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.

Single nucleotide polymorphisms of multidrug resistance gene 1 (MDR1) and risk of chronic myeloid leukemia

Tumor Biology ◽  
2014 ◽  
Vol 35 (11) ◽  
pp. 10969-10975 ◽  
Author(s):  
Kassogue Yaya ◽  
Dehbi Hind ◽  
Quachouh Meryem ◽  
Quessar Asma ◽  
Benchekroun Said ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Chronic Myeloid Leukemia ◽  
Single Nucleotide Polymorphisms ◽  
Resistance Gene ◽  
Myeloid Leukemia ◽  
Nucleotide Polymorphisms ◽  
Multidrug Resistance Gene ◽  
Single Nucleotide

Associations between varied susceptibilities of PfATP4 inhibitors and genotypes in Ugandan Plasmodium falciparum isolates

2021 ◽  
Author(s):  
Oriana Kreutzfeld ◽  
Stephanie A. Rasmussen ◽  
Aarti A. Ramanathan ◽  
Patrick K. Tumwebaze ◽  
Oswald Byaruhanga ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Single Nucleotide Polymorphisms ◽  
Ex Vivo ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Field Isolates ◽  
Frequent Mutations ◽  
Mixed Field ◽  
P Type

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda from 2016-2019. Median IC 50 s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many non-synonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%) and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92 and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

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 Single nucleotide polymorphisms in Plasmodium falciparum V type H+ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

2014 ◽  
Vol 24 ◽  
pp. 111-115 ◽  
Author(s):  
Irina Tatiana Jovel ◽  
Pedro Eduardo Ferreira ◽  
Maria Isabel Veiga ◽  
Maja Malmberg ◽  
Andreas Mårtensson ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

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.

Sign in / Sign up

Export Citation Format

Share Document