scholarly journals openPrimeR for multiplex amplification of highly diverse templates

2019 ◽  
Author(s):  
Christoph Kreer ◽  
Matthias Döring ◽  
Nathalie Lehnen ◽  
Meryem S. Ercanoglu ◽  
Lutz Gieselmann ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gene Segment ◽  
Pcr Primers ◽  
Proof Of Concept ◽  
Minimal Set ◽  
Multiplex Amplification ◽  
Variable Gene ◽  
Primer Sets ◽  
Correct Understanding ◽  
Intuitive Interface

AbstractTo study the diversity of immune receptors and pathogens, multiplex PCR has become a central approach in research and diagnostics. However, insufficient primer design against highly diverse templates often prevents amplification and therefore limits the correct understanding of biological processes. Here, we present openPrimeR, an R-based tool for evaluating and designing multiplex PCR primers. openPrimeR provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage. As proof of concept, we used openPrimeR to find optimal primer sets for the amplification of highly mutated immunoglobulins. Comprehensive analyses on specifically generated immunoglobulin variable gene segment libraries resulted in the composition of highly effective primer sets (oPR-IGHV, oPR-IGKV and oPR-IGLV) that demonstrated to be particularly suitable for the isolation of novel human antibodies.

A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

2019 ◽  
Vol 82 (2) ◽  
pp. 325-330 ◽  
Author(s):  
WANWAN LIU ◽  
XIAONAN WANG ◽  
JING TAO ◽  
BANGSHENG XI ◽  
MAN XUE ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Detection System ◽  
Meat Products ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Food Samples ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Multiplex Pcr Assay ◽  
Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

scholarly journals Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression

2012 ◽  
Vol 209 (12) ◽  
pp. 2199-2213 ◽  
Author(s):  
Sarah E. Powers ◽  
Malay Mandal ◽  
Satoshi Matsuda ◽  
Ana V. Miletic ◽  
Matthew H. Cato ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Gene Segment ◽  
Cyclin D3 ◽  
Cyclin D2 ◽  
Cycle Progression ◽  
Nuclear Compartment ◽  
Regulation Of Proliferation ◽  
Variable Gene ◽  
V Gene ◽  
Phosphoinositide 3 Kinase

Ubiquitously expressed D-type cyclins are required for hematopoiesis but are dispensable in other cell lineages. Furthermore, within different hematopoietic progenitor populations the D-type cyclins play nonredundant roles. The basis of this lineage and developmental specificity is unknown. In pro–B cells we demonstrate four distinct nuclear D-type cyclin compartments, including one cyclin D3 fraction associated with CDK4 and another phosphoinositide 3-kinase–regulated fraction not required for proliferation. A third fraction of cyclin D3 was associated with the nuclear matrix and repression of >200 genes including the variable (V) gene segments Igkv1-117, Iglv1, and Igh-VJ558. Consistent with different subnuclear compartments and functions, distinct domains of cyclin D3 mediated proliferation and Igk V gene segment repression. None of the cyclin D3 nuclear compartments overlapped with cyclin D2, which was distributed, unbound to CDK4, throughout the nucleus. Furthermore, compartmentalization of the cyclins appeared to be lineage restricted because in fibroblasts, cyclin D2 and cyclin D3 occupied a single nuclear compartment and neither bound CDK4 efficiently. These data suggest that subnuclear compartmentalization enables cyclin D3 to drive cell cycle progression and repress V gene accessibility, thereby ensuring coordination of proliferation with immunoglobulin recombination.

Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

1998 ◽  
Vol 61 (2) ◽  
pp. 141-145 ◽  
Author(s):  
HAU-YANG TSEN ◽  
LIANG-ZHAO JIAN ◽  
WAN-RONG CHI
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Skim Milk ◽  
Pcr Primers ◽  
Enterotoxigenic Escherichia Coli ◽  
Farm Animals ◽  
Target Cells ◽  
Heat Stable ◽  
Heat Labile

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT I- and ST II-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 100 cells per ml of the sample could be detected. Without the preculture step, 104 CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR System can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

scholarly journals A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

2009 ◽  
Vol 58 (8) ◽  
pp. 1045-1057 ◽  
Author(s):  
Lin Cai ◽  
Fanrong Kong ◽  
Qinning Wang ◽  
Huiping Wang ◽  
Meng Xiao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Rapid Identification ◽  
Reverse Line Blot ◽  
Discriminatory Power ◽  
Hybridization Assay ◽  
Coagulase Negative Staphylococci ◽  
Primer Sets ◽  
Mrsa Strains

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: Application for assessment of clonal composition

1999 ◽  
Vol 53 (2) ◽  
pp. 122-134 ◽  
Author(s):  
Y. Akatsuka ◽  
E.G. Martin ◽  
A. Madonik ◽  
A.A. Barsoukov ◽  
J.A. Hansen
Keyword(s):  
T Cell ◽  
Multiplex Pcr ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Rapid Screening ◽  
Gene Usage ◽  
Variable Gene ◽  
Clonal Composition

Selecting Degenerate Multiplex PCR Primers

2003 ◽  
pp. 512-526 ◽  
Author(s):  
Richard Souvenir ◽  
Jeremy Buhler ◽  
Gary Stormo ◽  
Weixiong Zhang
Keyword(s):  
Multiplex Pcr ◽  
Pcr Primers

Variable gene segment-specific N-insertions at the signal joint of T-cell receptor Vβ-Dβ recombinations

1998 ◽  
Vol 61 (2-3) ◽  
pp. 151-155 ◽  
Author(s):  
Yasuyoshi Kanari ◽  
Ryusuke Nakagawa ◽  
Hiroshi Arakawa ◽  
Hideo Yamagishi
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Gene Segment ◽  
Cell Receptor ◽  
Variable Gene ◽  
T Cell Receptor Vβ ◽  
Variable Gene Segment

scholarly journals Regulation of TCR δ and α repertoires by local and long-distance control of variable gene segment chromatin structure

2005 ◽  
Vol 202 (4) ◽  
pp. 467-472 ◽  
Author(s):  
Abbas Hawwari ◽  
Michael S. Krangel
Keyword(s):  
Chromatin Structure ◽  
Genetic Locus ◽  
Gene Segment ◽  
Chromatin Accessibility ◽  
Small Subset ◽  
Double Positive ◽  
Long Distance ◽  
Variable Gene ◽  
Distance Regulation ◽  
Variable Gene Segment

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4−CD8− (double negative [DN]) and CD4+CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of ∼100 variable gene segments contributes substantially to the adult TCRδ repertoire. Moreover, although most contribute to the TCRα repertoire, variable gene segments that are Jα proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRδ repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

scholarly journals Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (11) ◽  
pp. 6801-6807 ◽  
Author(s):  
Isabel Lopez ◽  
Fernanda Ruiz-Larrea ◽  
Luca Cocolin ◽  
Erica Orr ◽  
Trevor Phister ◽  
...  
Keyword(s):  
Lactic Acid ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Fungal Species ◽  
Bacterial Populations ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

Sign in / Sign up

Export Citation Format

Share Document