Production And Isolation Of The DNA Fragment
a) Restriction Endonucleases Digestion:
Endonucleases are enzymes that produce internal cuts called cleavage, in DNA molecules. A class of Endonucleases cleave the DNA only within or near those sites, which have a a specific base sequence; such Endonucleases are known as restriction Endonucleases, and the sites recognized by them are recognition sequences or sites.
There are four different types of restriction Endonucleases the type I are complex Endonucleases, that have recognition sequences of about 15 bp; they cleave the DNA about 1000 bp away from the 5’-end of the sequence “ TCA” located within the recognition site, the type II restriction Endonucleases are quite stable an induce cleavage either within or immediately outside their recognition sequences, the type III restriction Endonucleases cleave the DNA in the immediate vicinity of their recognition sequences.
b) Mechanical Shearing
The long, thin threads which constitute duplex DNA molecules are very rigid to be easily broken by shear forces in solution. Intense sonication with ultrasound can reduce the length to about 300 nucleotide pairs. More controlled shearing can be achieved by high-speed stirring in a blender. Breakage occurs at random with respect to DNA sequences.
c) Duplex cDNA synthesis
cDNA is the copy or complementary DNA produced by using mRNA as a template. In fact any RNA molecule can be used to produce a cDNA. Temin and Baltimore in 1970 discovered the enzyme reverse transcriptase obtained from avian myeloblastosis virus (AMV), the enzyme is used to produce DNA copy of RNA.
d) Direct chemical synthesis
The chemical synthesis of a gene utilizes chemical reagents for the various steps of the process. There are three major methods, i) phospodiester approach: in this method 3’-5’-OH groups of the deoxyribose moiety of nucleotides are suitably blocked and are selectively unblocked for joining of the additional nucleotides. ii) Phosphate trimester approach: it utilizes deoxyribose as starting material and involves step wise addition of mono and oligo nucleotides. iii) Phosphoramidite approach: it uses silica beads or controlled pore glass beads as solid support and an automated synthesis of oligonucleotides takes place in less than fifteen seconds. All the methods described above differ only in the protection of –OH group of phosphate residues.
e) Polymerase Chain Reaction (PCR).
PCR technique, developed by Kary Mullis in 1985 is used to get multiple copies of a the gene (or DNA) of interest is synthesized in vitro using two set of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA. and the enzyme DNA polymerase). The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template, if the process of DNA replication is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e. 1 billion copies are made. Thermostable DNA polymerase isolated from Thermus aquaticus makes such repeated amplification achievable. The amplified fragment if desired can now be used to ligate with a vector for further cloning.
Four different types of enzymes are used viz. nucleases, ligases, polymerases, and certain DNA modifying enzymes.
- For the production and isolation of the DNA fragment one of the following methods may be used:
- For the insertion of the gene into a suitable vector either of these methods are used:
- For further introduction of the recombinant DNA molecule into the host cell one of these methods is suitable:
- The end process of screening for the desired clones is carried out in one of the following manner:
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