Role of short tandem repeat DNA in forensic casework in the UK--past, present, and future perspectives

The analysis of short tandem repeat (STR) DNA sequences is of fundamental importance to forensic science because they have become the recognized standard in constructing national public databases. Consequently, considerable effort has been expended in developing multiplexed (one tube) reactions that analyze several loci in combination. The implementation of STRs in casework cannot take place without a full understanding of the systems used. The purpose of validation is to characterize multiplexes when one is challenged with forensic samples. For example, mixtures are often encountered that may be particularly difficult to interpret against a background of allelic artifacts. By increasing the number of PCR amplification cycles, it is possible to dramatically boost the sensitivity of the system so that just a handful of cells may be successfully analyzed. However, interpretation is much more complex because the origin of DNA profiles may be less certain and complicated by issues such as contamination, the potential for innocent transfer and a predominance of mixtures. This review provides a brief historical background of the development of STRs in forensic casework that culminated in the creation of national DNA databases. The development of guidelines to interpret complex DNA profiles, such as mixtures, is outlined. Finally, the recent innovation of low copy number DNA profiling is explained along with the special considerations needed to report in court.     

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.     

A graphical simulation model of the entire DNA process associated with the analysis of short tandem repeat loci

The use of expert systems to interpret short tandem repeat DNA profiles in forensic, medical and ancient DNA applications is becoming increasingly prevalent as high-throughput analytical systems generate large amounts of data that are time-consuming to process. With special reference to low copy number (LCN) applications, we use a graphical model to simulate stochastic variation associated with the entire DNA process starting with extraction of sample, followed by the processing associated with the preparation of a PCR reaction mixture and PCR itself. Each part of the process is modelled with input efficiency parameters. Then, the key output parameters that define the characteristics of a DNA profile are derived, namely heterozygote balance (Hb) and the probability of allelic drop-out p(D). The model can be used to estimate the unknown efficiency parameters, such as π_extraction. ‘What-if’ scenarios can be used to improve and optimize the entire process, e.g. by increasing the aliquot forwarded to PCR, the improvement expected to a given DNA profile can be reliably predicted. We demonstrate that Hb and drop-out are mainly a function of stochastic effect of pre-PCR molecular selection. Whole genome amplification is unlikely to give any benefit over conventional PCR for LCN.     

DNA profiling of spermatozoa by laser capture microdissection and low volume-PCR

Genetic profiling of sperm from complex biological mixtures such as sexual assault casework samples requires isolation of a pure sperm population and the ability to analyze low abundant samples. Current standard procedure for sperm isolation includes preferential lysis of epithelial contaminants followed by collection of intact sperm by centrifugation. While effective for samples where sperm are abundant, this method is less effective when samples contain few spermatozoa. Laser capture microdissection (LCM) is a proven method for the isolation of cells biological mixtures, even when found in low abundance. Here, we demonstrate the efficacy of LCM coupled with on-chip low volume PCR (LV-PCR) for the isolation and genotyping of low abundance sperm samples. Our results indicate that this method can obtain complete profiles (13-16 loci) from as few as 15 sperm cells with 80% reproducibility, whereas at least 40 sperm cells are required to profile 13-16 loci by standard 'in-tube' PCR. Further, LCM and LV-PCR of a sexual assault casework sample generated a DNA genotype that was consistent with that of the suspect. This method was unable, however, to analyze a casework sample from a gang rape case in which two or more sperm contributors were in a mixed population. The results indicate that LCM and LV-PCR is sensitive and effective for genotyping sperm from sperm/epithelial cell mixtures when epithelial lysis may be insufficient due to low abundance of sperm; LCM and LV-PCR, however, failed in a casework sample when spermatozoa from multiple donors was present, indicating that further study is necessitated.     

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The F?rster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.     

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

Development,characterization, and validation of a sensitive primate-specific quantification assay for forensic analysis

Accurate human-specific DNA quantification is essential for forensic casework analysis. In this work, we describe a microplate-based quantification assay that utilizes the PCR amplification of human-specific TH01 primers. This method enables the reliable quantification of human DNA samples from 0.2 to 40 ng, even in mixtures with nonhuman DNA. Analysis of samples can be semi-automated using 96-well microplates and a spreadsheet-based concentration calculator for high-throughput demands. We have used this quantification method with more than 15,000 forensic samples.     

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.     

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.     

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

DNA typing of primate major histocompatibility complex (Mhc)-DQA1 locus by PCR and dot blot hybridization.

Non-human primates (NHPs) are increasingly utilized as models to investigate different aspects of immune responses against self (autoimmunity) and foreign antigens. These animals provide valuable models for testing the efficacy of candidate vaccines against pathogens such as human immunodeficiency virus (HIV) and also fertility regulating agents (immunocontraceptives). In order to fully understand the effects of vaccination, it may be necessary to elucidate the immunogenetic background of these animals. The major histocompatibility complex (Mhc) molecules play an important role in the generation of effective immune responses. Serological techniques have been used in the identification of human leukocyte antigens (HLA) necessary for cross-matching organs and tissues for transplantation. However, the application of this technique for typing monkey Mhc alleles has been hampered by unavailability of well characterized immunological reagents. Polymerase chain reaction (PCR)-based techniques such as restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOP) have been extensively used for typing HLA-DP, DQ and DR alleles. A commercially available Kit (AmpliTypeR) designed for amplification and typing of HLA DQalpha alleles is routinely used in typing DNA samples for forensic casework. In the present study, we have evaluated this kit for possible application in routine typing of primate DQA1 alleles. Genomic DNA from ten African primate species (23 individuals) was isolated from peripheral blood lymphocytes and polymorphic second exon of DQA1 locus amplified using GH26 and GH27 PCR primers. The PCR products were hybridized on a nylon membrane containing immobilized sequence-specific oligonucleotide probes. Our results show seven of the nine probes hybridizing with primate DQA1 alleles, indicating that typing of equivalent primate alleles can be accomplished at lower stringency conditions. However, it may be necessary to design additional oligonucleotides probes (based on available primate DQA1 sequences) to improve the discriminating power of this kit for use in routine typing of Old World monkey DQA1 alleles.     

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.     

Use of propidium monoazide for live/dead distinction in microbial ecology

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.     

CYP2C19基因多态性PCR扩增体系的正交优化与验证

本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。     

Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.     

Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay.

Single nucleotide substitutions and unique insertions/deletions are the most common form of DNA sequence variation and disease-causing mutation in the human genome. Because of the biological and medical importance of these variations, a wide array of methods have been developed for their typing. We have applied an approach that combines the amplification of polymorphic regions by the polymerase chain reaction (PCR) with a system for typing diallelic variants using an oligonucleotide ligation assay (OLA). In this report, we describe a significant advance in this technology that permits the typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors. We demonstrate the specificity, sensitivity and ease of data interpretation with this system. Furthermore, we show that multiplex PCR/OLA not only increases the throughput of DNA typing but also increases its accuracy in typing diallelic sequence variations using an approach that can be broadly applied for human genome analysis (in evaluating genotype/phenotype links), in typing infectious agents and in forensic analysis.     

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.