Author: LaTanya Downer Jothi Malar Panandam, Kamariah Jamhari INTRODUCTION The Sambar is the largest deer native of the Indian subcontinent, southern China, and Southeast Asia. Its scientifically referred to as Rusa unicolor. Polymerase chain reaction (PCR) is used in molecular biology to amplify a single or a few copies of DNA and create millions of copies. Sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Molecular sexing is not only an attractive means to differentiate the sexes of sexually monomorphic species, but may be used to identify the gender of the killed animal from the meat and body fluid, as well as to identify the species. PROBLEM STATEMENT There is little genetic information about Sambar deer. I want to used this research opportunity to add information about the deer using molecular sexing.
OBJECTIVES To learn some techniques in Molecular Biology. To determine the amplification products and nucleotide sequences of the X and Y chromosomes linked Amelogenin genes (AMELX and AMELY) and the Y- linked DEAD-box RNA helicase Y gene (DBY) in the Sambar deer (Rusa unicolor). To compare the nucleotide sequences of AMELX and AMELY between the male and female Sambar deer.
METHODS & MATERIALS DNA EXTRACTION Blood Sample DNeasy Blood & Tissue Kit Biophotometer Centrifuge Used in the DNA extraction Used to check concentration of DNA Thermal Cycler POLYMERASE CHAIN REACTION (PCR) Used to amplify specific region of the DNA. Amelogenin Region and DBY Region Specific Protocol The DEAD-Box RNA Helicase Y (DBY / dby) region in Y chromosome of human and mouse.
Source: Mazeyrat et al., 1998 PRIMER SE 47 & 48 IS DESIGN BASE ON THE BOVINE AMELOGENIN LOCUS ELECTROPHORESIS SE 47 & 48 uses Metaphor Agarose gel (4%) DBY 8 uses Agarose gel Electrophoresis (1.6%)
Gel Documentation System GEL VIEWING Test product for electrophoresis PURIFICATION Expin Tm Combo GP
RESULTS
SE 47&48 Metaphor Agarose Gel DBY 8 Agarose Gel SEQUENCING BioEdit is a program used to view the nucleotide sequence of the PCR amplified and sequenced DNA fragments.
ClustalW2 is a general purpose multiple sequence alignment program for DNA or proteins. It is used to compare the sequences of PCR amplicons of different individuals and species, and in this case two sexes, i.e. of AMELX and AMELY.
CONCLUSION Primer SE 47 & 48 and DBY 8 can detect the sex of the animal.