A new tetra-primer ARMS-PCR for genotyping bovine kappa-casein polymorphisms

Fonseca, Pablo Augusto de Souza; Rosse, Izinara Cruz; Demiranda, M; Machado, M. A.; Verneque, R.S.; Peixoto, Maria Gabriela Campolina Diniz; Carvalho, Maria Raquel Santos

doi:10.4238/2013.december.11.3

Cited by 11 publications

(16 citation statements)

References 10 publications

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“…The DNA extraction method affects the outcome of T-ARMS PCR (Medrano and de Oliveira 2014). Various authors have undertaken different approaches to overcome these difficulties and successfully generated T-ARMS genotyping (Medrano and de Oliveira 2014; Fonseca et al 2013; Ahlawat et al 2014; Singh et al 2014). …”

Section: Resultsmentioning

confidence: 99%

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle

et al. 2016

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Fast and economical means of assaying SNP’s are important in diagnostic assays, especially when a large number of animals have to be screened for a genetic disease. This study was aimed at the development of a fast and economical screening assay for bovine leukocyte adhesion deficiency (BLAD) which is an important genetic disease of cattle industry. Four primers were designed where the outer primers amplify a 354 bp amplicon of CD18 gene carrying the polymorphism responsible for BLAD. The specifically designed inner primers in conjunction with the modified reaction mixture and cyclic conditions ensured amplification of either of wild or mutated alleles. Together with outer primers, the inner primers generated typical banding pattern in agarose gel which discriminated the normal animal against the carrier. We successfully used this protocol in 200 bulls for genotyping the BLAD allele which confirmed by sequencing, showing a cent percentage concordance. With the developed assay the need for restriction digestion or use of costly equipment viz. real time PCR was eliminated. This genotyping assay ensured fast and economical genotyping and could be adopted in every laboratory with a minimum equipment requirement of thermocycler and gel documentation system.

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Section: Resultsmentioning

confidence: 99%

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle

et al. 2016

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“…The present study reported the usefulness of T-ARMS PCR genotyping to detect CVM for the first time. T-ARMS PCR-based genotyping was published for other important mutations in livestock [ 25 – 28 ], including our report on Bovine Leukocyte Adhesion Deficiency (BLAD) testing [ 13 ].…”

Section: Discussionmentioning

confidence: 99%

Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle

et al. 2021

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Background DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. Results The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. Conclusions The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

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“…Thus, it can be concluded that this method is an efficient, as well as sufficient option for detection. 25 …”

Section: Discussionmentioning

confidence: 99%

Regulation of Lipocalin-2 oncogene and its impact on gene polymorphisms on breast cancer patients in Jeddah, Saudi Arabia

Linjawi

Al-Gaithy

Sindi

et al. 2018

SMJ

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Objectives:To identify the impact of Lipocalin-2 (LCN2) gene polymorphisms on breast cancer patients in Jeddah, Saudi Arabia.Methods:It is a case control study in which blood samples of participants from Medical Reference Clinics and King Abdulaziz University Hospital in Jeddah, Saudi Arabia have been taken between 2014 and 2016. This study recruited 128 participants (50% control, 50% patients) and used Tetra-Primer amplification-refractory mutation system-polymerase chain reaction method for the detection of missense SNP (rs11556770). The study measured LCN2 plasma protein expression by enzyme-linked immunosorbent assay technique.Results:The results have shown that 100% of the genotypes were normal allele (G/G). In contrast, the plasma level of LCN2 was considerably elevated among patients as compared to control (p=0.001), and higher in invasive ductal carcinoma patients (p=0.001). The LCN2 protein expression in plasma level was significantly elevated among patients, particularly who demonstrated invasive ductal carcinoma.Conclusion:There is no significant relationship between breast cancer patients and LCN2 gene polymorphisms (rs11556770).

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A new tetra-primer ARMS-PCR for genotyping bovine kappa-casein polymorphisms

Cited by 11 publications

References 10 publications

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle

Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle

Regulation of Lipocalin-2 oncogene and its impact on gene polymorphisms on breast cancer patients in Jeddah, Saudi Arabia

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