Isolation and cloning of ABCC3 gene from rasbora sarawakensis

Siti Nur Nabihah, Binti Zakariah (2015) Isolation and cloning of ABCC3 gene from rasbora sarawakensis. [Final Year Project Report] (Unpublished)

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Abstract

A TP-binding cassette (ABC) transporters are transmembrane proteins that play an important role in A TP hydrolysis which assist the molecules to cross the plasma membrane. A TP binding proteins consists of seven subfamilies i.e. ABCA to ABCG which are involved in different metabolic processes. Mutation of ABC genes leads human inherited disease such as pseudoxanthoma elasticum (PXE), adrenal deficiency and neuro degradation. Therefore, gene expression analysis is considered for further knowledge on how ABC gene is expressed. Besides, the aim of this research is to identify the expression of ABCC3 gene in Rasbora Sarawakensis and subsequently clone into pGEM®-T Easy Vector. Total RNA was initially isolated from whole fish homogenate using Tri reagent and phenol chloroform precipitation. Next, first strand cDNA were generated and ABCC3 transcript was amplified with peR using degenerate primers by targeting the conserved region of the gene. This yielded an approximately 607 bp amplicon which was then gel extracted and further cloned into pGEM®­ T Easy Vector. Transformation using in house prepared E.coli JM 109 yielded an efficiency of 108 transformants, with approximately 2 of blue and 5 of white colonies. Then, four white colonies were verified with colony peR and all shown the presence of insert. Further verification of insert using NotI restriction digestion was conducted which yielded two discreet bands. The plasmid minipreparation product was then sent for sequencing and the result was verified using BLAST BLAST analysis registered an E-value of 105 with 85% hjghest similarity to Danio rerio ABCC2 gene.

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