METHYLATION DETECTION IN H19 GENE IN BUFFALO [Bubalus bubalis) BY BISULFITE SEQUENCING
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Date
2011
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AAU, Anand
Abstract
It is increasingly clear that translation of the genetic code into proteins is not the only way that our genes influence our growth, development and health. Another system of influence on the expression of genes is referred to as epigenetics. Any biochemical modification of the DNA or chromatin that can account for epigenetics gene expression system, only one candidate mechanism that is site-specific DNA methylation, has received experimental support to date. Methylation of DNA in mammalian cells occurs at cytosine residues in CpG dinucleotides. Improper or absence of DNA methylation of imprinted gene may lead to abnormal growth or cancerous condition. The best candidate for the epigenetic mark at the H19/Igf-2/Ins-2 locus is paternal-specific DNA methylation of the H19 and its 5' flank. Methylation status of H19 influences the expression of IGF-2. Keeping in consideration that methylation status ofH]9 influences the IGF2 expression, present study was carried out to detect methylation status in H19 promoter in Jaffarabadi, Surti and Mehsani buffalo by bisulfite sequencing. DNA extraction from blood was carried out by phenolxhloroform method from seven sample of each breed which were subjected to bisulfite treatment by sodium bisulfite. Quality and quantity of DNA was checked by ND-1000 spectrophotometer and 0.8% agarose gel electrophoresis. Locus specific primers for normal DNA and for bisulfite treated DNA were used to amplify a gene fragment of H19 in three breeds of Bubalus bubalis. Purification of PCR product was carried out by eluting desired PCR product from agarose gel by using by QIAquick® Gel Extraction Kit and later the PCR products were ligated in pTZ57R/T vector of InsT/Aclone TM kit (Fermentas). Ligated recombinant vector was transformed in competent E. coli (DH5-α) cells. Recombinant colonies were identified by blue-white screening and white conies were subjected to Ml3 colony PCR for confirmation of positive recombinant clones. White colony containing recombinant clone was grown in LB broth and recombinant plasmids were isolated using QIAprep® Spin Miniprep Kit and used for cycle sequencing, that were later processed for sequencing. Multiple sequence alignment was done for all 21 samples using BioEdit v 7.0.7.1, which revealed no difference between all samples. Four clones from each bisulfite treated samples were sequenced and subjected to methylation study by BiQ Analyzer and BISMA softwares. In H19, 20 CpGs were revealed in all three breeds and were subjected to methylation study. In all three breeds, highest methylation per cent was found to be 94.12, while in some sample it showed no methylation at any CpGs site. The overall methylation per cent of HI9 gene in Jaffarabadi, Surti and Mehsani buffaloes were observed to be 50.19, 70.85 and 52.24, respectively. The incidence of mean per cent methylation in H19 gene in all three breeds was found to be 57.36. Unexpected nucleotide conversion (T>C, A>G and G>A) and deletion (A and G) after bisulfite sequencing were observed. Methylation of non CpG cytosine was observed which were found at CpA and, only in one animal at CpT. In some case, three CpG out of twenty were not analyzed due to non specific conversion of CG>TA. For estimation of association of milk production traits with methylation pattern, animals were grouped into high and low group with respect to milk yield and milk fat percentage. Statistical analysis indicated that there was no significant difference in high and low group with respect to milk yield and milk fat per cent among methylation pattern in all three breeds. Screenings of more number of animals are needed to get reliable data for estimation of association of milk production traits with methylation pattern.
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ANIMAL BIOTECHNOLOGY, A STUDY