模板DNA的STR分型的影响。方法模板DNA(9947A)的不同扩增用量(含低拷贝模板量),采用ProfilerPlus试剂盒,扩增循环数分别为28、30、32、34、38次,3100型基因分析仪(ABI,美国)检测结果。结果循环数从28次增至38次,模板DNA量最低检出量可从0.25ng减少至0.0312ng;先循环28次后,每反应加0.3μlAmpliTaqGoldDNA聚合酶,再循环6次较1次34个循环的检测灵敏度高,相当于1次38个循环的效果。结论增加PCR扩增循环数,可能影响低拷贝模板DNA的STR分型。     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Higher Capillary Electrophoresis Injection Settings as an Efficient Approach to Increase the Sensitivity of STR Typing

Abstract:  Evidentiary traces may contain low quantities of DNA, and regularly incomplete short tandem repeat (STR) profiles are obtained. In this study, higher capillary electrophoresis injection settings were used to efficiently improve incomplete STR profiles generated from low-level DNA samples under standard polymerase chain reaction (PCR) conditions. The method involves capillary electrophoresis with higher injection voltage and extended injection time. STR peak heights increased six-fold. Inherent to the analysis of low-level DNA samples, we observed stochastic amplification artifacts, mainly in the form of allele dropout and heterozygous peak imbalance. Increased stutter ratios and allele drop-in were rarely seen. Upon STR typing of 10:1 admixed samples, the profile of the major component did not become overloaded when using higher injection settings as was observed upon elevated cycling. Thereby an improved profile of the minor component was obtained. For low-level DNA casework samples, we adhere to independent replication of the PCR amplification and boosted capillary electrophoresis.     

A systematic analysis of PCR contamination.

In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.     

Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

A duplex primer set for the amplification of mitochondrial DNA HVI and HVII control regions was evaluated for the optimization of a DNA sequencing protocol suitable for forensic casework. HVI and HVII products, with the absence of non-specific products, could be detected by agarose gel electrophoresis when as little as 0.5 and 0.1pg of DNA were amplified for 34 and 38 cycles, respectively. Because HVI and HVII amplicons are co-synthesized in the duplex PCR, fewer steps are required (lessening the risk of cross contamination events) and more frugal use of precious extracted DNA samples is possible, both desirable features for forensic casework. The ABI Prism BigDyetrade mark version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.     

全基因组扩增在微量检材DNA分型中的应用

目的探索全基因组扩增技术对微量检材DNA分型的有效性。方法通过显微操作制备含1~20个细胞的模拟微量检材样本,在常规PCR-STR分型前加入全基因组扩增步骤,从等位基因不平衡、等位基因丢失、基因座丢失、伪等位基因(包含stutter峰)等方面探究PEP和MDA两种全基因组扩增方法对微量检材DNA分型的有效性。结果 MDA扩增效率高于PEP,但等位基因丢失和伪等位基因严重;PEP方法的正确分型率高于MDA,但小片段DNA优势扩增现象较严重。结论 MDA方法并不适合目前以STR分型为主导的法庭科学,当微量检材样本的绝对量相当少时,可以考虑使用PEP方法来扩大样本量,以满足重复检验的要求,但可能面临大片段DNA扩增失败的风险。     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

The successful DNA typing of samples following a thermal cycler power loss

An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.     

Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

Analysis of ancient human DNA and primer contamination: one step backward one step forward

The analysis of DNA from archaeological human remains is plagued by a unique set of methodological problems concerning contamination with modern exogenous DNA. Through an original approach, we propose complementary methods to identify all potential sources of contamination and complete guidelines for the validation of ancient human sequences. The study presented was conducted on non-European human samples (Polynesian and Amerindian) which were collected with all precautions during excavation. This permitted us to distinguish without ambiguity authentic and contaminant sequences. The samples' origins and histories were perfectly known, allowing us to trace all potential contamination sources and to determine the efficiency of precautions followed during all steps of the study. The data obtained confirm that precautions taken during sampling effectively prevent contamination. However, we demonstrate that human contamination can also be introduced during genetic analyses even if all precautions are strictly followed. Indeed, numerous human contaminations were detected in template-PCR products and negative controls, resulting in a striking diversity of contaminant mitochondrial DNA sequences. We argue that this contamination partly derives from the primers. To our knowledge, no previous experiment has been performed to investigate primers as a possible source of human contamination despite the fact that this specific type of contamination poses a real problem in terms of validating ancient human DNA studies. Finally, we confirm that the detection of contaminants in negative controls is clearly related to the number of PCR cycles used. This study enhances our understanding of contamination processes and confirms that, in reality, an absolutely contamination-free situation cannot be obtained. As a consequence, we propose improvements to the guidelines usually followed in the field in order to take the highly probable contamination of PCR reagents, including primers, into account.     

Genetic analysis of fingerprints—Could WGA or nested-PCR be alternatives to the increase of PCR cycles number?

In this work we aimed to compare the application of increasing PCR cycle number, whole genome amplification and nested-PCR on fingerprints genetic analysis. Results were compared for correct alleles, allele dropin and allelic dropout. We concluded that increasing the number of PCR cycles is yet the best way to attain the required sensitivity.     

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.