PCR/RFLP Assay for Copy Number of Mutant and Wild-Type Alleles

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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WITHDRAWN: Development of an RT-PCR-RFLP assay for the detection and differentiation of wild-type and vaccine strains of classical swine fever virus

Developmental & Comparative Immunology ◽

10.1016/j.dci.2012.05.002 ◽

2012 ◽

Author(s):

Jiming Hu ◽

Hu Shan ◽

Shubai Wang ◽

Peipei Yang ◽

Ying Wang ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Wild Type ◽

Rt Pcr ◽

Pcr Rflp ◽

Rflp Assay

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PCR-based Genotyping Assays to Detect Germline APC mutations Associated with Hereditary Gastrointestinal Polyposis in Jack Russell Terriers

10.21203/rs.3.rs-94557/v1 ◽

2020 ◽

Author(s):

Kyoko Yoshizaki ◽

Akihiro Hirata ◽

Hiroyuki Matsushita ◽

Naohito Nishii ◽

Mifumi Kawabe ◽

...

Keyword(s):

Mutant Allele ◽

False Negative ◽

Disease Onset ◽

Pcr Amplification ◽

Buccal Swab ◽

Wild Type ◽

Mutation Site ◽

Gastrointestinal Polyposis ◽

Pcr Rflp ◽

Rflp Assay

Abstract Background: The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell Terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline mutations in the APC gene (c.[462A>T; 463A>T]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC mutations were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs.Result: First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC mutations creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the mutation site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the mutation was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion: In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC mutations. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5′-UTR

Molecular and Cellular Probes ◽

10.1006/mcpr.2002.0414 ◽

2002 ◽

Vol 16 (3) ◽

pp. 209-216 ◽

Cited By ~ 12

Author(s):

N Siafakas ◽

P Markoulatos ◽

G Stanway ◽

G Tzanakaki ◽

J Kourea-Kremastinou

Keyword(s):

Molecular Classification ◽

Wild Type ◽

Rt Pcr ◽

Pcr Rflp ◽

Genetic Clusters ◽

Type Strains ◽

Rflp Assay

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PCR-RFLP Detection od PAI-2 Gene Variants: Prevelence in Ethnic Groups and Disease Relationship in patients Undergoing corony Angiography

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656084 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0955-0958 ◽

Cited By ~ 8

Author(s):

Carole A Foy ◽

Peter J Grant

Keyword(s):

Gene Variants ◽

Genotype Distribution ◽

Pima Indians ◽

Significant Difference ◽

Pcr Rflp ◽

History Of ◽

Caucasian Patients ◽

Clinical Conditions ◽

Rflp Assay ◽

Coronary Atheroma

SummaryPAI-2 is a fibrinolytic inhibitor produced predominantly by monocytes. Most PAI-2 is intracellular making study in clinical conditions difficult. Abnormalities in production may be associated with inflammation and fibrinolysis at sites of tissue damage such as the atherosclerotic plaque.PAI-2 gene variants have been described: variant A consists of Asn120, Asn404 and Ser413 and variant B consists of Asp120, Lys404 and Cys413. We designed a PCR-RFLP assay using primers spanning the region containing Asn/Lys404 and Ser/Cys413. Variant B contains an Mwol restriction site. We analysed 302 Pima Indians and 286 healthy Caucasian volunteers. To investigate relationships between genotype and vascular disease we analysed 333 Caucasian patients undergoing coronary angiography.Gene variant B was more common in the Pimas than in Caucasians (p <0.0001). There was no significant difference in genotype distribution between the volunteers and patients. In the patients there was no association between genotype and either a history of MI or extent of coronary atheroma.

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A 44-kb deleted-type copy number variation is associated with decreasing complement component activity and calf mortality in Japanese Black cattle

BMC Genomics ◽

10.1186/s12864-021-07415-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Shinji Sasaki ◽

Youko Miki ◽

Takayuki Ibi ◽

Hiroyuki Wakaguri ◽

Yuichi Yoshida ◽

...

Keyword(s):

Infectious Disease ◽

Copy Number Variation ◽

Copy Number ◽

Risk Allele ◽

Wild Type ◽

Japanese Black Cattle ◽

Complement Activity ◽

Categorical Trait ◽

Number Variation ◽

Calf Mortality

Abstract Background Calf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality. Results We identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1–180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease. Conclusions This study identified a deleted-type CNV associated with the mortality of 1–180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves.

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