Development of Tetra-primer Amplification Refractory Mutation System (ARMS) PCR for Detection of CHRNA3 rs8040868

BACKGROUND: Single nucleotide variations (SNV) have been mapped to be associated with several human conditions and diseases. To validate the association between SNV to certain human traits or diseases, a large number of subjects must be included. Thus, in need of a fast, relatively economic, and reliable genotyping method. This can be achieved through the use of tetra-primer amplification refractory mutation system polymerase chain reaction (Tetra-primer ARMS PCR). This study reports strategy to develop Tetra-primer ARMS PCR-based genotyping of CHRNA3 rs8040868.METHODS: The optimization of Tetra-primer ARMS PCR was done through these steps: identification of gene sequence and position of single mutation; designing outer and inner PCR primers; amplification of target gene fragments through PCR by using outer primer; confirming genotype of the PCR product by using sequencing; determining an optimum ratio of outer and inner primer; and determining optimum annealing temperature and cycles for the PCR program. The PCR products were run in 2% gel agarose electrophoresis and visualized under UV illumination.RESULTS: Outer and inner primer ratio of 1:3 with annealing temperature of 64.4°C and 40x cycles was found to be the most optimum condition. Tetra-primer ARMS PCR was able to confirm the results of the DNA sequence of 2 samples, confirming wild-type variants (TT allele) and the heterozygous mutant (CT allele).CONCLUSION: Tetra-primer ARMS PCR was able to genotype rs8040868 of the CHRNA3 gene.KEYWORDS: tetra-primer ARMS PCR, CHRNA3, rs8040868, genotyping

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A robust strategy for screening and confirmation of familial defective apolipoprotein B-100

Clinical Chemistry ◽

10.1093/clinchem/39.1.118 ◽

1993 ◽

Vol 39 (1) ◽

pp. 118-121 ◽

Cited By ~ 16

Author(s):

C D Mamotte ◽

F M van Bockxmeer

Keyword(s):

Apolipoprotein B ◽

Annealing Temperature ◽

Restriction Site ◽

Pcr Primers ◽

Test Methods ◽

Taq Polymerase ◽

Pcr Product ◽

Restriction Sites ◽

Pcr Products ◽

Polymerase Chain

Abstract The diagnosis of familial defective apolipoprotein B-100 (FDB) has been facilitated by the use of mutagenic polymerase chain reaction (PCR) primers to introduce restriction sites at the FDB gene locus. We describe a two-test strategy for diagnosing FDB that overcomes the potential for error in single-test methods based on such techniques. We introduce an Sau96I restriction site for PCR products of the normal apolipoprotein B allele. Incomplete digestion of the PCR product with Sau96I suggests an FDB heterozygote, although a false-positive result due to nonideal digestion conditions remains a possibility. In such cases we use a second test that introduces an ScaI restriction site in PCR products of the FDB allele. The diagnosis is confirmed if half of this product can be digested with ScaI. Both tests use 0.25 units of Taq polymerase and are robust with respect to annealing temperature (31-58 degrees C) and to Mg2+ concentration (1.0-3.2 mmol/L).

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A novel one-step amplification refractory mutation system PCR (ARMS-PCR) for differentiation of canine parvovirus-2 variants

Virus Genes ◽

10.1007/s11262-021-01861-w ◽

2021 ◽

Author(s):

Anusha Dema ◽

Vishweshwar Kumar Ganji ◽

Narasimha Reddy Yella ◽

Kalyani Putty

Keyword(s):

Canine Parvovirus ◽

Amplification Refractory Mutation System ◽

Arms Pcr ◽

Mutation System ◽

One Step

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Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v5i1.17020 ◽

2017 ◽

Vol 5 (1) ◽

pp. 81-85 ◽

Cited By ~ 1

Author(s):

Hemanta Kumari Chaudhary ◽

Mitesh Shrestha ◽

Prakash Chaudhary ◽

Bal Hari Poudel

Keyword(s):

Mycobacterium Tuberculosis ◽

Rpob Gene ◽

Amplification Refractory Mutation System ◽

Chain Reaction ◽

Arms Pcr ◽

Mutation System ◽

Mdr Tb ◽

Total Dna ◽

Polymerase Chain ◽

Rifampin Resistance

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

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Glucose 6 phosphate dehydrogenase deficiency and hemoglobinopathy in South Western Region Nepal: a boon or burden

BMC Research Notes ◽

10.1186/s13104-019-4762-6 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Narayan Gautam ◽

Bhagwati Gaire ◽

Trishna Manandhar ◽

Bishnu P. Marasini ◽

Niranjan Parajuli ◽

...

Keyword(s):

Sickle Cell ◽

G6pd Deficiency ◽

Sickle Cell Trait ◽

Amplification Refractory Mutation System ◽

Cellulose Acetate Electrophoresis ◽

Positive Trait ◽

Arms Pcr ◽

Phenotypic Method ◽

Mutation System ◽

Glucose 6 Phosphate Dehydrogenase

Abstract Objectives The study was carried out to optimize the phenotypic method to characterize the sickle cell trait (SCT), sickle cell anemia (SCA), and β-thalassemia (β-TT) suspected sample from tharu community of South Western province-5, Nepal. SCT and SCA were further evaluated by genotypic method employing amplification refractory mutation system (ARMS PCR). Moreover, Glucose 6 phosphate dehydrogenase (G6PD) was estimated in those hemoglobinopathy to observe its prevalence. The accurate and reliable method can play an important role in reduction of morbidity and mortality rate. Results The 100 suspected cases were subjected to phenotypic method adopting cellulose acetate electrophoresis and genotypic method using ARMS PCR which portraits (5%) SCA positive test showing HBS/HBS, (38%) SCT positive trait HBA/HBS and (36%) cases normal HBA/HBA. β-TT (21%) cases were confirmed by electropherogram. G6PD deficiency was observed in (40%) of SCA, (18.4%) of SCT, (4.8%) of β-TT and (2.8%) in normal cases. Increased G6PD were developed only in SCT (5.3%) and β-TT (4.8%). The study highlighted sickle cell disorder (SCD) and β-TT as the most common hemoglobinopathy coexisting with G6PD deficiency. Though hemoglobinopathy sometime could be protective in malaria but G6PD deficiency can cause massive hemolysis which may exacerbate the condition.

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A homogeneous method for genotyping with fluorescence polarization

Clinical Chemistry ◽

10.1093/clinchem/43.8.1336 ◽

1997 ◽

Vol 43 (8) ◽

pp. 1336-1341 ◽

Cited By ~ 23

Author(s):

Neil J Gibson ◽

Helen L Gillard ◽

David Whitcombe ◽

Richard M Ferrie ◽

Clive R Newton ◽

...

Keyword(s):

Fluorescence Polarization ◽

Simultaneous Detection ◽

Fluorescein Isothiocyanate ◽

Assay Method ◽

Amplification Refractory Mutation System ◽

Mutation System ◽

Pcr Products ◽

Homogeneous Method ◽

Rhodamine Dye ◽

Reaction Vessels

Abstract We combined the amplification refractory mutation system (ARMS™) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for ΔF508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.

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Multiplex amplification refractory mutation system PCR (ARMS-PCR) provides convenient method for differentiation of canine parvovirus vaccine and field strains

VirusDisease ◽

10.1007/s13337-018-0491-z ◽

2018 ◽

Vol 29 (4) ◽

pp. 565-568 ◽

Cited By ~ 1

Author(s):

Vikas Gupta ◽

Vishal Chander ◽

Soumendu Chakravarti ◽

Gaurav Kumar Sharma ◽

Javed Ahmed Malla ◽

...

Keyword(s):

Convenient Method ◽

Canine Parvovirus ◽

Amplification Refractory Mutation System ◽

Arms Pcr ◽

Multiplex Amplification ◽

Mutation System

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Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus

Infection Genetics and Evolution ◽

10.1016/j.meegid.2016.10.024 ◽

2016 ◽

Vol 46 ◽

pp. 59-64 ◽

Cited By ~ 6

Author(s):

Vishal Chander ◽

Soumendu Chakravarti ◽

Vikas Gupta ◽

Sukdeb Nandi ◽

Mithilesh Singh ◽

...

Keyword(s):

Canine Parvovirus ◽

Amplification Refractory Mutation System ◽

Arms Pcr ◽

Multiplex Amplification ◽

Mutation System

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Glucose 6 Phosphate Dehydrogenase Deficiency and Hemoglobinopathy in South Western Region Nepal: A Boon or Burden

10.21203/rs.2.14693/v1 ◽

2019 ◽

Author(s):

Narayan Gautam ◽

Bhagwati Gaire ◽

Trishna Manandhar ◽

Bishnu P Marasini ◽

Niranjan Parajuli ◽

...

Keyword(s):

Sickle Cell ◽

G6pd Deficiency ◽

Sickle Cell Trait ◽

Amplification Refractory Mutation System ◽

Cellulose Acetate Electrophoresis ◽

Positive Trait ◽

Arms Pcr ◽

Phenotypic Method ◽

Mutation System ◽

Glucose 6 Phosphate Dehydrogenase

Abstract Objectives: The study was carried out to optimize the phenotypic method to characterize the sickle cell trait (SCT), sickle cell anaemia (SCA) and β-thalassemia (β-TT) suspected sample from tharu community of South Western province-5, Nepal. SCT and SCA were further evaluated by genotypic method employing amplification refractory mutation system (ARMS PCR). Moreover, Glucose 6 Phosphate Dehydrogenase (G6PD) was estimated in those hemoglobinopathy to observe its prevalence. The accurate and reliable method can play an important role in reduction of morbidity and mortality rate. Results: The 100 suspected cases were subjected to phenotypic method adopting cellulose acetate electrophoresis and genotypic metod using ARMS PCR which portraits (5%) SCA positive test showing HBS/HBS, (38%) SCT positive trait HBA/HBS and (36%) cases normal HBA/HBA. β-TT (21%) cases were confirmed by electropherogram. G6PD deficiency was observed in (40%) of SCA, (18.4%) of SCT, (4.8%) of β-TT and (2.8%) in normal cases. Increased G6PD were developed only in SCT (5.3 %) and β-TT (4.8%). The study highlighted sickle cell disorder (SCD) and β-TT as the most common hemoglobinopathy coexisting with G6PD deficiency. Though hemoglobinopathy sometime could be protective in malaria but G6PD deficiency can cause massive hemolysis which may exacerbate the condition.

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