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Research Paper> BIOL1133 Biological Sciences Laboratory Course. Practical 1 DNA Sequencing and Analysis. Plasmid DNA no.: 16

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Abstract Automated sequencing with dye-terminator was used in this practical. Different length of DNA fragments was synthesized with fluorescently labeled ddNTP complementary to the template DNA. T... he DNA fragments were then separated by capillary electrophoresis and computer found out the sequence. The sequence was then matched with GenBank using BLAST. Finally the sample DNA sequence was found to be Mus musculus zinc finger protein (Sna) mRNA, complete cds. Introduction DNA, i.e. deoxyribonucleic acid, is one of the most important molecules for life. It controls the functioning as well as development of living things by carrying genetic information and expresses as protein to influence the biological status of the living organisms. Technique of DNA sequencing is important in acquiring the order of nitrogenous base in DNA. There are many DNA sequencing techniques nowadays, ranging from Chemical method (Maxam and Gilbert 1977) to Dideoxy method (Sanger). In this experiment, automated sequencing with dye-terminators was used to acquire the DNA sequence of an unknown gene which had been previously cloned into the given plasmid DNA, and the gene was identified accordingly. The experiment was divided into two main parts. The first part was to perform automated sequencing with dye-terminators, which makes use of the special helical structure of DNA. Nucleotides are the basic component of DNA, consisting of three components, a 5-C deoxyribose, a nitrogenous base and a phosphate group attached to the 5’-end. The nucleotides are linked together by the phosphate group and the hydroxyl group at the 3’-end of the next nucleotide (Saenger & Wolfram 1984). Several components required for the automated sequencing method are included in the substrates of the dye terminator, namely the given plasmid DNA, template DNA, primer, DNA polymerase, free normal dNTPs and small amount of fluorescently-labeled ddNTPs. ddNTPs, i.e. dideoxy nucleotides, are nucleotideswithout 2’ and 3’ hydroxyl group to prevent continuous elongating once added to the end of a DNA strand. Such dideoxy sequencing is based on the primer-initiated synthesis of DNA strands complementary to the template DNA of unknown sequence (George et al. 1977). The primer would first attach to the template DNA, and then elongation will carry out under the presence of free nucleotides and the effect of polymerase. The elongation will stop at different lengths due to the presence of four types of dideoxy nucleotides, generating different sizes of DNA fragments. The elongation was performed under the following condition: [Show More]

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