LAMP on human SRY DNA sequence emerging a simple technique for sexual investigation from minimal blood and other less tissue specimens Varunee Musikawata, Noppawon Polpanicha, Pennapa Manitchotpisita, Panun Karnjanapooma, Narong Arunrutb,c, Wansika Kiatpathomchaib,c, Gun Anantasomboona,* a Department of Medical Sciences (Anatomy and Biochemistry Units), Faculty of Science, Rangsit University, Laghok, Paholyothin Rd., Patumthani 12000; bBIOTEC, Science Park, Paholyothin Rd., Patumthani 12120; cCentex Shrimp, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand.
*Corresponding author, e-mail:
[email protected]
We applied the Loop-mediated Isothermal Amplification (LAMP) for sexual differentiation with specimens of human tissues. LAMP of DNA is a relatively new advantage DNA amplification technique, which due to its simplicity of single-tube procedure, rapidity, and low cost. In LAMP, the target sequence is amplified at a constant temperature of 60-65 °C using either two or three sets of primers and a Bst DNA polymerase (1). It has the potential to be used as a simple screening for infectious disease in the field, diagnosis of hereditary disease, as well as investigation of target genomic sequence. Two primer sets for SRY (Sex-determining region Y) (2) gene upon the short arm of Y chromosome were designed and tested with minimal samples (less than 10 ng of total DNAs) extracted from the male and the female blood and oral epithelia. Result revealed the positive LAMP reactions on only male specimens with high specificity and sensitivity, equivalent to the result from nested-PCR amplification. Although, the sexual differentiation using PCR base on SRY gene has been commonly used in medical science, athlete, and forensic science but it need for expensive thermal-cycler and gel-electrophoresis. We have planned to combine a condition of visible bio-reporter (3) for direct notification when the positive LAMP reaction was occurred. As be mentioned, LAMP with visible bio-reporter can obviate the usage of PCR thermalcycler, consume minimal time and skill of worker, so it proves to be a suitable tool for field investigation. Keywords: LAMP, nested-PCR, sexual determination, SRY gene, human
Selected References: 1. Notomi T., et al. Loop-mediated Isothermal Amplification of DNA. Nucleic Acids Res. 2000; 28(12): e63. 2. Haqq C.M., et al. Molecular Basis of Mammalian Sexual Determination: Activation of Müllerian Inhibiting Substance Gene Expression by SRY. Science. 1995; 266(5190): 1494–500. 3. Assawapong S., et al. Detection of Shrimp Taura Syndrome Virus by Loop-mediated Isothermal Amplification Using a Designed Portable Multi-channel Turbidimeter. J. Virol. Methods. 2011; 175(2): 141-148.
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Fig. 1 Gel electrophoresis exhibiting LAMP inspection from human DNA specimens. Positive characteristic of LAMP-SRY amplification is specifically observed as smear amplified products with all of the male specimen extracts: fresh blood (m1); hair follicles (m2); and oral epithelia (m3), whereas the negative results are occurred with all of DNAs that collected from the female blood (f1) and hair follicles (f2). Lane M is DNA ladder.
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