Biochip-based approach for comprehensive pharmacogenetic testing

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Anna Yu. Ikonnikova ◽  
Marina A. Filippova ◽  
Sergey A. Surzhikov ◽  
Victoria O. Pozhitnova ◽  
Ruslan E. Kazakov ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Genetic Factors ◽  
Wide Spectrum ◽  
Group 12 ◽  
Er Positive ◽  
One Step ◽  
Polymerase Chain ◽  
Step Polymerase Chain Reaction ◽  
Positive Breast Cancer

AbstractObjectivesIndividual sensitivity to many widely used drugs is significantly associated with genetic factors. The purpose of our work was to develop an instrument for simultaneous determination of the most clinically relevant pharmacogenetic markers to allow personalized treatment, mainly in patients with cardiovascular diseases.MethodsMultiplex one-step polymerase chain reaction (PCR) followed by hybridization on a low-density biochip was applied to interrogate 15 polymorphisms in the following eight genes: VKORC1 –1639 G>A, CYP4F2 1297 G>A, GGCX 2374 C>G, CYP2C9 *2,*3 (430 C>T, 1075 A>C), CYP2D6 *3,*4, *6, *9, *41 (2549delA, 1846 G>A, 1707delT, 2615_2617delAAG, 2988 G>A), CYP2C19 *2,*3,*17 (681 G>A, 636 G>A, −806 C>T), ABCB1 (3435 C>T), SLCO1B1 *5.ResultsTwo hundred nineteen patients with cardiovascular diseases (CVD) and 48 female patients with estrogen receptor (ER)-positive breast cancer (BC) were genotyped. Of the 219 CVD patients, 203 (92.7%) carried one or more actionable at-risk genotypes based on VKORC1/CYP2C9, CYP2C9, CYP2C19, SLCO1B1, and CYP2D6 genotypes. Among them, 67 patients (30.6%) carried one, 58 patients (26.5%) carried two, 51 patients (23.3%) carried three, 26 patients (11.9%) carried four, and one patient (0.4%) carried five risk actionable genotypes. In the ER-positive BC group 12 patients (25%) were CYP2D6 intermediate or poor metabolizers.ConclusionsThe developed biochip is applicable for rapid and robust genotyping of patients who were taking a wide spectrum of medications to optimize drugs and dosage and avoid adverse drug reactions in cardiology, oncology, psychiatry, rheumatology and gastroenterology.

Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method

1999 ◽  
Vol 9 (3) ◽  
pp. 327-332 ◽  
Author(s):  
Gen-Fu Chen ◽  
Yong-Ming Tang ◽  
Bridgett Green ◽  
Dong-Xin Lin ◽  
F. Peter Guengerich ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gene Polymorphism ◽  
Low Frequency ◽  
Polymerase Chain Reaction Method ◽  
Chain Reaction ◽  
Reaction Method ◽  
One Step ◽  
Polymerase Chain ◽  
Cyp2a6 Gene ◽  
Step Polymerase Chain Reaction

Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene

2005 ◽  
Vol 51 (2) ◽  
pp. 85-90 ◽  
Author(s):  
Ivano de Filippis ◽  
Carlos R.S. do Nascimento ◽  
Maysa B.M. Clementino ◽  
Andrea B. Sereno ◽  
Cristina Rebelo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cerebrospinal Fluid ◽  
Neisseria Meningitidis ◽  
Rapid Detection ◽  
Chain Reaction ◽  
One Step ◽  
Polymerase Chain ◽  
Step Polymerase Chain Reaction

Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil

Plant Disease ◽  
2007 ◽  
Vol 91 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Tiesen Cao ◽  
Jalpa Tewari ◽  
Stephen E. Strelkov
Keyword(s):  
Ribosomal Rna ◽  
Plasmodiophora Brassicae ◽  
Rrna Gene ◽  
Infested Soil ◽  
Resting Spores ◽  
One Step ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Total P ◽  
Step Polymerase Chain Reaction

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.

scholarly journals A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (12) ◽  
pp. 2505
Author(s):  
Hiroki Hayashi ◽  
Tsutomu Kishi
Keyword(s):  
Dna Sequences ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Epitope Tagging ◽  
One Step ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The One ◽  
Step Polymerase Chain Reaction

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

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