DNA Primer Sets Research Articles

Development of Primers Specific for Detection of Litylenchus crenatae, the Causal Agent of Beech Leaf Disease, in Plant Tissue.

Beech leaf disease (BLD), an emerging threat to American beech (Fagus grandifolia) in the northern United States and Canada, was recently confirmed to be caused by the nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae). Consequently, there is a need for a rapid, sensitive, and accurate method for detecting L. crenatae for both diagnostic as well as control purposes. This research developed a new set of DNA primers that specifically amplify L. crenatae and allow for accurate detection of the nematode in plant tissue. These primers have also been used in quantitative PCR (qPCR) to determine relative differences in gene copy number between samples. This primer set provides an improved, effective tool for monitoring and detecting L. crenatae in temperate tree leaf tissue which is necessary to understand the spread of this emerging forest pest and to develop management strategies.

Molecular evolution of the Pi-d2 gene conferring resistance to rice blast in Oryza.

The exploitation of plant disease resistance (R) genes in breeding programs is an effective strategy for coping with pathogens. An understanding of R gene variation is the basis for this strategy. Rice blast disease, caused by the Magnaporthe oryzae fungus, is a destructive disease of rice. The rice blast resistance gene Pi-d2 represents a new class of plant R gene because of its novel extracellular domain. We investigated the nucleotide polymorphism, phylogenetic topology and evolution patterns of the Pi-d2 gene among 67 cultivated and wild rice relatives. The Pi-d2 gene originated early in the basal Poales and has remained as a single gene without expansion. The striking finding is that susceptible Pi-d2 alleles might be derived from a single nucleotide substitution of the resistant alleles after the split of Oryza subspecies. Functional pleiotropy and linkage effects are proposed for the evolution and retention of the disease-susceptible alleles in rice populations. One set of DNA primers was developed from the polymorphic position to detect the functional nucleotide polymorphism for disease resistance of the Pi-d2 gene based on conventional Polymerase Chain Reaction. The nucleotide diversity level varied between different domains of the Pi-d2 gene, which might be related to distinct functions of each domain in the disease defense response. Directional (or purifying) selection appears dominant in the molecular evolution of the Pi-d2 gene and has shaped its conserved variation pattern.

The forensic landscape and the population genetic analyses of Hainan Li based on massively parallel sequencing DNA profiling.

Due to the formation of the Qiongzhou Strait by climate change and marine transition, Hainan island was isolated from the mainland southern China during the Last Glacial Maximum. Hainan island, located at the southernmost part of China and separated from the Leizhou Peninsula by the Qiongzhou Strait, laid on one of the modern human northward migration routes from Southeast Asia to East Asia. The Hlai language-speaking Li minority, the second largest population after Han Chinese in Hainan island, is the direct descendants of the initial migrants in Hainan island and has unique ethnic properties and derived characteristics; however, the forensic-associated studies on Hainan Li population are still insufficient. Hence, 136 Hainan Li individuals were genotyped in this study using the MPS-based ForenSeq™ DNA Signature Prep Kit (DNA Primer Set A, DPMA) to characterize the forensic genetic polymorphism landscape, and DNA profiles were obtained from 152 different molecular genetic markers (27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iiSNPs). A total of 419 distinct length variants and 586 repeat sequence sub-variants, with 31 novel alleles (at 17 loci), were identified across the 58 STR loci from the DNA profiles of Hainan Li population. We evaluated the forensic characteristics and efficiencies of DPMA, demonstrating that the STRs and iiSNPs in DPMA were highly polymorphic in Hainan Li population and could be employed in forensic applications. In addition, we set up three datasets, which included the genetic data of (i) iiSNPs (27 populations, 2640 individuals), (ii) Y-STRs (42 populations, 8281 individuals), and (iii) Y haplogroups (123 populations, 4837 individuals) along with the population ancestries and language families, to perform population genetic analyses separately from different perspectives. In conclusion, the phylogenetic analyses indicated that Hainan Li, with a southern East Asia origin and Tai-Kadai language-speaking language, is an isolated population relatively. But the genetic pool of Hainan Li influenced by the limited gene flows from other Tai-Kadai populations and Hainan populations. Furthermore, the establishment of isolated population models will be beneficial to clarify the exquisite population structures and develop specific genetic markers for subpopulations in forensic genetic fields.

Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories

Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines.A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or “barcodes” are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.

Abstract 1380: Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring

Abstract We have developed a new molecular assay that utilizes digital PCR to detect and quantify cancer mutations within poor quality and limited quantity samples such as archival tissue DNA and circulating tumor DNA (ctDNA). This digital PCR assay is highly sensitive and is capable of detecting the targeted mutant fraction at an individual DNA molecule resolution. Thus, this assay is ideal for applications where DNA is in low abundance. An example is where DNA is shed from tumors that can be extracted from the plasma fraction of routine blood draws. The resulting circulating DNA (ctDNA) is extremely low in concentration, which makes it a prime candidate for our highly sensitive molecular assay. Our assay incorporates small amplicon PCR primers and can be configured for nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. Mutation quantitation relies on two DNA primer sets that are identical with the exception of the mutant or wild type specific base at the 3’ end of the “detecting” primer sets, to amplify the genomic region of interest. Through the addition of artificial 5’ non-complementary tails to our mutant and wild type specific “detection” primers, we are able to consistently differentiate between droplets that contain the mutant or wild type alleles based upon their differential amplicon lengths. The synthetic amplicon extension tails minimize bias within the PCR reaction by allowing our primers to target an identical region of genomic DNA, with the exception of the single nucleotide variant specific base at the 3’ end of the detecting primer. The dPCR technology allows the standard PCR reaction to be partitioned into 20,000 independent wells; we can then assay each individual droplet to assess whether the individual DNA molecule partitioned into the droplet is “wild-type” or the target “mutant”. The resulting data provides an absolute count of mutant and wild-type templates in a given patient sample. We have optimized this new molecular assay technology for quantitatively measuring clinically actionable mutations including BRAF V600E, KRAS G12D among others. We have successfully validated the sensitivity, specificity and reproducibility of our assays through controlled cell line DNA mixed dilution samples ranging from 66% mutant to 0.1% mutant in each of our optimized primer sets. Our assay improves the limit of detection to seven DNA molecules containing a mutation among a total number of 7,000 genome equivalents. Because our assay is sensitive down to the single DNA molecule resolution, we have also been able to reduce the amount of clinical sample DNA required to determine the presence of clinically actionable mutations such as those occurring in KRAS or BRAF. We are testing this extremely low cost, highly sensitive diagnostic technology for detecting nearly any cancer mutation from longitudinal samples of ctDNA. Citation Format: Christina M. Wood, Billy Lau, Laura Miotke, Hanlee P. Ji, Stephanie Greer. Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1380.

Achromobacter xylosoxidans Endophthalmitis Diagnosed by Polymerase Chain Reaction and Gene Sequencing

Purpose: To report a patient with Achromobacter xylosoxidansendophthalmitis that was diagnosed using polymerase chain reaction and gene sequencing. Methods: Case report. A patient with culture-negative endophthalmitis underwent an anterior chamber tap. Polymerase chain reaction was performed on the aqueous sample using the 16S ribosomal DNA primer set to detect and amplify bacterial DNA. The amplified DNA was sequenced and compared to archived sequences in a gene library using the BLAST search program. Results: A 214-base pair gene sequence was amplified and matched with the gene sequence for A. xylosoxidans. Antimicrobial treatment was instituted with resolution of hypopyon, anterior chamber cells, and vitreous cells one month after treatment. Conclusions: Polymerase chain reaction of 16S rDNA combined with gene sequencing may be an alternative method of diagnosing culture-negative bacterial endophthalmitis.

Development of Mitochondrial DNA Primers for Use with Homarid Lobsters

DNA primer sets were developed for the amplification of complete mitochondrial genomes for both European and American lobsters in 4 suitable-sized segments. Optimal conditions for polymerase chain reaction routine screening were established. The 4 segments were screened with 24 restriction endonucleases in a test population sample, covering the whole distribution of the European lobster, and restriction patterns of each enzyme were revealed. A segment of 3000 bp comprising part of cytochrome oxidase I gene, the genes cytochrome oxidase II and III, subunits 6 and 8 of ATPase, subunit 3 of the NAD dehydrogenase, and various transfer RNAs, was found to be the most polymorphic. A number of enzyme patterns in each segment differentiated European and American lobsters. Extra bands were observed, indicating heteroplasmy phenomena, which were verified with various approaches. Furthermore, a primer set that enables 1-step ampli fication of the complete mitochondrial genome of the European lobster was established.

Periodic expression of an ecdysteroid-induced nuclear receptor in a lepidopteran cell line (IAL-PID2)

A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10 −6 M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH-5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle.

Use of a monoclonal antibody and genomic fingerprinting by repetitive-sequence-based polymerase chain reaction to identify Xanthomonas populi pathovars

Diagnostic tools to facilitate the rapid and specific identification of Xanthomonas populi pv. populi, the cause of bacterial canker of poplar, were evaluated. A monoclonal antibody, MAb 13E7, that reacted in immunofluorescence tests with representative strains of physiological races of X. populi pv. populi as well as with the related X. populi pv. salicis and showed less cross-reaction with other xanthomonads than did a polyclonal antibody, was produced. The Xanthomonas strains were also characterized by rep-PCR (repetitive-sequence-based polymerase chain reaction) genomic fingerprinting, using three DNA primer sets (BOX, ERIC, and REP). Computer-assisted phylogenetic analysis of the linearly combined fingerprints clearly distinguished the strains of X. populi pv. populi. Fingerprint clusters of the two X. populi pathovars had ~60% similarity while the X. populi group had <20% similarity to the other xanthomonads. Des outils diagnostiques visant à faciliter l'identification rapide et spécifique du Xanthomonas populi pv. populi, agent causal du chancre bactérien du peuplier, ont été évalués. L'anticorps monoclonal MAb 13E7 qui a été produit a réagi, au cours des épreuves d'immunofluorescence, avec des souches représentatives des races physiologiques du X. populi pv. populi ainsi qu'avec le pathovar apparenté X. populi pv. salicis. De plus, il a montré une réaction croisée moins intensive qu'un anticorps polyclonal avec les autres xanthomonades. Les souches de Xanthomonas ont également été caractérisées par empreinte génomique par réaction en chaîne de la polymérase basée sur une séquence répétitive à l'aide de trois ensemble d'amorces d'ADN (BOX, ERIC et REP). L'analyse phylogénique assistée par ordinateur des empreintes linéairement combinées a permis de distinguer clairement les différentes souches du X. populi pv. populi. La similarité des groupes d'empreintes des deux pathovars du X. populi était d'environ 60%, tandis que celle du groupe du X. populi était de moins de 20% par rapport aux autres xanthomonades.

PCR-Based Identification of Postmortem Microbial Contaminants—A Preliminary Study

Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.

Polymerase Chain Reaction Amplification of Genetic Loci from Diseased Channel Catfish Found Dead in Ponds

Abstract As part of a selective breeding program for farm-raised channel catfish Ictalurus punctatus, we screened diseased fish to identify genetic markers linked to disease resistance or susceptibility. Because many diseased fish in ponds are not detected until after death, we investigated the utility of DNA isolated from diseased channel catfish found dead in ponds. Channel catfish (4–25 g) diagnosed with enteric septicemia of catfish or saprolegniasis were sampled 24–48 h postmortem from infected ponds. Control fish were killed by anesthetic overdose and sampled immediately. Total DNA isolated from liver, muscle (with skin), and caudal fin was quantified and analyzed for degradation. Yield of purified DNA, measured as micrograms of DNA per milligram of tissue, was significantly (P < 0.05) lower in diseased fish than in controls. Two sets of DNA primers were used to amplify a portion of the channel catfish growth hormone gene and the mitochondrial D-loop region with the polymerase chain reaction. Degrad...

Cassette labeling for facile construction of energy transfer fluorescent primers

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.

We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.