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. The DNA fragments wer
...
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. The 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]
dfdfefe.png)
