scholarly journals Fingerprinting of fecB gene in five Egyptian sheep breeds

2009 ◽  
Vol 25 (3-4) ◽  
pp. 205-212 ◽  
Author(s):  
Hanafy el ◽  
Saadani el
Keyword(s):  
Point Mutation ◽  
Restriction Enzyme ◽  
Base Pair ◽  
Genomic Dna ◽  
Restriction Site ◽  
Wild Type ◽  
Specific Primer ◽  
Sheep Breeds ◽  
Pcr Rflp ◽  
Pcr Products

Recently, many aspects of FecB gene, including reproductive endocrinology, organs development and body mass have been studied. FecB has an additive effect on litter size and ovulation. The present investigation was carried out to study polymorphism by forced PCR-RFLP of FecB gene in five Egyptian local sheep breeds and its comparison with other foreign sheep breeds. Genomic DNA was isolated from a total of 100 animals of Egyptian sheep breeds namely Rahmani, Ossimi, Awassi, Barki and Awassi x Barki crossbred. Forced PCR of the FecB gene 190 base pair (bp) was amplified using specific primer designed to introduc a point mutation in the resulting PCR products with FecB carrier sheep containing an AvaII restriction site (G|GACC), whereas products from noncarriers lacked (of) this site. Digestion of FecB gene 190 base pair with AvaII restriction enzyme resulted in non carrier 190 bp band (wild type) in all the animals belonging to the five Egyptian breeds studied revealing absence of this restriction site in those five breeds. .

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.

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 Two methods for genotyping a 4-base deletion in the canine ABCB1 gene

2019 ◽  
Vol 31 (6) ◽  
pp. 889-892
Author(s):  
Carolina A. Silvestro ◽  
Liliana A. Soria ◽  
Adriana Conte ◽  
Graciela Marrube
Keyword(s):  
Molecular Detection ◽  
High Resolution Melting ◽  
Fragment Length Polymorphism ◽  
Genetic Disorders ◽  
Chemotherapeutic Agents ◽  
Specific Method ◽  
Wild Type ◽  
Abcb1 Gene ◽  
Pcr Rflp ◽  
Pcr Products

A 4-bp deletion (c.230_233delATAG) of the ABCB1 gene, frequently found in various dog breeds, results in intolerance to certain drugs routinely used in veterinary medicine, including many chemotherapeutic agents and macrocyclic lactones. The use of rapid and reliable genetic testing is fundamental for early detection of the mutation and prevention of undesirable toxicoses. We developed and compared 2 genotyping tests: PCR–high-resolution melting (PCR-HRM) and PCR–restriction-fragment length polymorphism (PCR-RFLP) to identify the 4-bp deletion in the ABCB1 gene of canine breeds. Amplified PCR products were sequenced in order to confirm different genotypes. Both techniques were efficient in discriminating homozygous wild-type, homozygous mutated, and heterozygous ABCB1 genotypes, and proved to be reproducible and economical methods. The HRM analysis, a sensitive and specific method for the molecular detection of genetic disorders, does not require labeled probes, processing, or separations after PCR.

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 412-415 ◽  
Author(s):  
Zhong-Nan Yang ◽  
T Erik Mirkov
Keyword(s):  
Restriction Enzyme ◽  
Genomic Dna ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Anchored Pcr ◽  
Pcr Products ◽  
Left And Right ◽  
The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

scholarly journals Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human health.

1970 ◽  
Vol 60 (4) ◽  
Author(s):  
Martina Miluchová ◽  
Michal Gábor ◽  
Anna Trakovická
Keyword(s):  
Diabetes Mellitus ◽  
Genomic Dna ◽  
Total Population ◽  
Human Consumption ◽  
Effective Number ◽  
Immune Systems ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Beta Casein ◽  
Commercial Kit

The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta casein can result in the production of bioactive beta casomorphin-7 (BCM-7); this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit, and used in order to estimate beta-casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37 animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2-0.5405 were the most frequent, while homozygotes A1A1-0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7, and thus is safe for human consumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds to the effective number of alleles (1.7071).

scholarly journals PCR-based Single-strand Conformation Polymorphism (SSCP) Analysis to Clone Nine Aquaporin Genes in Cucumber

2002 ◽  
Vol 127 (6) ◽  
pp. 925-930 ◽  
Author(s):  
Jiahua Xie ◽  
Todd C. Wehner ◽  
Mark A. Conkling
Keyword(s):  
Dna Sequence ◽  
Restriction Enzyme ◽  
Genomic Dna ◽  
Sequence Variation ◽  
Single Strand ◽  
Sscp Analysis ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Pcr Products ◽  
Dna Pcr

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.

Detection of polymorphism of Calgranulin A gene in Indian Murrah Buffalo

2015 ◽  
Author(s):  
Sourabh Sulabh ◽  
Archana Verma ◽  
I. D. Gupta ◽  
S. Rajesh Kumar
Keyword(s):  
Immune System ◽  
Genetic Variants ◽  
Genomic Dna ◽  
Annealing Temperature ◽  
Rflp Analysis ◽  
Clinical Mastitis ◽  
Murrah Buffalo ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Calgranulin A

Calgranulin A (S100A8) gene is one of the important candidate genes, which affects the host disease resistance by enhancing the immune system. Present study was undertaken with the objectives to identify polymorphism in Calgranulin A gene and to associate identified genetic variants with the incidence of clinical mastitis in Murrah buffalo. Genomic DNA was isolated from 100 randomly selected lactating Murrah. Two sets of primers were designed to amplify targeted regions of the gene. PCR products were obtained at annealing temperature of 63.8oC and were of 449 and 489 bp for respective primer set. PCR-RFLP analysis was carried out using HinfI for contig I and AluI and MboII restriction endonucleases for contig II. Both the contigs revealed monomorphism with frequency of the only prevailing A allele as 1.00. It was not feasible to analyze association with the incidence of mastitis, as no genetic variants were observed in the animals studied.

scholarly journals Survey of mutations in prolificacy genes in Santa Ines and Morada Nova sheep

2017 ◽  
Vol 69 (4) ◽  
pp. 1047-1053
Author(s):  
G.M.L. Holanda ◽  
J.C. Oliveira ◽  
D.M.F. Silva ◽  
S.S.N. Rocha ◽  
V. Pandolfi ◽  
...  
Keyword(s):  
Restriction Site ◽  
Fragment Length Polymorphism ◽  
Nucleotide Sequencing ◽  
Chain Reaction ◽  
Specific Primers ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Santa Inês ◽  
Restriction Fragment Length

ABSTRACT Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.

scholarly journals The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene

2001 ◽  
Vol 169 (2) ◽  
pp. R1-R6 ◽  
Author(s):  
CJ Souza ◽  
C MacDougall ◽  
C MacDougall ◽  
BK Campbell ◽  
AS McNeilly ◽  
...  
Keyword(s):  
Point Mutation ◽  
Genomic Dna ◽  
Genome Mapping ◽  
Kinase Domain ◽  
Ovulation Rate ◽  
Wild Type ◽  
Coding Region ◽  
Antral Follicles ◽  
Gene Carriers

Genetic variations in ovulation rate which occur in different breeds of sheep provide useful models to explore the mechanisms regulating the development of antral follicles. The Booroola gene, an autosomal mutation that affects ovulation rate, has been known for over two decades and despite intensive research it has not yet been identified. Using resources from human genome mapping and known data about gene linkage and chromosome location in the sheep, we selected the gene encoding the Bone Morphogenetic Protein receptor (BMPR) type 1 B (ALK-6) as a candidate site for the mutation. The BMPR1B gene in the human is located at the region linked with the Booroola mutation, syntenic to chromosome 6 in the sheep. A fragment of the sheep BMPR1B gene was cloned from an ovarian cDNA and the deduced aminoacid (AA) sequence is over 98% homologous to the known mammalian sequences. cDNA and genomic DNA from 20 Booroola genotypes were screened and two point mutation were found in the kinase domain of the receptor, one at base 746 of the coding region (A in the ++ to a G in FF animals) which results in a change from a glutamine in the wild type to a arginine in the Booroola animals. Another point mutation was identified at position 1113, (C to A) but this mutation does not change the coding aminoacid. The first mutation was confirmed in genomic DNA from 10 ewes from an independent Brazilian flock which segregates the Booroola phenotype. In all instances homozygous FecB gene carrier (n=11) had only the 746 A to G mutation, non gene carriers (n=14) had only the wild type sequence and heterozygote gene carriers (n=5) had both sequences. This mutation in the subdomain 3 of the kinase domain could result in an alteration in the expression and/or phosphorylation of SMADs, resulting in the phenotype characteristic of the Booroola animals which is the 'precocious' development of a large number of small antral follicles resulting in increased ovulation rate.

scholarly journals G1 point mutation in growth differentiation factor 9 gene affects litter size in Sudanese desert sheep

2021 ◽  
Vol 14 (1) ◽  
pp. 104-112
Author(s):  
Amani Z. Abdelgadir ◽  
Lutfi M. A. Musa ◽  
Khaleel I. Jawasreh ◽  
Aubai. O. Saleem ◽  
Faisal El-Hag ◽  
...  
Keyword(s):  
Point Mutation ◽  
Litter Size ◽  
Reference Sequence ◽  
Wild Type ◽  
Mutant Type ◽  
Type Allele ◽  
Sheep Breeds ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Gdf9 Gene

Background and Aim: Sudanese desert sheep encompass different sheep breeds named according to the different Sudanese tribes that rear them such as the Dubasi, Shugor, and Watish sheep. The objectives of this study were to screen for G1 point mutation in the polymorphic growth differentiation factor 9 (GDF9) gene, investigate its association with litter size, and construct the phylogeny of the different tribal breeds that belong to the Sudanese Desert sheep tribal types. Materials and Methods: Genomic DNA was extracted from whole blood of three tribal Desert sheep breeds (Dubasi, Watish, and Shugor) using the guanidine chloride method. Polymerase chain reaction-restriction fragment length polymorphism with HhaI restriction enzyme and sequencing techniques was used for genotyping the GDF9 locus for possible mutations associated with litter size in the three desert sheep tribal types. Results: G1 mutation in GDF9 caused the replacement of Arginine by Histidine at residue 87. The wild type allele (A) had the highest frequency, whereas the mutant type allele (a) had the lowest in all the sequenced subtypes. The genotype frequencies of the wild type ewes (AA) were higher than the heterozygous (Aa) and the mutant type (aa) frequencies in the three studied desert sheep types. No significant differences were found in the allele frequency between the three tribal types. Litter size was significantly influenced by the genotypes of GDF9 gene, parities, and subtypes (p≤0.01, 0.01, and 0.05, respectively). In the Watish sheep type, heterozygous sheep in their second parity recorded the highest litter size. Sequence alignment of GDF9 gene samples with the database entry indicated that all three tribal types were similar and identical to the reference sequence. The phylogenetic tree revealed that Shugor is the common ancestor of the studied types and Watish is more closely related to Shugor than Dubasi. This result mi ght partly explain the lower reproductive performance of Dubasi compared to Watish and Shugor. Conclusion: The presence of one copy of GDF9 gene increased litter size in the studied Sudanese Desert sheep. This locus may be used as a biomarker for litter size improvement through genotypic selection and allele or gene introgression.

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