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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1060-1063 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1061Research Institute (NDRI) for presence of FXIdeficiency.Materials and MethodsBlood samples were collected fromrandomly selected 200 Karan Fries cattle(Holstein Friesian x Tharparkar crossbred)maintained at cattle yard of NDRI, Karnal. DNAextraction was done using Phenol-chloroformmethod as described by Sambrook et al. (12) withfew modifications. Gene-specific oligonucleotideprimers (Forward 5’ CCC ACT GGC TAGGAA TCG TT 3’ ; Reverse 5’ CAA GGC AATGTC ATA TCC AC 3’) for bovine factor XI genewere synthesized as described by Marron et al.,(8) to amplify exon 12 (244 and/or 320 bp) ofFXI geneThe PCR amplification was performedin programmed Thermal cycler (MJ research).22 ul of PCR reaction mixture was mixedthoroughly by vortexing with 3 ul template DNA(50ng/ul) and PCR amplification was performedin a final volume of 25 ul. The PCR reactionmixture was incubated in thermal cycler initiallyat 94 o C for 2 minutes followed by 30 cycles of94 o C for 30 seconds, 55 o C for 1 minute, 72 o Cfor 1 minute and a final extension of 72 o c for 10minutes to obtain PCR product of amplifiedDNA. The PCR products were checked byelectrophoresis on 1.5% agarose gel at 100 voltsfor 30 minutes to verify the amplification of targetregion. Genotyping of animals of animals wascarried out by 2.5% Agarose gel horizontalelectrophoresis at 100 volts for 40 minutes. Thegels were stained with 1% ethidium bromidesolution (@ 1µl/100 ml of gel) and scored for thepresence of single and double bands forhomozygous and heterozygous individualrespectively. The gel photograph was taken bygel documentation systemResult and DiscussionThe genotyping of Karan Fries cattlerevealed only one band of 244 bp in all the animalsas shown in Plate 1 indicating that there is no 76-bp insertion in exon 12, similar results wereobtained in Indian crossbred cattle by Patel etal., (11). However, they found two Holstein cattleas carrier with 77bp insertion within exon 12 ofthe FXI gene (Genbank accession numbersDQ438908 and DQ438909). The representativesamples of Karan Fries obtained by PCR analysiswere custom sequenced. Sequence data wasanalyzed using Chromas (Ver.5, http://www.technelysium.com.au/chromas.html).Multiple sequence alignments were performed tostudy nucleotide and amino acid changes amongKaran fries and reference sequence of Bostaurus, which also indicated that there was no76-bp or 77bp insertion in exon 12. There wasneither homozygous mutant nor heterozygouscarrier individual, hence, it is concluded that thereLane 1-16 : 244 bp monomorphicLane M : 100 bp ladderFig. 1. Multiple sequence alignment of nucleotidesof FXI Exon 12 of Karan Fries and Bos taurusAzad et al
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1060-1063 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1062is no polymorphism of FXI gene in the studiedanimals of Karan Fries breed of cattle maintainedat NDRI, Karnal. However, one nucleotidechange from G to A at position 105 in Karan Frieswas observed when compared with Bos taurusreference sequence and similar substitution wasalso seen in chromatograph (Figure 1 and 2). Thisnon synonymous mutation resulted into a changeof amino acid from Arginine (R) in Bos taurus toGlutamine (Q) in Karan fries in second codon(CGA TO CAA) as revealed by in siliconucleotide translation and the alignments of Karanfries and Bos taurus (Figure 3). The contigscontaining exon 12 of Factor XI gene weresubjected to basic local alignment search(BLAST) to know the sequence similarity withthe corresponding regions of other species.Fig. 2. Chromatogram of FXI Exon 12 showing nucleotidechangeFig. 3. Multiple sequence alignment of insilico translated amino acids sequences of FXI exon12 of Karan Fries and Bos taurusBLAST analysis of Factor XI gene for exon 12regions of Karan Fries cattle showed 96 to 98%sequence identity with buffalo and Bos TaurusReferences1. Brush, P. J., Anderson, P. H. and Gunning,R. F. (1987). Identification of factor XIdeficiency in Hostein-Friesian cattle inBritain. Vet. Rec., 121: 14–17.2. Coomber, B. L., Galligan, C. L. and Gentry,P. A. (1997). Comparison of in vitrofunction of neutrophils from cattle deficientin plasma factor XI activating and fromnormal animals. Vet. Immunol.Immunopathol., 58: 121–1313. Gentry, P. A. and Ross, M. L. (1993).Coagulation factor XI deficiency in Holsteincattle: Expression and distribution of factorXI activity. Can. J. Vet. Res., 58: 242–247.4. Ghanem, M. E. and Nishibori, M. (2008).Genetic Description of Factor XIDeficiency in Holstein Semen in WesternJapan. Reprod. Domestic. Anim., 44 (5):792-6.5. Gurgul, A., Rubioe, D. and Sota, E (2009).Identification of carriers of the mutationcausing coagulation factor XI deficiency inPolish Holstein-Friesian cattle. J. Appl.Genet., 50 (2): 149-152.6. Kunieda, M., Tsuji, T., Abbasi, A. R., Khalaj,M., Ikeda, M Miyadera, K., Ogawa, H.and Kunieda, T. (2005). Mamm. Genome16: 383–3897. Liptrap, R. M., Gentry, P. A., Ross, M. L.and Cummings, E. (1995). Preliminaryfindings of altered follicular activity inHolstein cows with coagulation factorXI deficiency. Vet. Res. Commun. 19: 463-471.Factor XI Gene Deficiency
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