Touchdown PCR Research Articles

Development of a new comprehensive HIV-1 genotypic drug resistance assay for all commercially available reverse transcriptase, protease and integrase inhibitors in patients infected with group M HIV-1 strains

Comprehensive PCR assays for the genotypic drug resistance analysis of all HIV-1 antiretroviral agents (reverse transcriptase, protease and integrase inhibitors) are increasingly in demand due to introduction of integrase inhibitors in the first line regimens and the increasing presence of non-B HIV-1 clades around the world. This study focused on the development and evaluation of a new PCR-based assay for the amplification and sequencing of the entire HIV-1 pol region of major circulating group M HIV-1 strains in Europe for genotypic drug resistance analysis. The comprehensive touchdown PCR assay developed in this study utilized HIV-1 RNA extracted from the plasma of blood samples of consenting HIV-1 infected patients in Cyprus, collected from 2017 to 2019. The HIV-1 pol region was amplified by touchdown PCR for both the primary RT-PCR and the secondary PCR steps. Successful PCR amplicons were determined by population DNA sequencing, using the Sanger method and the genotypic drug resistance analysis was performed with the Stanford University HIV Drug Resistance Database Program. The newly developed assay successfully amplified the entire HIV-1 pol region (2844 nucleotides long) of 141 out of 144 samples of group M HIV-1 subtypes and recombinant strains of the Cyprus HIV-1 Transmission Cohort Study (CHICS) isolated from 2017 to 2019 and genotypic analyses were conducted for all currently available HIV-1 reverse transcriptase, protease and integrase inhibitors. The drug resistance, epidemiological and demographic data of these study subjects will be expanded upon in the CHICS (L.G. Kostrikis et al., manuscript in preparation for publication). The newly developed HIV-1 genotypic drug resistance assay would benefit clinical settings, and research focusing on the world-wide spread of HIV-1 drug-resistant strains, especially in geographic regions characterized by polyphyletic HIV-1 infections.

Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts.

Background:In burn patients, wound healing is often accompanied by hypertrophic scar (HS) development, resulting in both functional and aesthetic problems. HSs are characterised by abundant presence of myofibroblasts that contribute to overproduction of extracellular matrix (ECM) that is regulated by the TGF-β signalling pathway. Studies have shown that inhibition of TGF-β receptors in fibrotic diseases reduces the fibrotic load. In the present study, we aim to inactivate ALK5, also known as TGF-β receptor I, in human HS fibroblasts by exon skipping using antisense oligonucleotides (AONs).Methods:HS biopsies were used to isolate and set up fibroblast monocultures. AONs targeting ALK5 were supplemented to the fibroblast cultures to induce exon skipping, while pharmacological ALK5 inhibition was induced using SB431542. AON delivery in HS fibroblasts was examined using immunofluorescence (IF), while TGF-β signalling downstream targets, such as Smad2/3, PAI-1, ACTA2, COL1A1 and COL3A1, were analysed using touchdown polymerase chain reaction (PCR), quantitative PCR (qPCR), IF or western blotting.Results:Our data clearly demonstrate that AONs were successfully delivered in the nuclei of HS fibroblasts and that functional exon skipping of ALK5 took place as confirmed with touchdown PCR and qPCR. In addition, exon skipping affected the expression of ECM-related genes, such as type I/III collagens, PAI-1 and CCN2. Moreover, AON treatment did not affect the migration of HS fibroblasts in a model for wound healing.Conclusion:Exon skipping is a promising tool to modulate the TGF-β signalling pathway in HS. This would open a therapeutic window for the treatment of patients suffering from HSs.

Optimization of overlap extension PCR for efficient transgene construction

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

Linkage analysis of hearing impairment in families of Bannu Distric.

To link congenital hearing loss with known loci to establish a platform for future research. The cross-sectional study was conducted from February 2016 to March 2017 in Bannu, Khyber Pakhtunkhwa, Pakistan, and comprised families with Pashtun ethnicity having at least 2 individuals suffering from congenital hearing loss. Deoxyribonucleic acid from whole blood samples was extracted by salting-out method. Amplification was done through touchdown polymerase chain reaction to see any possible linkage to already reported deafness loci. Linkage analysis was carried out using microsatellite markers for each locus. Genotyping of the samples was done and haplotypes were accordingly generated to either include or exclude the linked / unlinked regions. Of the 4 families, family PKDF 1620 showed linkage with DFNB12/CDH23 (D10S1432, D10S606, and D10S1694) and family PKDF 1625 had linkage with DFNB3/MYO15A (D17S2196, D17S2207 and D17S2206). Families PKDF1623 and PKDF1624 showed no linkage with any of the prevalent reported loci in Pakistan . Linkage to DFNB12 and MYO 15 showed heterogeneity of congenital deafness.

Genetic linkage analysis of DFNB22 in families with autosomal recessive non-syndromic hearing loss in Khuzestan province

Background and aims: Hearing loss (HL) is the most common sensorineural disorder affecting 1 in 1000 newborns. Autosomal recessive non-syndromic hearing loss (ARNSHL), which is the most common cause of severe HL, is caused by mutations in more than 80 loci. The OTOA gene located on DFNB22 is a rare cause of the disease and the gene studied less in Iranian ARNSHL families. Hence, limited information is available on the frequency and type of OTOA mutations in different populations. In this study, we investigated the role of DFNB22 locus in ARNSHL patients in Khuzestan province, Iran. Materials and Methods: In this descriptive-experimental study, 23 large families with pre-lingual ARNSHL from Khuzestan province were enrolled. Mutations in GJB2 were excluded by DNA sequencing followed by linkage analysis. Homozygosity mapping of DFNB22 was conducted using 6 short tandem repeat polymorphic markers via touch-down PCR and polyacrylamide gel electrophoresis. Homozygosityby-descent was identified by calculating two-point and multi-point LOD score and haplotype reconstruction. Results: Families were negative for GJB2 mutations. Genotyping the STRP markers, haplotype reconstruction, and two-point and multiplepoint LOD scores did not show homozygosity-by-descent in any of the pedigrees. Conclusion: Our findings suggest that OTOA mutations might not contribute significantly to the molecular pathophysiology of ARNSHL in Khuzestan province. However, extending the sample size can illuminate the role of this gene in Khuzestan province.

Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A; -B; -C; -DRB1 and -DQB1 loci using sequencing-based typing

Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature.

Antimicrobial Susceptibility, Biotypes and Phylotypes of Clinical Cutibacterium (Formerly Propionibacterium) acnes Strains Isolated from Acne Patients: An Observational Study.

IntroductionThe aim of this study was to investigate the distribution of antimicrobial susceptibility, biotypes and phylotypes of clinical Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes) isolates as well as the relationship among demographic factors, C. acnes biotypes and phylotypes.MethodsCutibacterium acnes was collected from the skin lesions of acne patients who visited the dermatologic department of Huashan Hospital in Shanghai from October 2016 to March 2017. The agar dilution method was conducted to determine the minimum inhibitory concentrations (MICs) of C. acnes, the fermentation test to identify biotypes and then multiplex touchdown polymerase chain reaction (PCR) to identify phylotypes.ResultsOf the 63 C. acnes strains we isolated, 18 (28.6%), 31 (49.2%) and 4 (6.3%) strains were resistant to clindamycin, erythromycin and moxifloxacin, respectively; no strains were resistant to tetracycline, minocycline, fusidic acid or β-lactam, while metronidazole was completely resisted; 3 strains showed multidrug resistance (MDR). Biotype III (BIII) was the major biotype (50.8%) followed by BI and BV (both 15.9%), BII (12.7%) and lastly BIV (4.8%). IA1 was the predominant phylotype (71.4%) followed by IA2 (19.0%), II (4.8%), IB (3.2%) and IC (1.6%), while III was not detected. Significant differences were observed in the severity of disease: different degrees of acne severity reflected different biotype and phylotype distributions, and the biotype distribution of mild acne was different from that of moderate acne; the phylotype distribution of moderate acne varies from that of severe acne, too. Additionally, there was no significant difference in the distribution of biotypes or phylotypes between resistant and susceptible strains.ConclusionErythromycin and clindamycin resistances are the most common in clinical C. acnes strains; BIII is the predominant biotype and IA1 is the major phylotype of C. acnes, which are mainly related to disease severity.

Abstract 3422: Label-free circulating tumor cells isolation platform for lung cancer EGFR mutation identification and colorectal cancer with KRAS mutation detection

Abstract Circulating tumor cell becomes an urgent issue in personalized cancer treatment studies. AS the liquid biopsy, many previous reports showed the number and the characteristic of CTC are very important for patient prognosis. In cancer therapy, specific gene mutation in primary tumor decides the therapy strategy and drug response. However, the patient who cannot perform an operation or take biopsy would become a risk factor for treatment and affect therapy outcome. Hence, drug treatable related gene mutation detection by CTC is necessary. Platforms for mutation detection in tissue were well established but less useful for CTC. Most of them were lower efficacy for rare cell or DNA. In order to solve this dilemma, scientists use next-generation sequence (NGS) or droplet digital PCR (ddPCR) for gene mutation detection. But, in most clinical situations, we only have to follow-up some specific gene mutation, instead of many mutant types. Thus, we eager to develop a fast and low-cost methodology for drug treatable related gene mutation detection by CTC. In this study, 4mL blood was collected by EDTA vacuum blood collection tube from 37 patients. Whole blood was processed with RBC lyse steps, then added CD45 and glycophorin A antibodies conjugated with the nanoparticle to remove leukocyte and remained RBC. Further, Genomic DNA was extracted from negative selection cell mixture. An ARMS-PCR (Amplification-refractory mutation system PCR) combined with a touchdown PCR program was used for EGFR mutations identification. We enrolled 31 lung cancer patients. Nineteen patients’ CTC were matched with tissue mutation results (14 mutant and 5 wild-types), and 12 unmatched. In those 12 patients, 2 patients with mutant tissue but wild-type CTC due to their blood were drawn at disease-free status after long-term therapy, 4 patients with wild-type tissue but mutant CTC were still had tumor burden after therapy and need to more long-term follow-up. Overall, we have a mutation correlation rate of (19 + 2) / 31 = 68% between CTC and primary tumor. Furthermore, an LNA-PCR (Locked nucleic acid PCR) system was designed for colorectal cancer KRAS codon 12 and codon 13 mutations detection. In this platform, modified RNA was designed for blocked wild-type DNA amplification in PCR. We enrolled 6 patients with primary tumor mutant positive, 5 of 6 (83%) patients were also mutant positive in their CTC sample. In conclusion, an easy CTC isolation platform and PCR design could hugely help cancer patient mutation detection. Only less than 4 hours and 100 USD were needed for drug treatable gene mutation detection by CTC. Citation Format: Hung-Chih Lin, Yu-Lin Yang, Chia-Hsun Hsieh. Label-free circulating tumor cells isolation platform for lung cancer EGFR mutation identification and colorectal cancer with KRAS mutation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3422.

Characterization of retroviral vector derived DNA-isoforms by PCR and sequencing

For the analytical surveillance of genetic engineering operations suitable methods for characterization of used (recombinant) cell lines and used transfer vector plasmids are needed. The purpose of the described method is the identification of retroviral DNA-isoforms based on lenti-or gammaretrovirus (HIV-1 or MLV). The primers, used in an optimized touch-down PCR protocol, specifically amplify transfer-vector encoded vector genomes and proviruses integrated into stably transduced vertebrate cell lines, distinguishing them from the background of endogenous retroviral sequences. The resulting PCR products were isolated by preparative agarose electrophoresis and subsequently used for sequence identification by Sanger sequencing. In addition, the amplification and sequencing of PCR products corresponding to the HIV-1- and MLV-based vector-genomes was successful from residual transfer vector plasmids in cell culture supernatants and basic polyethylene glycol-precipitated vector preparations. The methods were validated using 19 samples (cell lines, plasmids and viral vector preparations). No false-positive PCR results were observed, while both retroviral DNA-isoforms were detected with the respective assay. The successful implementation in routine analysis is presented using two routine samples.

Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.

The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with Taq DNA polymerase.

Linkage analysis of hearing impairment in families of Bannu district

To link congenital hearing loss with known loci to establish a platform for future research.The cross-sectional study was conducted from February 2016 to March 2017 in Bannu, Khyber Pakhtunkhwa, Pakistan, and comprised families with Pashtun ethnicity having at least 2 individuals suffering from congenital hearing loss. Deoxyribonucleic acid from whole blood samples was extracted by salting-out method. Amplification was done through touchdown polymerase chain reaction to see any possible linkage to already reported deafness loci. Linkage analysis was carried out using microsatellite markers for each locus. Genotyping of the samples was done and haplotypes were accordingly generated to either include or exclude the linked / unlinked regions.Of the 4 families, family PKDF 1620 showed linkage with DFNB12/CDH23 (D10S1432, D10S606, and D10S1694) and family PKDF 1625 had linkage with DFNB3/MYO15A (D17S2196, D17S2207 and D17S2206). Families PKDF1623 and PKDF1624 showed no linkage with any of the prevalent reported loci in Pakistan .Linkage to DFNB12 and MYO 15 showed heterogeneity of congenital deafness.

301. The Use of Multiplex Touchdown PCR to Genotype Cutibacterium (Propionibacterium) acnes Isolated from Periprosthetic Shoulder Infections

BackgroundAs biogeographic surveys of the human skin microbiome have shown that C. acnes is a major component of the residential axillary microflora, the organism is frequently isolated from synovial tissue and joint aspirates obtained from patients with suspected periprosthetic shoulder infections. We hypothesized that multilocus sequence typing (MLST) applying a prior validated rapid high through-put multiplex PCR protocol would segregate C. acnes into distinctive phylogroups associated with periprosthetic infections compared with commensal strains.Methods C. acnes collected between 2015 and 2017 were correlated with the presence or absence of infection in a detailed retrospective chart review. To determine the C. acnes genotype, bacterial genomic DNA isolated from a single patient isolate served as template in a six locus multiplex touchdown PCR assay using organism-specific primers targeting six genes (16S rRNA, ATPase, sodA, Fic toxin, aspD and recA). Isolates were classified as a contaminant in the absence of multiple positive cultures from an anatomic site and without corresponding clinical, laboratory and histopathologic correlates of infection. The assignment of a diagnosis of prosthetic joint infection (PJI) conformed to the definition recommended by the IDSA Clinical Practice Guidelines of PJI.ResultsOf the C. acnes recovered from 94 patients, 14 (14.9%) were from patients with shoulder implants of which shoulder PJI was present in 10 individuals (10.6% of the total). The remaining 84 (89.4%) isolates were retrieved from a variety of tissue and fluid samples of which the majority (65.5%) were deemed as contaminants. Overall, phylogroups IA1, IB, and II predominated (79.8%). Although a similar genetic profile was present in all of the shoulder isolates, no phylogroup association was detected with PJI (P < 0.72). No genetic difference was present in the lineage of strains not causing PJI compared with those responsible for PJI (P < 0.25).ConclusionOur results mirror those from a previous investigation using a less robust four gene MLST PCR based scheme that showed a lack of a phylogenetic association with shoulder PJI. Our results are a reflection of the phylogroup composition of the circulating C. acnes sequence types in our community.Disclosures All authors: No reported disclosures.

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis.

Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced Leishmania spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for Leishmania major, L. donovani, L. venezuelensis, and L. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of L. major (Seidman and Friedlin), and L. venezeuelensis. These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

Development of a touch-down multiplex PCR method for simultaneously rapidly detecting three novel insertion/deletions (indels) within one gene: an example for goat GHR gene

The multiplex polymerase chain reaction (PCR) method is fast and accuracy for screening several polymorphism loci in a single reaction in large samples. Therefore, this study aimed to identify three novel insertion/deletion (indel) loci in goat growth hormone receptor (GHR) gene by using multiplex PCR method and the genotypes in three indel loci are identified in 918 individuals from three Chinese goat breeds, as well as to evaluate its associations with growth traits. After validating the accuracy by taking the conventional PCR method, the concordance between these two methods was 100%. Moreover, P14 locus polymorphism was significantly associated with six growth traits of Hainan black goats. Briefly, an economic multiplex PCR method is presented to simultaneously and accurately detect three indel polymorphisms within goat GHR without the use of any special equipment, thus improving the experimental efficiency and fast obtaining the experimental result for indel genotyping.

Molecular cloning of novel transcripts of the adaptor-related protein complex 2 alpha 1 subunit (AP2A1) gene, using Next-Generation Sequencing

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation.

ISOLATION AND SEQUENCE ANALYSIS OF A NOVEL PARTIAL VACUOLAR Na+/H+ ANTIPORTER cDNA FROM Capparis orientalis, Lycium shawii AND Zygophyllum album

Egyptian native (nondomesticated) plant species from different families such as Capparis orientalis, Lycium shawii and Zygophyllum album were collected from North Western coast of Marsa Matrouh Governorate, Egypt. Plant vacuolar Na+/H+ antiporter candidate gene from tolerant plant species; several are localized on the tonoplast, plays an important role in several plant species (Halophytes and xerophytes) under abiotic stress. Then, once the genes will be identified from tolerant plant species, the overall goal of this study is to identify partial the vacuolar antiporter NHX1 candidate gene. According to NHX1 family homologous sequence conservative region; one degenerate oligonucleotide primer pair was used to amplify core (partial middle) fragment of cDNAs vacuolar Na+/H+ antiporter with about size of 600 bp, approximately. Touchdown PCR program (TD-PCR) of cDNAs were success to increase specificity, sensitivity and yield to amplify core cDNA of vacuolar Na+/H+ antiporter gene. Sequence analysis provided us with a novel partial fragment length of cDNAs about 548 bp, 557 bp and 557 bp of a novel CoNHX1, LsNHX and ZaNHX was deposited in GenBank database with NCBI, GenBank accession no. KJ452345.1, KJ452346.1 and KJ452347.1 and amino acid sequences about 182 a.a, 185 a.a and 185 a.a with GenBank accession no. AHY19036.1, AHY19037.1 and AHY19038.1, respectively. BLASTN of sequences result and phylogenetic relationship analysis indicated that all were clustered into the vacuolar Na+/H+ antiporter group. The deduced amino acid sequences showed high identities with other plant vacuolar-type Na+/H+ antiporters. Taken together, these results suggest that CoNHX, LsNHX and ZaNHX are new members of the vacuolar Na+/H+ antiporter family. The ultimate goal of this study provided a basic foundation information about a Novel partial vacuolar Na+/H+ antiporter gene to develop 5` and 3` RACE technique (Rapid amplification cDNA Ends).

Touchdown Polymerase Chain Reaction (PCR)

"Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs. In touchdown PCR the temperature selected for the annealing step is initially set 5°C-10°C higher than the calculated Tm of the primers. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2°C-5°C below the calculated Tm of the primers. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target-for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers.

Improved Conventional PCR Assay for Detecting Tetracapsuloides bryosalmonae DNA in Fish Tissues.

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonaeDNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies, and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than did PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples, while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length.

Interlaboratory comparison of fig (Ficus carica L.) microsatellite genotyping data and determination of reference alleles

Microsatellites have been identified as the marker of choice in plant genotyping projects. However, due to length discrepancies obtained between different laboratories for the same allele, interlaboratory comparison of fingerprinting results is often a difficult task. The objectives of this study were to compare genotyping results of two laboratories, to evaluate genetic parameters of microsatellite markers and to determine reference allele sizes for fig cultivars from the Istrian peninsula.Genotyping results of ninety fig (Ficus carica L.) accessions were comparable between the laboratories despite differences observed when comparing electropherograms of different capillary electrophoresis systems. Differences in lengths of the same alleles were detected due to different PCR methods and laboratory equipment, but the distances between alleles of the same locus were preserved. However, locus FSYC01 exhibited one allele dropout which led to misidentification of 28 heterozygotes as homozygote individuals suggesting this locus as unreliable. Allele dropout was assigned to the tail PCR technology or to a touchdown PCR protocol.Genotypes of twenty-four reference cultivars from the Istrian peninsula were confirmed by both laboratories. These results will contribute to the usage of markers with greater reliability, discrimination power and consequently, to more reliable standardization with other fig genotyping projects.

ReToAd: simple method for the rapid replacement of promoters to improve protein production.

To develop a method for fast replacement of promoters to improve protein production. A method (entitled retreat to advance or "ReToAd"), which includes a deleting PCR and a touchdown PCR, was validated by replacing seven IPTG-inducible promoters with enhanced green fluorescent protein (eGFP). The seven promoters were fully recovered by sequencing only 30 clones. The activity of E. coli harboring ω-transaminase (ω-TA) was increased from 112 U/mg cells (T7 promoter) to 147 U/mg cells (Trc promoter) by combining ReToAd and screening experiments. After screening a library comprising glutamate dehydrogenase (GDH) expressed by different promoters, the activity of E. coli cell harboring Trc-promoter-expressed GDH was ~31-fold higher than that of T7-promoter-expressed GDH. The "ReToAd" for in situ rapid replacement of promoters was developed and optimized, and one round of "ReToAd" can be completed within 3days.