scholarly journals Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation

1996 ◽  
Vol 42 (9) ◽  
pp. 1391-1397 ◽  
Author(s):  
T Pastinen ◽  
J Partanen ◽  
A C Syvänen
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Dna Templates ◽  
Single Nucleotide ◽  
Large Numbers ◽  
Dna Sequence Variation ◽  
Hla Dqa1 ◽  
Efficient Screening

Abstract We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

scholarly journals Multiplex Genotyping of Cytochrome P450 Single-Nucleotide Polymorphisms by Use of MALDI-TOF Mass Spectrometry

2007 ◽  
Vol 53 (5) ◽  
pp. 933-939 ◽  
Author(s):  
Ashish Misra ◽  
Jun-Yan Hong ◽  
Sobin Kim
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Affinity Purification ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Base Extension

Abstract Background: Polymorphisms in cytochrome P450 (CYP450) genes contribute to interindividual differences in the metabolism of xenobiotic chemicals, including the vast majority of drugs, and may lead to toxicity and adverse drug reactions. Studies on these polymorphisms in research and diagnostic settings typically involve large-scale genotyping and hence require high-throughput assays. Methods: We used the previously developed solid-phase capture–single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes. Results: We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13. Conclusions: The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.

scholarly journals Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms

1994 ◽  
Vol 22 (20) ◽  
pp. 4167-4175 ◽  
Author(s):  
Theo T. Nikiforov ◽  
Robert B. Rendie ◽  
Philip Goelet ◽  
Yu-Hui Rogers ◽  
Michael L. Kotewicz ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Phase Method ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Solid Phase Method

Single nucleotide polymorphism (SNP) markers for benzimidazole resistance in veterinary nematodes

Parasitology ◽  
2007 ◽  
Vol 134 (8) ◽  
pp. 1077-1086 ◽  
Author(s):  
G. VON SAMSON-HIMMELSTJERNA ◽  
W. J. BLACKHALL ◽  
J. S. McCARTHY ◽  
P. J. SKUCE
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Building Blocks ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Principal Mechanism ◽  
Tubulin Isotype ◽  
Veterinary Importance

SUMMARYResistance to the benzimidazole class of anthelmintics in nematodes of veterinary importance has a long history. Research into the mechanisms responsible for this resistance is subsequently at a more advanced stage than for other classes of anthelmintics. The principal mechanism of resistance to benzimidazoles is likely to involve changes in the primary structure of β-tubulins, the building blocks of microtubules. Specifically, point mutations in the β-tubulin isotype 1 gene leading to amino acid substitutions in codons 167, 198, and 200 of the protein have been associated with resistance in nematodes. These single nucleotide polymorphisms offer a means of detecting the presence of resistance within populations. In this mini-review, we focus on the prevalence and importance of these polymorphisms in three groups of nematodes: trichostrongylids, cyathostomins, and hookworms. A brief overview of existing strategies for genotyping single nucleotide polymorphisms is also presented. The CARS initiative hopes to exploit these known polymorphisms to further our understanding of the phenomenon of BZ resistance.

Allele-specific, nested, one tube PCR: application to Pfmdr1 polymorphisms in Plasmodium falciparum

Parasitology ◽  
1999 ◽  
Vol 119 (1) ◽  
pp. 1-6 ◽  
Author(s):  
I. S. ADAGU ◽  
D. C. WARHURST
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Single Nucleotide Polymorphisms ◽  
Resistance Gene ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Multidrug Resistance Gene ◽  
Single Nucleotide ◽  
Reliable Detection ◽  
Allele Specific

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P. falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

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