scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
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 ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
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 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.

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

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.

Validation study of direct sequencing and PCR-invader for detecting EGFR mutations in non-small cell lung cancer (NSCLC)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8094-8094
Author(s):  
Y. Naito ◽  
K. Goto ◽  
H. Kenmotsu ◽  
Y. Nishiwaki ◽  
K. Kubota ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
The Other ◽  
Egfr Mutations ◽  
Wild Type ◽  
Highly Sensitive ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 19 Deletion ◽  
Exon 19

8094 Background: EGFR mutation is both a predictive and prognostic factor for NSCLC treated with EGFR-TKI. Although new, highly sensitive methods for detecting EGFR mutations are currently available, these methods have not been validated. Methods: To validate direct sequencing and PCR-invader for detecting EGFR mutation, we analyzed 124 NSCLC by both methods concomitantly. Tumor tissues were obtained by surgical resection. Formalin-fixed paraffin-embedded specimens were prepared to analyze EGFR mutation. In direct sequencing, Exon 18, 19, and 21 of the EGFR gene were amplified, and PCR amplification products were sequenced directly (Mitsubishi Chemical Medience Corporation). PCR-invader was performed using the invasive cleavage of probe oligonucleotides to detect 10 mutations including Exon 18, 19, 20, 21 (BML incorporation). Results: EGFR mutations were detected in 51 patients (41%) by direct sequencing and 56 (45%) by PCR-invader. Discordance between two methods was found in 12 patients (10%). Exon 19 deletion was detected in 18 and 22 patients respectively. Exon 21 L858R was detected in 30 and 32 patients respectively. Each mutation in exon 19 deletions or L858R detected by direct sequencing could also be identified by PCR-invader. Overall 45 mutations were concordant by both methods. In two patients who received gefitinib, one patient with wild type by both methods did not respond to gefitinib. On the other hand, the other patient expressing Exon 19 deletion by PCR-invader but regarded as wild type by direct sequencing responded to gefitinib monotherapy. Conclusions: Discrepancy between two methods for detecting EGFR mutation was demonstrated and PCR-invader seems to be more sensitive. Further investigation including other highly sensitive methods is currently underway. No significant financial relationships to disclose.

scholarly journals Evaluation of PCR in Detection of Mycobacterium tuberculosis from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays

1998 ◽  
Vol 36 (6) ◽  
pp. 1512-1517 ◽  
Author(s):  
Giulia Marchetti ◽  
Andrea Gori ◽  
Lidia Catozzi ◽  
Luca Vago ◽  
Manuela Nebuloni ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
False Negative ◽  
Pcr Amplification ◽  
Positive Culture ◽  
Single Copy ◽  
High Rate ◽  
Antigen Gene ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of themtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 μg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

scholarly journals A Missense Mutation rs781536408 (c.2395G>A) of TYK2 Affects Splicing and Causes Skipping of Exon18 in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Suqing Chen ◽  
Peilin Wu ◽  
Bin Wu ◽  
Chenye Lin ◽  
Junhong Chen ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Disease Onset ◽  
Hek293t Cell ◽  
Pcr Amplification ◽  
Wild Type ◽  
Mutant Type ◽  
Whole Exome ◽  
Construction Strategies

TYK2 variants can impact disease onset or progression. In our previous study, we identified abnormal splicing that happened near rs781536408 in the TYK2 gene. The purpose of this research was to examine the effect of the mutation on alternative splicing in vivo and in vitro. Whole exome sequencing was performed to identify the mutations followed by bidirectional Sanger sequencing. Then the minigene analysis was carried out based on HeLa and HEK293T cell lines. The results showed that rs781536408 (c.2395G>A, p.G799R) was homozygous in the patient, but heterozygous in parents. PCR amplification confirmed the abnormal splicing in the somatic cells of the patients, but not in the parents. Sanger sequencing results showed that there was a skipping of exon18 near the mutation. For minigene analysis, there was no difference between the wild-type and the mutant type in the two minigene construction strategies, indicating that mutation c.2395G>A had no effect on splicing in vitro. Combining the results of in vivo, we speculated that the effect of the mutation on splicing was not absolute, but rather in degree.

scholarly journals An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 794-799 ◽  
Author(s):  
K. B. Duttweiler ◽  
G. Y. Sun ◽  
J. C. Batzer ◽  
T. C. Harrington ◽  
M. L. Gleason
Keyword(s):  
Agar Plate ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Its Region ◽  
Morphological Description ◽  
Sooty Blotch ◽  
Pcr Rflp ◽  
Sooty Blotch And Flyspeck ◽  
Rflp Assay

A restriction fragment length polymorphism (RFLP)-based technique was developed to identify members of the sooty blotch and flyspeck (SBFS) disease complex on apple because these fungi are difficult to identify using agar-plate isolation and morphological description. The method includes polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using a fungal-specific forward primer (ITS1-F) and an SBFS-specific reverse primer (Myc1-R), followed by digestion of the PCR product by the HaeIII restriction enzyme. When applied to previously identified isolates of 24 SBFS-causing species in nine genera, the PCR-RFLP assay produced 14 unique banding patterns. Different genera never shared the same RFLP pattern. To evaluate performance in vivo, the technique was applied to DNA extracted directly from SBFS colonies on apple fruit from three Iowa orchards. The primers amplified the rDNA of only SBFS fungi, with the exception of a Cladosporium sp.; however, its RFLP banding pattern was distinct from those of SBFS fungi. The majority (60%) of SBFS colonies in the in vivo trial were identified to genus by RFLP analysis. The PCR-RFLP assay greatly streamlined the identification process by minimizing the need for culturing, indicating its value as a tool for field studies of the SBFS complex.

scholarly journals Identifikasi Mutasi FecX Pada Gen BMP15 dan Pengaruhnya Terhadap Sifat Prolifik pada Kambing Lokal di Kabupaten Lombok Barat

2019 ◽  
Vol 1 (1) ◽  
pp. 1
Author(s):  
Rahmat Agus Hidayat ◽  
Sulaiman Ngongu Depamede ◽  
Maskur Maskur
Keyword(s):  
Litter Size ◽  
Mutant Allele ◽  
Secondary Data ◽  
Wild Type ◽  
Banding Patterns ◽  
Average Litter Size ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Visually Based ◽  
Its Polymorphism

The aims of this study were to identify the mutations of FecX gene in the local goats and to analyze its polymorphism as well as its influence on the prolific nature of the local goats in West Lombok Regency, Indonesia. The study was conducted in the Immunobiology Laboratory of Mataram University, using DNA isolated from 100 blood samples of local female goats which have given birth of once to three times. The methods used were PCR-RFLP method and the PCR products were digested with HinfI restriction enzyme (G|ANTC) then analyzed visually based on DNA banding patterns on 2% agarose gels. The frequency of allele and genotype obtained, were then analyzed through a comparison with the secondary data of litter size obtained from the local goat keepers information. The results showed that the gene mutation of FecXG produced two alleles: "wild-type" (+) sized of 110 bp and 31 bp, and the mutant allele (G) of 141 bp with the allele frequency of 0,965 and 0,035 respectively. Combinations of alleles in the gene BMP15 produced two genotypes, namely (a) genotype ++ (110 bp/110 bp) with a frequency of 0.93, with the average litter size of 1.59 ± 0.319, and (b) genotype G + (141bp/110 bp), with a frequency of 0.07 and with the average litter size of 1.65 ± 0.202. The results of this study indicated that mutation occurred in BMP15 gene, i.e. FecXG gene, the gene responsible for the prolificacy of animals studied. Furthermore there was a correlation between polymorphism of FecXG gene and the prolific nature of the local goats, which was predicted to lead the divergence in litter size of each local goat genotype  

scholarly journals Polymorphisms of TLR4 Asp299Gly and TNF-α -308G/A in Leptospirosis

2016 ◽  
Vol 2 (1) ◽  
pp. 17
Author(s):  
Nur Farhanah ◽  
Muhammad Hussein Gasem ◽  
Sultana MH Faradz
Keyword(s):  
Mutant Allele ◽  
Fragment Length Polymorphism ◽  
Wild Type ◽  
Chain Reaction ◽  
Healthy Group ◽  
Tnf Α ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Severe Leptospirosis ◽  
Increased Susceptibility

AbstractBackground : TLR4 Asp299Gly and TNF-α -308G/A polymorphisms have been shown to be associated with increased susceptibility and severity of infection. TLR4 Asp299Gly polymorphism could affect the host’s ability to respond to leptospira sp. TNF-α -308G/A polymorphism, is associated with the high producer of TNF-α.Methods : Total of 36 leptospirosis patients (IgM anti leptospira and MAT positive) and healthy individual with the equal number were included. The polymorphisms were determined  by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using site spesific restriction enzyme.Results : Distribution of  homozygous wild-type TLR4 Asp299Gly polymorphism was higher in both of groups ( 94.5:97.2%.) and homozygous mutant allele was absent. There was not significantly difference of  TLR4 Asp299Gly in leptospirosis patients and healthy group ( ρ=1.00; OR 0.5; 95%CI, 0.04-5.6) and between mild and severe leptospirosis (ρ=0.54; OR 1.54 ; 95% CI, 1.20-1.98). The presence of homozygous wild-type TNF-α -308G/A polymorphism was higher between leptospirosis patients and healthy group (100:94.5%) andhomozygous mutant allele was not found in both of the groups. No significantly different of TNF-α -308G>A polymorphism between leptospirosis patient and healthy group (ρ=0.49).Conclusions : In this study, the polymorphisms of TLR4 Asp299Gly and TNF-α -308G/A were not associated with the susceptibility and severity of leptospirosis. Keywords : Leptospirosis, TLR4 Asp299Gly polymorphism, TNF-α -308G/A polymorphism

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

BioTechniques ◽  
1996 ◽  
Vol 21 (6) ◽  
pp. 1055-1060 ◽  
Author(s):  
Joanne S. Finch ◽  
G. Tim Bowden
Keyword(s):  
Copy Number ◽  
Wild Type ◽  
Pcr Rflp ◽  
Rflp Assay
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