Tag Archives: PCR Genotyping
Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR
Genet Mol Biol. 2012 Dec;35(4 (suppl)):955-9. Epub 2012 Dec 18.
de Souza Godinho FM, Bock H, Gheno TC, Saraiva-Pereira ML.
Source
Laboratório de Identificação Genética, Centro de Pesquisa Experimental e Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, RS, Brazil. ; Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive inherited disorder caused by alterations in the survival motor neuron I (SMN1) gene. SMA patients are classified as type I-IV based on severity of symptoms and age of onset. About 95% of SMA cases are caused by the homozygous absence of SMN1 due to gene deletion or conversion into SMN2. PCR-based methods have been widely used in genetic testing for SMA. In this work, we introduce a new approach based on TaqMan(®)real-time PCR for research and diagnostic settings. DNA samples from 100 individuals with clinical signs and symptoms suggestive of SMA were analyzed. Mutant DNA samples as well as controls were confirmed by DNA sequencing. We detected 58 SMA cases (58.0%) by showing deletion of SMN1 exon 7. Considering clinical information available from 56 of them, the patient distribution was 26 (46.4%) SMA type I, 16 (28.6%) SMA type II and 14 (25.0%) SMA type III. Results generated by the new method was confirmed by PCR-RFLP and by DNA sequencing when required. In conclusion, a protocol based on real-time PCR was shown to be effective and specific for molecular analysis of SMA patients.
Master Mix Evaluation
In order to improve the productivity of the genotyping processing, we think it is important to find the efficient master mixes for the PCR which leads to save our labors.
The points to pick up the right master mix we are looking at are
1. Pre-mixed and Ready to Use
2. Available on the first cycle program, within 1 hour in total
Finally we chose and tested three Master Mixes which meet our demands as following.
a. Fast Cycling PCR (Qiagen)
b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific)
c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific)
Evaluation
a. Fast Cycling PCR (Qiagen) : Basically it was good, but poor yields for 1kb or longer amplicons found, even followed the instruction. There is not the 2-step protocol available in the instruction. One hour with 35 cycles. The most asset in this master mix is that almost of reactions work on the same melting temperature, at 60 degree C.
b. Phusion High-Fidelity PCR Master Mix (Thermo Scientific) : The 2-step protocol did not work for us. The 3-step protocol takes over 1 hour, so that it does not meet our demand.
c. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) : Basically it was as good as the Qiagen’s, and better yields for longer amplicons found than Qiagen’s. On the other hand, the 2-step protocol did not work for us as well. One hour with 35 cycles. We need to optimize the melting temperature on demands and this point is not as good as the Qiagen’s.
Three multiplex snapshot assays for SNP genotyping in candidate innate immune genes
BMC Res Notes. 2013 Feb 7;6(1):54. [Epub ahead of print]
Esteves LM, Bulhões SM, Brilhante MJ, Mota-Vieira L.
Abstract
ABSTRACT:
BACKGROUND: Innate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases. This immune response has been reported to be genetically diverse, due to polymorphisms coded by different genes. For this reason, our purpose was to develop a multiplex assay that allows the genotyping of candidate single nucleotide polymorphisms (SNPs) in innate immune genes.
FINDINGS:
We developed three multiplex PCR panels coupled with the minisequencing (SNaPshot) technique (multiplex PCR, multiplex primer extension, and capillary electrophoresis). The panels were tested in a sample set composed of 100 anonymous DNAs from healthy blood donors living in Sao Miguel Island (Azores, Portugal). Sixteen relevant SNPs among nine genes of the innate immune system — IL1alpha, IL1beta, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14 — were genotyped and validated by direct sequencing, with the exception of one that was undetected by minisequencing. We suggest that these panels can be used in future studies for detection of risk gene variants in several populations and/or diseases.
CONCLUSIONS:
In summary, we propose a multiplex assay that is able to identify the most frequent candidate SNPs in innate immune genes, using a medium scale genotyping platform. The assays can be used to evaluate the risk gene variants in populations of various geographic origins.
Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton
J Biosci. 2013 Mar;38(1):167-72.
Rao PS, Kumar PS, Sonti RV.
Source
Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India.
Abstract
Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.
An HPV testing algorithm comprising a combination of the L1 broad-spectrum SPF10 PCR and a novel E6 high-risk Multiplex type-specific genotyping PCR
J Clin Microbiol. 2013 Jan 30. [Epub ahead of print]
van Alewijk D, Kleter B, Vent M, Delroisse JM, de Koning M, van Doorn LJ, Quint W, Colau B.
Source
DDL Diagnostic Laboratory, Visseringlaan 25, Rijswijk, The Netherlands.
Abstract
HPV epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1 based SPF(10) PCR DEIA LiPA system and a novel E6 based multiplex type-specific system (MPTS123) using Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n=860) and cervical biopsies (n=355) were tested with a focus on HPV detected by the MPTS123 assay (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, -6 and -11). Among the HPV positive samples, identification of individual HPV genotypes was compared. When all MPTS123 targeted genotypes were taken together a good overall agreement was found (κ = 0.801, 95% CI: 0.784-0.818) with identification by SPF(10) LiPA, but significantly more genotypes (P<0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV-16, -35, -39, -45, -58, and -59. An alternative type-specific assay was evaluated, based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed similar results as the expanded MPTS123 Luminex assay.These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs offers a highly accurate method for the analysis of HPV infections and diminishes the rate of false-negative results, which may be particularly useful for epidemiological and vaccine studies.
The case for genetic monitoring of mice and rats used in biomedical research
Mamm Genome. 2013 Jan 12. [Epub ahead of print]
Fahey JR, Katoh H, Malcolm R, Perez AV.
Source
Laboratory Animal Health Services Department, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME, 04609, USA, jim.fahey@jax.org.
Abstract
Currently, there is the potential to generate over 200,000 mutant mouse strains between existing mouse strains (over 24,000) and genetically modified mouse embryonic stem cells (over 209,000) that have been entered into the International Mouse Strain Resource Center (IMSR) from laboratories and repositories all over the world. The number of rat strains is also increasing exponentially. These mouse and rat mutants are a tremendous genetic resource; however, the awareness of their genetic integrity such as genetic background and genotyping of these models is not always carefully monitored. In this review, we make a case for the International Council for Laboratory Animal Science (ICLAS), which is interested in promoting and helping academic institutions develop a genetic monitoring program to bring a level of genetic quality assurance into the scientific interchange and use of mouse and rat genetically mutant models.
A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
PLoS Negl Trop Dis. 2013 Jan;7(1):e2017. doi: 10.1371/journal.pntd.0002017. Epub 2013 Jan 17.
Boubaker G, Macchiaroli N, Prada L, Cucher MA, Rosenzvit MC, Ziadinov I, Deplazes P, Saarma U, Babba H, Gottstein B, Spiliotis M.
Source
Institute of Parasitology, University of Bern, Bern, Switzerland ; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland ; University of Monastir, Faculty of Pharmacy, Department of Clinical Biology B, Laboratory of Parasitology and Mycology, Monastir, Tunisia.
Abstract
Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.
Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping
Biotechnol Lett. 2012 Dec 15. [Epub ahead of print]
Sysol JR, Kempf C, Helton MN, Dong Y, Zhu D, Sun H, Garcia JG, Machado RF, Chen J.
Source
Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA, jsysol2@uic.edu.
Abstract
Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, “crude” DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.
Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities
J Vet Med Sci. 2012 Dec 10. [Epub ahead of print]
Ayabe H, Ikeda S, Maruyama S, Shioyama S, Kikuchi M, Kawaguchi A, Yamada T, Ikeda T.
Source
Plasma Team, Production Department, CHARLES RIVER LABORATORIES JAPAN, Inc.
Abstract
We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.