Detection of breast cancer susceptibility: Gene 1 (BRCA1) using optimal conditions of Polymerase Chain Reaction (PCR)
Al Rahbi, Zakiya Amer Said
Cardiff Metropolitan University
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Background: BRCA1 is a tumor suppressor that functions in controlling cell growth, maintaining genomic stability and has been linked to early-onset breast and ovarian cancer. The human BRCA1 gene comprises 24 exons and encodes a 1863-amino acid protein, which contain a highly conserved RING figure domain at the amino terminus and two BRCT (BRCA1 carboxyl-terminal) repeats at the carboxyle terminus. More than 850 clinical mutations in the BRCA1 gene have been identified, many of which are associated with an increased risk of breast cancer. Aim & Methods: The present study was designed to optimise the conditions necessary for the detection of the BRCA1 gene using the molecular techniques of polymerase chain reaction (PCR) and agarose gel electrophoresis. The first objective of this study was to design BRCA1 gene specific primers with the aid of primer design software (NCBI) to eliminate the complications introduced with primer-dimers and secondary structure. The second objective was to isolate DNA from Adherent and non-Adherent cells using Chelex resin. The third and the final objective of this study was optimisation of PCR conditions for BRCA1 gene and housekeeping gene β-Actin using different parameters e.g. (annealing temperature, MgC12 concentration, template DNA amount, buffer, primers, dNTPs and cell cycle number). Results: The finding showed that the optimal conditions were determined included DNA template (100ng β-Actin, 150ng BRCA1), Annealing temperature (61 oC β-Actin, 65.2 BRCA1), MgC12 concentration (2.0 mM β-Actin, 4.0 mM BRCA1), cell cycle number (30 & 33 β-Actin, no amplified products for BRCA1), Buffer concentration (1.5 µL BRCA1), dNTPs concentration (0.080 & 0.100 mM BRCA1) and finally Primers (0.2 mM BRCA1). Conclusions: Optimised the detection of the presence of exon-15 of the BRCA1 gene and this approach may be useful in the detection of the gene or its mutations within clinical samples. At last, the validation of experiment was performed under the optimized condition, and the expectant results were produced.
BSc (Hons) Biomedical Science
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