scholarly journals Genetic Screening of Deficiency of Uridine Monophosphate Synthase in Holstein Freisian Crossbred Cattle

2017 ◽  
Vol 13 (02) ◽  
Author(s):  
Sherly Ignetious ◽  
Shrikant Joshi ◽  
Ranjit Aich ◽  
Shefali Macwan
Keyword(s):  
Chromosome 1 ◽  
Normal Allele ◽  
Jugular Veins ◽  
Blood Samples ◽  
Normal Individuals ◽  
Holstein Friesian ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Endonuclease Enzyme ◽  
Uridine Monophosphate

Fifty Holstein Friesian (H.F) crossbredcattle were screened for Deficiency of Uridine Monophosphate Synthase (DUMPS) using PCR-RFLP. Blood samples were collected from jugular veins in 2 ml capacity vaccutainers containing 2 mg/ml (K2 EDTA). DNA was isolated from the blood samples by using whole blood extraction kit. PCR was performed for amplification of polymorphic region of Uridine Monophosphate Synthase (UMPS) gene (108 bp) on bovine chromosome 1. The PCR products of 108 bp were digested with AvaI endonuclease enzyme. The normal allele in unaffected cattle produced three fragments of 53 bp, 36 bp and 19 bp. No animal was found carrier for UMPS gene. The genotype frequency of normal individuals and the gene frequency of normal allele were found to be one.

scholarly journals Molecular Detection and Differentiation of Theileria lestoquardi, T. ovis and T. annulata in Blood of Goats and Ticks in Kermanshah Province, Iran

2019 ◽  
Author(s):  
Mozhgan Rahmani-Varmale ◽  
Mousa Tavassoli ◽  
Bijan Esmaeilnejad
Keyword(s):  
Nested Pcr ◽  
Molecular Detection ◽  
Ixodid Ticks ◽  
Hyalomma Anatolicum ◽  
Blood Samples ◽  
Theileria Lestoquardi ◽  
Blood Smears ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Theileria Spp

Background: This study was carried out to identify Theileria spp. infections in goats and ticksin Kermanshah Prov­ince, western Iran from May–Sep 2015. Methods: For differentiation of different Theileria spp. both blood and tick samples were examined by nested PCR-RFLP. Results: Light microscopy of blood smears revealed Theileria spp. infection in 22 (5.5%), while 68 (17%) of blood samples were positive using nested PCR. Out of 68 positive samples, 85.3% (58/68) and 11.7% (8/68) were respec­tively positive for Theileria ovis and T. lestoquardi. Mixed infection was detected in 3% (2/68) cases. Overall, 420 ixodid ticks belong to seven different hard ticks species were collected from goats. Rhipicephalus turanicus 112 (26.7%), R. sanguineus 95 (22.6%), R. bursa, 91(21.7%), Hyalomma anatolicum, 55(13.1%), H. excavatum 27(6.4%), H. marginatum, 22(5.3%) and Dermacentor marginatus, 18(4.2%) were the main tick species infesting goats. The PCR products obtained from ticks were subjected to the differentiation of Theileria species. Respectively, 2 and 8 pools of H. marginatum and R. turanicus salivary glands were infected with T. ovis and T. lestoquardi. In addition, T. annulata and T. lestoquardi infection weredetected in three pools of H. anatolicum. Conclusion: This is the first report of goats and collected ticks to Theileria spp infection in Iran. The results suggest that T. ovis has a higher prevalence than T. lestoquardi. It is also postulated H. marginatum, R. turanicus and H. ana­tolicum might play an important role in the field as a vector of Theileria spp in this area.

Identification of Point Mutation in Exon 3 of Leptin Gene in Munjal Sheep

2021 ◽  
Author(s):  
Sandeep Kumar ◽  
S.P. Dahiya ◽  
Ankit Magotra ◽  
Yogesh C. Bangar ◽  
Asha Rani Garg
Keyword(s):  
Point Mutation ◽  
Small Sample Size ◽  
Genomic Region ◽  
Vital Role ◽  
Small Sample ◽  
Resource Population ◽  
Candidate Mutation ◽  
Body Development ◽  
Pcr Rflp ◽  
Pcr Products

Background: Leptin is a varied hormone which plays vital role in body development by regulating the balance between food intake and energy expenditure by signaling to the brain. Leptin has diverse effect on controlling appetite, energy metabolism, growth, reproduction, body composition and immunity. The present study was aimed to screen candidate point mutation (g.332G greater than A) in the targeted genomic region of leptin gene in Munjal sheep. Methods: A total of 50 Munjal sheep were selected and genomic DNA was isolated in Automated Maxell RSC DNA/ RNA purification system by using Maxwell RSC whole blood DNA kit. Reported set of primers was used to amplify 463bp fragment encompassing targeted region (exon 3) of leptin gene. PCR-RFLP was performed to genotype targeted point mutation in our resource population. PCR products were digested by Cail 1 restriction enzyme to genotype g.332G greater than A (at 332th nucleotide of exon 3 leptin gene) non-synonymous mutation (Arg to Gln). Result: All studied samples resolved into monomorphic banding pattern, revealed only AA (463bp single band bp) genotype. The absence of candidate mutation in our resource population might be due to small sample size.

scholarly journals Analysis of KatG Ser315Thr Mutation in Multidrug Resistant Mycobacterium tuberculosis and SLC11A1 Polymorphism in Multidrug Resistance Tuberculosis in Central Development Region of Nepal Using PCR-RFLP Technique: A Pilot Study

1970 ◽  
Vol 1 (1) ◽  
pp. 14-21 ◽  
Author(s):  
Raunak Shrestha ◽  
Rubin Narayan Joshi ◽  
Kriti Joshi ◽  
Bal Hari Poudel ◽  
Bhupal Govinda Shrestha
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Blood ◽  
Diagnostic Technique ◽  
Molecular Diagnostic ◽  
Multi Drug Resistance ◽  
Base Line ◽  
Blood Samples ◽  
Pcr Rflp ◽  
Development Region ◽  
Whole Blood Samples

Ser315Thr mutations in genes encoding the mycobacteria catalase-peroxidase (KatG) has been associated with the major resistance to isoniazid (INH) in Mycobacterium tuberculosis (MTB). Also G/C polymorphisms in INT4 region of the solute carrier family 11 member 1 gene (SLC11A1) and susceptibility towards tuberculosis (TB) has been demonstrated worldwide. 24 drug resistant MTB culture positive samples and 24 whole?blood samples were collected from different TB patients of Central Development Region of Nepal in 2009. A Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) assay was carried out in order to investigate Ser315Thr KatG mutation and G/C polymorphism in INT4 region. 4 (16.67%) samples out of 24 MTB culture samples demonstrated the Ser315Thr KatG mutation whereas none of the 24 whole blood samples were found to contain G/C polymorphism in INT4. Though no significant correlation could be found between INT4 polymorphism and TB susceptibility, overall scenario of Nepal cannot be drawn from this data. Molecular diagnostic technique such as PCR-RFLP can be used in a robust scale to carry out base line studies in the TB population of Nepal. Key words: Multi?drug resistance; Tuberculosis; PCR; RFLP Nepal Journal of Biotechnology. Jan. 2011, Vol. 1, No. 1 : 14-21

PCR-RFLP for detection of Fusarium graminearum genotypes with resistance to phenamacril

Plant Disease ◽  
2020 ◽  
Author(s):  
Jiao-Sheng Li ◽  
Luo-Yu Wu ◽  
Hui Zhang ◽  
Xiu-Shi Song ◽  
Jian-Xin Wang ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Conventional Method ◽  
Resistance Mutations ◽  
Head Blight ◽  
Fusarium Asiaticum ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Resistant Strains ◽  
Codon Mutation

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and Fusarium asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in vitro lab experiments. In this study, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217, and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L) or 1649 bp (for mutation of E420K) in myosin-5 gene were amplified respectively by appropriate primer pairs. Restriction enzyme KpnⅠ, TasⅠ or DraⅠ was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L and E420K, respectively. KpnⅠ digested the 841 bp PCR products of phenamacri-resistant strains with codon mutation A135T into two fragments of 256 bp and 585 bp. In contrast, KpnⅠ did not digest the PCR products of sensitive strains. TasⅠ digested the 802 bp PCR products of phenamacril-strains with codon mutation S217L into three fragments of 461 bp, 287bp and 54 bp. In contrast, TasⅠ digestion of the 802 bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515bp and 287bp. DraⅠ digested the 1649 bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 bp and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three resistant mutation genotypes of F. graminearum to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

scholarly journals FEATURES OF DIAGNOSIS OF NECROBACTERIOSIS OF COWS BY PCR-RFLP

2020 ◽  
pp. 26-37
Author(s):  
O.D. Biriukova ◽  
T.M. Suprovych ◽  
M.P. Suprovych ◽  
S.V. Laiter-Moskaliuk ◽  
I.O. Chornyi
Keyword(s):  
Molecular Genetic ◽  
Experimental Sample ◽  
Exon 2 ◽  
Blood Samples ◽  
Restriction Fragments ◽  
Black And White ◽  
Pcr Rflp ◽  
Dairy Breed ◽  
Established Technique ◽  
Gene Exon

Molecular genetic markers can detect polymorphism at the DNA level. This feature determines the possibility of their widespread use in genetics and breeding. Alleles of the BoLA-DRB3 gene (exon 2) can act as such markers if a statically significant association between the disease and the allele is established. The presence of such DNA markers in the genotype of animals makes it possible to judge the likelihood of disease in postnatal ontogenesis immediately after the birth of a heifer, based on which we can conclude about the conditions of further use of the animal in the main herd. According to the results of studying the polymorphism of the BoLA-DRB3 gene in cows of the Ukrainian black and white dairy breed resistant and susceptible to necrobacteriosis, four "informative" alleles were revealed. Two of them *03 and *22 are associated with resistance, and the other two - *16 and *23 with susceptibility to necrobacteriosis. The presence of these alleles in the genotype of the animal is determined by testing performed by PCR-RFLP. The method is time consuming, labor intensive and costly. To simplify it, the following technique is proposed. Restriction fragments of alleles *03, *16, *22 and *23 for endocluases RsaI, XhoII and HaeIII have the following DNA patterns: bbb, jbd, mba and nba. Due to the peculiarity of the restriction fragments, which is that endonuclease XhoII reveals in these alleles only one pattern b with length of 284 bp, the process of determining informative alleles can be simplified. Isolation of DNA from blood samples and amplification of a fragment of the BoLA-DRB3.2 gene with a size of 284 bp is carried out according to the established technique. Next, the restriction of the fragment by endonuclease XhoII and sampling having a pattern b. Selected samples are treated with RsaI endonuclease and only those with patterns b, j, m and n remain. The next step is to restrict the selected samples with HaeIII endonuclease and select heifers with bbb (*03) and nba (*23) genotypes. After the first restriction, blood samples without pattern b are eliminated from the experimental sample; after the second – two alleles with patterns RsaI + XhoII jb (*16) and mb (*22) are unambiguously determined, after the third – genotypes bbb and nba, which correspond to alleles *03 and *23. In total, only 75% of blood samples are typed, which reduces the material consumption, time and cost of work to identify heifers genetically susceptible (resistant) to necrobacteriosis.

scholarly journals Effect of G2548A polymorphism in the leptin gene on the BMI level in human population

2016 ◽  
pp. 5-10
Author(s):  
Kritína Candráková ◽  
Anna Trakovická
Keyword(s):  
Human Population ◽  
Genetic Relatedness ◽  
Heterozygous Genotype ◽  
Dominant Form ◽  
Snp Polymorphism ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Agarose Electrophoresis ◽  
Prevalence Of Obesity ◽  
Lep Gene

The polymorphism in leptin (LEP 2548A) seems to influence obesity among others genes. The aim of this study is to investigate the effect of the G2548A polymorphism on body mass index. We included 79 people from Slovakia with some genetic relatedness and used barrels kit to isolate the genomic DNA from an adenoblast swab- from the salivary. PCR products were amplified by pursued polymorphisms and G2548A, we restriction-analyzed them and then we identified the specific fragments describing the presence of chosen SNP polymorphism by the agarose electrophoresis, to analyze SNP polymorphism by PCR-RFLP method. The LEP gene had increased frequency of G allele (0.5506). The most common genotype occurring in the gene LEP was heterozygous genotype (AG) and the least frequent genotype in LEP was AA (0.1899). Taking the age into account the BMI is higher if the G allele occurs in the LEP gene. Moreover, if the G allele genotype was situated in dominant form, then the highest average BMI was present. According to the results we can assume that the AA genotype (LEP) has a protective effect on the prevalence of obesity compared to the other genotypes.

scholarly journals Identifikasi Keragaman Gen FTO Pada Bangsa Sapi Potong Indonesia

2019 ◽  
Vol 6 (2) ◽  
pp. 232
Author(s):  
Sutikno Sutikno ◽  
Rudy Priyanto ◽  
Cece Sumantri ◽  
Jakaria Jakaria
Keyword(s):  
Beef Cattle ◽  
Genetic Markers ◽  
Direct Sequencing ◽  
Nucleotide Position ◽  
Fto Gene ◽  
Blood Samples ◽  
Pcr Rflp ◽  
Gene Functions ◽  
Hardy Weinberg Equilibrium ◽  
Cattle Blood

ABSTRAK Gen FTO berfungsi sebagai regulasi homeostasis, deposisi lemak dan pengaturan obesitas. Penelitian ini bertujuan untuk mengidentifikasi polimorfisme SNP g.125550A>T di ekson 3 gen FTO pada bangsa sapi potong Indonesia. Sampel darah diperoleh dari 209 ekor sapi, terdiri atas sapi bali (44), madura (20), Pesisir (20), katingan (20), Peranakan ongole (PO) (22), Pasundan (20), Sumba Ongole (SO) (11), brahman (20), simental (15), dan limousin (18). Polimorfisme gen FTO dianalisis menggunakan metode PCR-RFLP (HpyCH4III) dan direct sequencing. Hasil genotiping SNP g.125550A>T adalah polimorfik (genotipe AA, AT, dan TT) pada sapi madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, dan limousin. Frekuensi alel A dan T masing-masing adalah 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 dan 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31. Nilai Ho dan He masing-masing adalah 0,60-0,14 dan 0,44-0,18 serta dalam keseimbangan Hardy-Weinberg (P>0.05). Sementara pada sapi bali bersifat monomorfik hanya bergenotipe AA. Hasil sekuensing SNP g.125550A>T ditemukan mutasi tranvesi A menjadi T pada posisi nukleotida  g.125550. Berdasarkan hasil penelitian ini, dapat disimpulkan bahwa SNP 125550A>T gen FTO beragam dan berpotensi dijadikan marka genetik untuk kualitas daging pada bangsa sapi potong Indonesia.Kata Kunci: gen FTO, PCR-RFLP, Sapi, SNP g.125550A>TABSTRACTThe FTO gene functions as regulation of homeostasis, fat deposition and regulation of obesity. This study aimed to identify the polymorphism of SNP g.125550A>T in exon 3 of FTO gene in Indonesian beef cattle. Blood samples were collected from 209 cattle, including bali (44), madura (20), pesisir (20), katingan (20), PO (22), pasundan (20), SO (11), brahman (20), simental (15), and limousin (18). Polymorphism of the FTO gene was analyzed using PCR-RFLP (HpyCH4III) and direct sequencing methods. The results of genotyping SNP g.125550A>T was polymorphic (AA, AT and TT genotypes) in madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, and limousin cattle. The frequency of A and T alleles were 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 and 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31 respectively. The values of Ho and He were 0,60-0,14 and 0,44-0,18 respectively and in Hardy-Weinberg equilibrium (P>0,05). While in Bali cattle was monomorphic (AA genotype). Results of sequencing SNP g.125550A>T of the FTO gene found a transverse mutation A to T at the nucleotide position g.125550. As a result of this study, it can be concluded that SNP 125550A>T of the FTO gene was diverse and potentially used as genetic markers for meat quality in Indonesian beef cattle.Keywords: cattle, FTO gene, PCR-RFLP, SNP g.125550A>T.

scholarly journals Genetic Polymorphism of SCD1 Gene of Holstein-Friesian Cows in Indonesia

2019 ◽  
Vol 24 (2) ◽  
pp. 56
Author(s):  
Ari Sulistyo Wulandari ◽  
HD Rahayu ◽  
SD Volkandari ◽  
N Herlina ◽  
S Anwar ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Genetic Polymorphism ◽  
Breeding Strategy ◽  
Holstein Friesian ◽  
Pcr Rflp ◽  
Hardy Weinberg Equilibrium ◽  
Molecular Selection ◽  
Friesian Cattle ◽  
Selection Of ◽  
Friesian Cows

<p class="abstrak2">Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Polymorphism of SCD1 gene in Holstein-Friesian (HF) cows could be used as a basis of molecular selection of cattle in order to increase their productivity. The aim of this study was to investigate the polymorphism of SCD1 gene of Holstein-Friesian cows in Indonesia. A total of 162 blood samples of HF cows were collected from four different locations i.e. Bogor, Sukabumi, Tasikmalaya and Enrekang districts. Genotyping of SCD1 gene used PCR-RFLP method with NcoI restriction enzyme. The result showed that three genotypes (AA, AV and VV) and two alleles (A and V) have successfully found and polymorphic. A allele was dominant in all populations (0.63) and in Hardy Weinberg Equilibrium. The highest A allele was found in Sukabumi (0.78) and the lowest was in Bogor (0.55). Heterozigosity observed and expected reached 0.471 and 0.470, respectively. In conclusion, genetic polymorphism was found in all population with dominant of A allele. This finding can be used as a early genetic information of Holstein-Friesian cattle in Indonesia and to build breeding strategy for improving of productivity especially improving of healthy fat milk. </p><p class="abstrak2"><span><br /></span></p>

scholarly journals Identification of restriction enzyme in the FSHR gene of indonesian local cattle

2021 ◽  
Vol 888 (1) ◽  
pp. 012024
Author(s):  
P W Prihandini ◽  
A Primasari ◽  
M Luthfi ◽  
D Pamungkas ◽  
A P Z N L Sari ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Restriction Site ◽  
Follicle Stimulating Hormone ◽  
Restriction Enzymes ◽  
Future Studies ◽  
Fshr Gene ◽  
Pcr Rflp ◽  
Mapping Analysis ◽  
Pcr Products ◽  
Enzyme Mapping

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

scholarly journals Detection of the CD18 gene mutation as a marker of BLAD genetic disorder of Holstein-Friesian cattle in West Java

2020 ◽  
Vol 18 (2) ◽  
pp. 116
Author(s):  
Mukh Fajar Nasrulloh ◽  
Ari Sulistyo Wulandari ◽  
Indriawati Indriawati ◽  
Endang Tri Margawati ◽  
Slamet Diah Volkandari
Keyword(s):  
Gene Mutation ◽  
Leukocyte Adhesion ◽  
Genetic Disorder ◽  
Rflp Analysis ◽  
West Java ◽  
Holstein Friesian ◽  
Pcr Rflp ◽  
Dairy Cattle Breeding ◽  
Disorder Identification ◽  
Cluster Of Differentiation

<p class="MDPI17abstract"><strong>Objective: </strong>Bovine Leukocyte Adhesion Deficiency (BLAD) is a genetic disorder in Holstein-Friesian (HF) cattle that have a significant economic impact on dairy cattle breeding. Mutation c.383A&gt;G in Cluster of Differentiation Molecule 18 (CD18) gene was known as related to BLAD. This study aimed to detect the CD18 gene mutation that causes BLAD of HF cattle in the West Java Province.</p><p class="MDPI17abstract"><strong>Methods: </strong>The genomic DNA was extracted from 88 blood samples of HF cattle from Cibungbulang-Bogor (n=34), Ciampea-Bogor (n=31), and Sukabumi (n=23) by the GB300 DNA extraction kit, Geneaid<sup>TM</sup>. The CD18 gene mutation was detected by PCR-RFLP analysis using the <em>Taq</em>I restriction endonuclease.<strong></strong></p><p class="MDPI17abstract"><strong>Results: </strong>All samples in this study produced two fragments DNA, i.e., 359 bp and 260 bp with the monomorphic homozygote genotype (BB). HF cattle with BB genotype indicated as normal cattle and did not carry BLAD.<strong></strong></p><strong>Conclusions: </strong>All samples in the three populations of West Java were free of BLAD genetic disorder. Identification of BLAD on HF cattle in other places, including bulls in the Center of Semen Production in Indonesia was needed to prevent this genetic disorder.

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