Browsing by Author "Nugapola, N.W.N.P."

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    Development of Standard DNA Size Markers for Short Tandem Repeat Loci BM1818 and BM1824 of Cattle DNA Typing in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2013) Nugapola, N.W.N.P.; Bulumulla, P.B.A.I.K.; Fernando, T.S.R.; Illeperuma, R.J.
    Cattle are the most common type of livestock animal raised for meat, dairy, as draft animals and various other purposes. Sri Lankan cattle population mainly consist of indigenous cattle “Sinhala batu cattle”, European pure breeds (Jersey, Friesian, Ayrshire), Indian cattle breeds (Sahiwal, Sindhi, Tharparkar), and their crosses. Identification and traceability of cattle and their products is extremely important for proven of ownership, exploration of pedigree and animal disease control. Traditional identification methods are less effective and associated with many economic, cultural and ethical concerns and are not reliable methods of establishing maternity or paternity of cattle. Deoxyribo Nucleic Acid (DNA) based identification is based on the unique, unalterable, inherited DNA profile of an individual animal as an identifier. Testing the Short Tandem Repeats (STR) of DNA in establishing the identity and parentage is widely used for humans in Sri Lanka. However DNA based identity testing methods are needed to be developed for cattle in order to use it as an accurate method than conventional methods to address problems of cattle identity and traceability. Currently there is no proper procedure of DNA based of identification in Sri Lanka due to the unavailability of standard DNA size markers and there is an urgent need of developing standard DNA size markers for cattle STR (allelic ladders) for accurate genotyping of an individual. Therefore the present study was conducted to develop standard DNA size markers for STR loci BM1818 and BM1824. Methodology Selection of loci Two bovine specific STR markers (BM1818, BM1824) recommended by the Food and Agriculture Organization (FAO) and the International Society for Animal Genetics (ISAG) (FAO, 2011) and previous studies done by Goor and Van de (2011) were selected for this study. Sample collection and DNA extraction Blood samples (n=38) and tissue samples (n=12) were randomly collected from unrelated animals from different areas of the country according to the recommendations provided by FAO 100 extraction protocol described by Phillips (2009) with minor modifications. DNA from tissue samples was extracted by ChargeSwitch Forensic DNA Purification Kit (Invitrogen™, Life Technologies Corporation) according to the kit’s manual. PCR amplification PCR amplification of extracted DNA samples was carried out with 5 µL of genomic DNA in a 50 µL reaction volume, consisted of 5 µL of 10X STR buffer (DreamTaq Green Buffer - Thermo Scientific), 5 µL of each dNTP (Guangzhou Geneshun Biotech Ltd.), 5 µL of primer (FAO, 2011 and Goor and Van de 2011) (Integrated DNA Technology), 0.30 µL of Taq DNA polymerase (DreamTaq-Thermo scientific) and 30 µL of sterilized distilled water. The PCR reaction was carried out in a Veriti 96 well thermal cycler (Applied Biosystem, USA) under the following PCR cycling conditions (Sodhi et al., 2006): 2 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at annealing temperature (56 to 62 °C) of each primer, 1 min at 72 °C, and final extension of 10 min at 72 °C. Subsequent to PCR, the products were subjected to 3 % w/v agarose gel electrophoresis to verify the success of the PCR reaction. Then the PCR products were subjected to 7 % denaturing polyacrylamide gel electrophoresis. Amplified products were detected using DNA Silver Staining procedure described by Bassam et al. (1991) and Promega technical manual. Allelic ladder construction Allelic ladders were constructed by two methods as follows (1) DNA samples that can contribute to maximum number of polymorphic alleles to each locus were selected, pooled and amplified by PCR reaction (100 µL reaction volume), (2) PCR products that can contribute to maximum number of polymorphic alleles to each locus were selected and pooled to create the allelic ladders. Results and Discussion According to the Table 1, allelic range of the BM1818 locus was from allele 11 to 21. The highest frequency was shown by allele No.18 at a percentage of 35.0 percent. Allele 11 had the lowest frequency with 1.3 percent. When considering BM1824 locus, allelic range was from allele 11 to 19. The highest frequency was shown by allele No.13 at a percentage of 34.1 percent. Two alleles (11 and 18) showed the lowest allelic frequency of 1.2 percent.
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    Development of Standard DNA Size Markers for Short Thandem Repeat Loci BM1818 and BM1824 of Cattle DNA Typing in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2013) Nugapola, N.W.N.P.
    Cattle; the most common livestock animal reared in the world and also true in Sri Lanka. When considering Sri Lanka, National cattle herd is mainly consist of indigenous cattle "Sinhala batu cattle", European pure breeds (Jersey, Frisian, Ayrshire), Indian cattle breeds (Sahiwal, Sindhi, Tharparkar), and their crosses. Identification and traceability of cattle and their products is extremely important for proven of ownership, exploration of pedigree, record keeping and animal disease control. Current identification methods are based on tattooing, branding and ear-tagging of an animal. However tagging devices can become separated from an animal thereby compromising identity and traceability. Moreover, tattooing is not a reliable method of establishing maternity or paternity of cattle, often cause significant financial consequences for the herd owner in instances of proven of ownership. Deoxyribo Nucleic Acid (DNA) based identification relies on using the unique, unalterable, inherited DNA profile of an individual animal as an identifier. Testing the Short Tandem Repeats of DNA (STR) in establishing the identity and parentage is widely used for humans in Sri Lanka. However DNA based identity testing methods needed to be developed for cattle in order to use it as an accurate method than conventional cattle identification in order to address problems of cattle identity and traceability. A total of 38 blood samples and 12 tissue samples were collected, DNA was extracted by Chelex® 100 (blood) and Charge Switch® - Forensic DNA purification kit (tissue). STR analysis done for loci BM1818, BM1824, BM2123, CSRM60, CSSM66, ETH10, HAU27, ILSTS006, INRA023, TGLAl26. Standard DNA size markers for BM1818 (PIC = 0.77) and BM1824 (PIC = 0.71) were constructed for Sri Lankan cattle population since they were identified as the most polymorphic among panel of 10 loci. Allelic frequencies for each allele of the BM1818 and BM1824 loci were calculated. BM1818 locus has nine alleles and allele 18 was found with highest frequency (35 %). BM1824 locus has eight alleles and allele 13 was the most frequent allele (34.1 %). Newly constructed DNA size markers (allelic ladders) were successfully applied in cattle maternity tests and the results of these STR analyses have been accepted by courts. Key words: - Identity Test, Parentage Analysis, Microsatellite, Cattle DNA, BM1818, BM1824.