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Method Validation for Detection of Factor V Leiden Mutation by Real Time PCR and RFLP Analysis

Abhijit V. Sahasrabudhe 1, Dharmendra Mishra 2, Deepa S.3, and Harshada Deshpande 4
1. DSPM's K V Pendharkar College of Arts, Science and Commerce, MIDC, Dombivli (E), Thane, India.
2. DNA lab, Basmati Export Development Foundation, Meerut, UP, India.
3. Department of Biotechnology, Padmashree Dr. D.Y. Patil University.
4. Department of Biotechnology, G.H. Raisoni College, Yashwantrao Chavan Maharashtra Open University, Nasik.
Abstract—Factor V Leiden mutation is the most common factor for venous thrombosis and it is associated with the increased risk of pregnancy loss. It¡¯s a single point mutation at nucleotide 1691 (G 㰰〶䘾 A) in exon 10 of Factor V gene, which produces an Arg 506 Gln substitution (R506Q). Although conventional sequencing is a widely used method to detect point mutation but it has several drawbacks. Here we report a rapid and reliable Real Time PCR based method to detect the Factor V Leiden mutation. We have standardized blood DNA isolation with few modifications in SDS based method. The key features are during blood DNA isolation use of hazardous chemicals such as phenol, chloroform: isoamylalcohol etc. was avoided. Without the use of RNase it was possible for us to isolate pure and sufficient amount of DNA which proved to be amenable to Real Time PCR analysis. A desired amplicon of 249 bp was produced. Restriction digestion was carried out using enzyme Mnl I which cut at 163 bp and 49 bp.

Index Terms—Factor V, RaPC, Point mutation, SYBR green, Real Time PCR, MnlI

Cite: Abhijit V. Sahasrabudhe, Dharmendra Mishra, Deepa S., and Harshada Deshpande, "Method Validation for Detection of Factor V Leiden Mutation by Real Time PCR and RFLP Analysis," International Journal of Life Sciences Biotechnology and Pharma Research, Vol. 1, No. 4, pp.143-152, October 2012.
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