SINGLE NUCLEOTIDE EXTENSION ASSAY FOR CHARACTERIZATION OF DGAT1 LOCUS IN MEHSANA BUFFALO

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dc.contributor.advisorJoshi, C. G.
dc.contributor.authorPATIL, RAHUL CHAITRAM
dc.date.accessioned2018-07-18T04:41:35Z
dc.date.available2018-07-18T04:41:35Z
dc.date.issued2009
dc.description.abstractIndia, home ground of water buffalo (Bubalus bubalis) has 11 recognized breeds adapted to different climatic zones. The immense importance of this species due to contribution of more than 55 per cent to the total milk production and making country as number one milk producer in the world. Tremendous variation in production traits provokes buffalo genomic research to identify genes underlying the variability of milk production traits that could be useful in effective breeding programs. Present study was carried out with enormous interest in genotyping of Diacylglycerol acyltransferase 1 (DGATl) locus of Mehsana buffalo. The DGATl gene plays crucial role in triglyceride synthesis in the mammary gland which is proved by mice lacking both copies of DGATl gene are completely devoid of milk secretion and became a functional candidate gene for lactation traits. In the present study, five SNPs of DGATl gene of Indian water buffalo (GenBank-accession number DQ886485) with nucleotide position 1179, 1195 and 3096 (intron 1), 5545 (intron 2) and 6067 (intron 3) were selected for screening 64 Mehsana buffalo samples with the help of single nucleotide extension assay. According to principle of assay, unlabeled primers are hybridized to the DNA template just adjacent to respective SNP site and primer is extended by one base by DNA polymerase with fluorescence-labeled ddNTP terminators and further separated by capillary electrophoresis. Genomic DNA samples of Mehsana buffalo were subjected to DGATl specific PCR amplification using appropriate primer pairs and PCR products of expected size were successfully amplified at annealing temperature dO^C and then electrophoresed on 2 per cent agarose along with the MassRular Low range DNA ladder. Purified PCR products were subjected to single nucleotide primer extension with respective target DNA template and optimized under thermal cycling condition of armealing at 60°C and extension at 65°C for 25 cycles. Along with test samples, positive and negative control was also processed. All the SNaPshot PCR products then treated with CIAP and subjected to capillary electrophoresis on ABI PRISM 310 Genetic Analyzer along with LIZ 500 size standard for further analysis. The type of nucleotide present confirmed by the signal colour observed and length of final product obtained, by comparing with the size standard. The final length of each test primer extension product was judged by repeatedly running a single primer reaction and then determined consistently observed length of particular primer. Further (multiplex SNaPshot reaction was carried out using multiple primers with optimum concentration to determine the position and type of SNPs in single reaction. All the samples were found homozygous in both groups for SNP 1179, 1195 and 3096 with genotype AA, CC and CC respectively. This indicated that these alleles were fixed. Both the variants at nucleotide position 5545 (C and T) and 6067 (T and C) were observed. Allelic frequency was checked for both these SNPs and were found 0.85 (CC) and 0.15 (TT) for SNP 5545 while 0.57 (CC) and 0.43 (TT) for SNP 6067. Statistical analysis showed no significant association of these five SNPs with milk production traits like milk yield and milk fat percentage. All studied SNPs belonged to intronic regions however, may not be involved in manifestation of the traits.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/5810060668
dc.keywordsSINGLE NUCLEOTIDE EXTENSION ASSAY, CHARACTERIZATION OF DGAT1 LOCUS, MEHSANA BUFFALOen_US
dc.language.isoenen_US
dc.publisherAAU, Ananden_US
dc.research.problemSINGLE NUCLEOTIDE EXTENSION ASSAY FOR CHARACTERIZATION OF DGAT1 LOCUS IN MEHSANA BUFFALOen_US
dc.subAnimal Genetics and Breedingen_US
dc.subjectA STUDYen_US
dc.themeSINGLE NUCLEOTIDE EXTENSION ASSAY FOR CHARACTERIZATION OF DGAT1 LOCUS IN MEHSANA BUFFALOen_US
dc.these.typeM.V.Sc.
dc.titleSINGLE NUCLEOTIDE EXTENSION ASSAY FOR CHARACTERIZATION OF DGAT1 LOCUS IN MEHSANA BUFFALOen_US
dc.typeThesisen_US
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