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Abstract:

The purpose of the experiment was to incorporate the DNMT2 gene into pBluescript plasmid that has Hind lll restriction site. DNMT2 codes for a protein which adds a methyl group to cytosine and studies have shown that its overexpression can cause cell death via apoptosis. But as such no serious anomaly is reported. Cloning of the insert into vector was successful and gel analysis confirmed that the gene had been incorporated. The orientation was right. Therefore, the DNMT2 was successfully cloned into pBluescript II KS+.

Introduction:

DNA methylation is a mechanism of gene regulation and incorrect methylation is associated with various diseases, especially cancers. Cytosine methylation plays important part in various biological processes. Addition of methyl on cytosine happens on the opposite strands of cytosine residues. The gene that was supposed to be cloned in this experiment was the DNA Methyl Transferase 2 (DNMT2). DNMT2 gene, also known as tRNA (cytosine-5-)-Methyl transferase, was initially assigned as a member of DNA methyl transferase family of enzymes due to its extensive homology with DNA-(cytosine C5)-methyl tranferase (Yoder and Bestor, 1998). The enzyme catalyzes methylation of cytidine at the carbon-5 position, forming 5-methyl cytosine, commonly abbreviated as 5 mC. They catalyze the addition of methyl at position 38 in tRNAAspGUC (Goll et al. 2006), but its other biological functions remain unknown (Okano et al. 1998). The gene is present in humans, mice and other mammals as well. DNMT2 codes for a protein similar in structure to DNA methyl transferase but it does not show transferase activity. The protein binds to DNA which suggests that it might have specific sequence in the genome. Hence DNMT2 gene does not methylate DNA but methylates tRNA. The cytosine at position 38 in the non-coding loop of aspartic acid tRNA is methylated. (McCabe et al., 2012) and is found on the short arm of chromosome 10 in humans (10p13) (European Bioinformatics Institute, 2017). Stress-induced cleavage of tRNA is DNMT2 dependent (Jeltsch et al, 2006) and DNMT2 mediated methylation protects tRNA protects against ribonuclease cleavage (Goll et al, 2006) In order to further investigate about its potential functions in humans, cloning the gene in a suitable vector to provide an ample amount is important and also to make sure that the gene has been cloned accurately before analyzing it. To clone the gene, it is amplified first using PCR, then integrated into the plasmid, pBluescript II KS+ and then transcribed and expressed in an E. coli colony. Employing a blue/white selection method makes it easy to know which E.coli contains the cloned DNA. This is because this plasmid contains a multiple splicing site (MSS) and an ampicillin-resistance gene within it. This sequence codes for LacZ’ gene in Escherichia coli. Using a particular Restriction enzyme, EcoRI in this case, the linear DNMT2 insert and vector can be cut to produce complementary overhangs. Restriction enzyme recognition sites are palindromic, so cutting with the restriction enzyme, the insert, and vector DNAs can bind together. First, the vector is digested then DNMT2 insert is subject to PCR to amplify the number of molecules. The goal of this experiment was to clone the DNMT2 (insert) gene into pBlueScript (vector) to produce recombinant plasmid.

Methodology

All steps of PCR cloning begin from designing primers, performing PCR Amplification, sequencing PCR products, analyzing the sequenced data and finally assessing gene expression. The sequence of DNMT2 gene is amplified with PCR primers, in which there is a proper restriction enzyme site. The DNMT 2 gene is cloned into the plasmid pBluescript which has 2961 base pairs and has 21 multiple cloning sites (MCS) and each one is specific for one restriction enzyme. Its MCS ranges from 626-791 bp. Flanking the MCS are RNA polymerase promoters. The choice of RNA promoter determines which strand of the insert (Gene of interest) cloned into the MCS will be transcribed. In this case, EcoR1 restriction enzyme is used to insert and clone the gene. But before starting the process, PCR primers that have proper restriction enzyme sites, need to be designed. Two primers, forward and reverse have to be synthesized. According to the given sequence….