Standard PCR Conditions Research Articles

PCR optimization for the detection of bunchy top virus of abaca (Musa textilis Nee) in Eastern Visayas Philippines

The bunchy top virus in Eastern Visayas has serverly reduced abaca production. Early and accurate detection of plant viral pathogens is an essential and crucial component for disease management. At present, there are no standard PCR conditions in the Eastern Visayas region for detecting the bunchy top virus at an early stage using PCR. Thus, optimization for the detection was carried out to assist in disease management. Different annealing temperatures (57, 60 and 65oC), gel concentrations (1, 1.5 and 2%), and running conditions (80, 90 and 100 volts) were tested using My TaqTM DNA Polymerase (Bioline, USA). The annealing temperatures of 57oC and 60oC resulted in DNA amplification as indicated by the presence of bands but absence of bands at 65oC. The higher voltages of 90 and 100 volts resulted in smears and distorted DNA bands with 1% and 1.5% agarose; thus 2% agarose gel was used to resolve small DNA fragments (100bp to 3kb). Electrophoresis using 80 volts for 45min successfully separated the DNA bands. The amplification of the product with internal control primers indicated the absence of PCR inhibitors in the abaca-extracted DNA samples. This confirmed the negative PCR reaction as indicative of the absence of the virus. The optimized PCR conditions could be applied by students and researchers for the early detection of bunchy top virus in the National Abaca Research Center Germplasm collection and the region.

A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37.

Spinocerebellar ataxia 37 (SCA37) is caused by an (ATTTC)n insertion in a polymorphic ATTTT repeat in the non-coding region of DAB1. The non-pathogenic alleles have a configuration [(ATTTT)7-400], whereas pathogenic alleles have a complex structure of [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90]. Molecular diagnosis of SCA37 is laborious because about 7% of the pentanucleotide repeat alleles in DAB1 are larger than 30 units and, thus, fail to amplify with standard PCR conditions, resulting in apparently homoallelism or in complete lack of PCR amplification in several cases. The molecular test currently available requires long-range PCR and sequencing analysis for the detection and characterization of these large alleles. We developed a simple assay capable of rapidly detecting the presence or absence of large pentanucleotide repeat sizes. This assay is based on repeat-primed PCR followed by high-throughput capillary electrophoresis. Combining the standard PCR with RP-PCR allows completion of the diagnosis in more than 80% of individuals, minimizing the number of samples that require long-range PCR followed by Sanger sequencing analysis. This assay meets many of the requirements for pre-screening of large cohorts of affected individuals.

Optimisation of DNA recovery and analysis of urine samples stored on FTA® card

Denial of ownership of urine samples submitted by athletes for doping control has led to an increasing need to incorporate DNA analysis into testing protocols. As there are often large volumes of samples to process, a streamlined sample collection and analysis protocol suitable for urine samples that is quick, reliable, and economical would be highly advantageous. FTA® cards have been employed extensively for preparing and preserving genetic material for long periods of time at room temperature. This study reports the possibility of using FTA® cards to store urinary DNA, which can be used directly as templates in STR analysis for human identification. Urine samples from six male and female volunteers were collected. The sample preparation steps for harvesting cells from urine, such as minimum urine volume required and centrifugal speed, were optimised, and suitable sizes of the FTA® card punches were determined. The results showed that regardless of the sample donor’s sex, a complete DNA profile could be reproducibly generated from a single 2.0-mm punch of the 3-mL urine samples that had been deposited on FTA® cards using the standard PCR conditions for AmpFLSTR® Identifiler® Plus. The developed protocol could become an integral part of a guideline for authentication of urine samples submitted for doping control or forensic toxicological analysis, where cost per sample and storage space are factors of consideration.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia

Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found.We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time.The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM DisclosuresNo relevant conflicts of interest to declare.

Minifish mtDNA has abundance of repeat sequences and inefficient replication in vitro.

Paedocypris is a newly described minifish genus endemic to Southeast Asia. Besides a tiny adult size of ~8 mm in length, minifish feature fragmentary habitats of acidic peat blackwater swamps, an unusual reproduction mode, truncated development and one of the smallest known genomes. A complete sequence is absent for the minifish mitochondrial DNA (mtDNA). Here we report the complete mtDNA sequence and its unusual feature in the minifish P. progenetica (Pp). We show that the Pp mtDNA is a circular molecule of 17,382 bp in length and has the same number of similarly oriented genes as in other vertebrates. Specifically, it comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 D-loop. Surprisingly, the D-loop is elusive for amplification by standard PCR conditions. The D-loop possesses a 28-bp dinucleotide TA repeat and more intriguingly, up to 25 copies of a 34-bp tandem repeat sequence. These tandem repeats predict the formation of paired regions. Hence, besides a generally conserved mtDNA with other vertebrates, the Pp mtDNA features an unusual D-loop and compromised DNA replication in vitro.

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms.

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

Primer Based Approach for PCR Amplification of High GC Content Gene:MycobacteriumGene as a Model

The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.

Development and characterization of 96 microsatellite markers suitable for QTL mapping and accession control in an Arabidopsis core collection

BackgroundTo identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping.ResultsIn this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions.ConclusionThe set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions.

Standardization and reproducibility of random marker based analysis of micropropagated crimson beebalm

Genetic analysis of the stability in micropropagated plant material are required to be perform by effective and reproducible techniques. When no specific DNA based markers are available for certain species, such as for crimson beebalm, standardized and optimized protocols can be used even for random based markers. Here we report for the first time the optimization of PCR conditions for RAPD analysis for the purposes of the establishment of fast, reliable and reproducible method for screening of genetic stability in Monarda dydima, L. micropropagated cultures. RAPD protocol was optimized based on the use of 50 or 100 ng of template DNA and annealing temperature of 36?C, resulted optimal amplification. Setting the standardized PCR conditions, primers used in experiments generated reproducible and distinguishable fingerprinting patterns for micropropagated cultures of crimson beebalm and is suitable for further analysis of studying genetic variation of this medicinal plant.

First Evidence of a Binucleate Rhizoctonia as the Causal Agent of Dry Rot Canker of Sugar Beet in Nebraska.

Sugar beet (Beta vulgaris L.) is the primary source of domestic sucrose in the United States. In 2011, a sugar beet field in Morrill County, NE, was noted with wilting and yellowing symptoms suggestive of Rhizoctonia root and crown rot (RRCR), an important disease of sugar beet primarily caused by Rhizoctonia solani anastomosis group 2-2 (4). While the foliar symptoms were consistent with RRCR, the symptoms on the root were not. Root symptoms consisted of localized, dry, sunken lesions covering brown spongy tissue penetrating deeply into taproots. The surface tissues of the cankers distinctively produced a series of concentric circles. These root symptoms are inconsistent with RRCR, but are suggestive of a rarely occurring disease known as dry rot canker (DRC). DRC was first identified from Utah in 1921 (1), and assumed at the time to be caused by an uncharacterized strain of R. solani. It has since been sporadically but empirically noted from most western sugar beet growing states (4), but little is known about the pathogen or disease due to its infrequent appearances. To investigate the etiology of this disease, necrotic lesion borders were excised from diseased taproots, surface disinfested in 1% (v/v) sodium hypochlorite for 90 s, rinsed with distilled water for 90 s, and after drying on sterile tissue paper, placed on half-strength potato dextrose agar (½PDA) and incubated at 25 to 27°C. After 24 to 36 h, Rhizoctonia-like fungal growth was readily observed emerging from tissue pieces. Resulting colonies were tan to light brown. The ITS region of the rDNA was amplified from 4 isolates obtained from 4 distinct lesions and roots using the ITS1 and ITS4 primers (3) with standard PCR conditions, and sequenced (GenBank KC842197 to KC842200). The ITS regions were 100% identical between the 4 isolates and 96% (E-value = 0.0) identical to binucleate Rhizoctonia and Ceratobasidium sp. AG-F (e.g., JF519832, FR734295, JF705217). Hyphal cells were observed to be binucleate after staining 48-h-old cultures with lactophenol blue. Therefore, these isolates were identified to be a binucleate Rhizoctonia group AG-F based on morphological and molecular characteristics. Although distinct from DRC, a similar phenomenon has been recently reported from China implicating binucleate Rhizoctonia species with seedling disease in sugar beets (2). To determine pathogenicity of DRC isolates, 1- and 2-month-old sugar beet plants grown in 10 cm pots (5 plants per pot with 4 replications per isolate) were inoculated with all 4 isolates by placing 3 mycelial plugs (8 mm diameter) taken from the leading edge of ½PDA plates onto the soil surface of each pot. PDA plugs were utilized as controls. After ~3 weeks, root lesions resembling DRC were observed and isolates were recovered and identified from diseased plants as described above. No symptoms developed on control plants. To our knowledge, this is the first formally confirmed report of DRC on sugar beets in more than 75 years from the Western Hemisphere. The original investigator suspected that the isolates he found inducing this disease were different from typical R. solani isolates based on different symptoms (1). Our results, based on different symptoms but also with distinct molecular, biological, and pathogenicity traits, validate those suspicions while also fulfilling Koch's postulates with binucleate Rhizoctonia AG-F pathogenic to sugar beet that is distinct from the more common R. solani.

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.

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini‐barcode

Bees (Apidae), of which there are more than 19900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century-old museum specimens and shown to be useful as mini-barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode.

Cost-Effective and Scalable DNA Extraction Method from Dried Blood Spots

Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA

Background454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies.ResultsWe constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions.ConclusionStandard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.

Advances towards a Marker-Assisted Selection Breeding Program in Prairie Cordgrass, a Biomass Crop

Prairie cordgrass (Spartina pectinata Bosc ex Link) is an indigenous, perennial grass of North America that is being developed into a cellulosic biomass crop suitable for biofuel production. Limited research has been performed into the breeding of prairie cordgrass; this research details an initial investigation into the development of a breeding program for this species. Genomic libraries enriched for four simple sequence repeat (SSR) motifs were developed, 25 clones from each library were sequenced, identifying 70 SSR regions, and primers were developed for these regions, 35 of which were amplified under standard PCR conditions. These SSR markers were used to validate the crossing methodology of prairie cordgrass and it was found that crosses between two plants occurred without the need for emasculation. The successful cross between two clones of prairie cordgrass indicates that this species is not self-incompatible. The results from this research will be used to instigate the production of a molecular map of prairie cordgrass which can be used to incorporate marker-assisted selection (MAS) protocols into a breeding program to improve this species for cellulosic biomass production.

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Aim Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX and use of Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% of the read depth of the reported true allele. These artifacts appeared to be PCR crossovers between the DRB1 and DRB3/4/5 sequences. The crossovers usually had one or more mismatches with the IMGT database but occasionally matched a very rare allele (e.g. DRB1 ∗ 03:42). We performed a study to determine if these sequences were in vitro artifacts and to determine if some rare DRB alleles in the IMGT database might, in fact, be in vitro artifacts that had been submitted. Methods To test for in vitro artifact formation during late cycles of genomic PCR, we amplified the DRB loci from 21 homozygous cells lines under both our standard PCR conditions (35 cycles) and a reduced number of cycles (28), followed by 454 sequencing. Using the same PCR conditions, we also sequenced the DRB loci of four samples, each of which had previously been identified as having a novel DRB allele that was subsequently submitted to the IMGT database. Results For all homozygous 21 cell lines, amplification for 35 cycles gave low abundance sequences as possible “second alleles.” In 36% of the cases, these artifactual sequences corresponded to named alleles. Such sequences were not detected with amplification for 28 cycles. In the case of 4 samples with rare alleles previously submitted to the IMGT, we found that 3 were true alleles. The fourth, while not a crossover product, contained a sequencing error which was revealed by our 454 HLA genotyping assay. Conclusions Formation of in vitro crossover products of the DRB loci can occur in late cycles of PCR. PCR conditions have been identified that minimize generation of these in vitro artifacts. Clonal sequencing on the 454 GS FLX is a valuable method for revealing both these artifacts and traditional sequencing errors.

Genetic diversity and bottleneck analysis of Konkan Kanyal goats

Konkan Kanyal goats are medium-sized animals reared for meat purpose. They are predominantly black in colour with white markings in a specific pattern and are found in the Kudal, Sawantwadi, Dodamarg, Malvan and Vengurle Talukas of Sindhudurg district of Konkan region of Maharashtra state. The mean body weight ranges from 30 kg in adult females to 35 kg in males. The age at first kidding, kidding interval and twinning of these goats is 546 days, 240 days and 35 percent respectively. The average daily milk yield and lactation yield were found to be 0.6 and 69 kg, respectively. Characterization of Konkan Kanyal goats was carried out using a panel of 25 microsatellite markers. The genomic DNA of 50 unrelated goats was isolated and PCR amplified under standardized PCR conditions. The amplified DNA was used for genotyping by the automated DNA Sequencer ABI 3730. The analysis of data revealed that the effective number of alleles ranged from 1.17 to 8.94 and the number of observed alleles ranged from 5 to 19. A total of 255 alleles were observed with a mean of 10.2 alleles/locus. The observed heterozygosity ranged from 0.13 to 0.95, while the expected heterozygosity ranged from 0.14 to 0.89, indicating the heterogeneous nature of the population distributed in the breeding tract. The mean polymorphism content was 0.79. The qualitative graphical method based on the allele frequency spectra detected no shift in the frequency distribution of alleles and a normal L-shaped curve was observed where the alleles with the lowest frequencies were found to be most abundant. Based on the phenotypic and genetic variability, Konkan Kanyal goats appear to be distinct from other goats of Maharashtra state.

DNA mixture genotyping by probabilistic computer interpretation of binomially-sampled laser captured cell populations: Combining quantitative data for greater identification information

Two person DNA admixtures are frequently encountered in criminal cases and their interpretation can be challenging, particularly if the amount of DNA contributed by both individuals is approximately equal. Due to an inevitable degree of uncertainty in the constituent genotypes, reduced statistical weight is given to the mixture evidence compared to that expected from the constituent single source contributors. The ultimate goal of mixture analysis, then, is to precisely discern the constituent genotypes and here we posit a novel strategy to accomplish this. We hypothesised that LCM-mediated isolation of multiple groups of cells ('binomial sampling') from the admixture would create separate cell sub-populations with differing constituent weight ratios. Furthermore we predicted that interpreting the resulting DNA profiling data by the quantitative computer-based TrueAllele® interpretation system would result in an efficient recovery of the constituent genotypes due to newfound abilities to compute a maximum LR from sub-samples with skewed weight ratios, and to jointly interpret all possible pairings of sub-samples using a joint likelihood function. As a proof of concept, 10 separate cell samplings of size 20 recovered by LCM from each of two 1:1 buccal cell mixtures were DNA-STR profiled using a specifically developed LCN methodology, with the data analyzed by the TrueAllele® Casework system. In accordance with the binomial sampling hypothesis, the sub-samples exhibited weight ratios that were well dispersed from the 50% center value (50±35% at the 95% level). The maximum log(LR) information for a genotype inferred from a single 20 cell sample was 18.5 ban, with an average log(LR) information of 11.7 ban. Co-inferring genotypes using a joint likelihood function with two sub-samples essentially recovered the full genotype information. We demonstrate that a similar gain in genotype information can be obtained with standard (28-cycle) PCR conditions using the same joint interpretation methods. Finally, we discuss the implications of this work for routine forensic practice.