Short Notes On Enzymes of rDNA Technology
Enzymes used in rDNA Technology
DNA ligase
DNA ligase is isolated from E.coli and Bacteriophage commercially and used in recombinant DNA technology. The enzyme DNA ligase joins the DNA fragments with a cloning vector. DNA ligases can form a phosphodiester bond at a single-strand break in DNA, a reaction between a 3′-OH group and a 5′-monophosphate.
Reverse transcriptase
RT is used to synthesize complementary strands (cDNA) from mRNA template. It is also known as RNA-dependent DNA polymerase. It is isolated from a retrovirus.
Restriction endonuclease
Restriction endonuclease enzymes recognize and cut DNA strands at specific sequences called restriction sites. These enzymes are isolated from a wide variety of microorganisms. Endonuclease enzymes degrade foreign genomes when they enter a microbial cell but the host cell's own DNA is protected from its endonuclease by methylation of bases at the restriction site. There are 3 types of restriction endonuclease:
- Type I Restriction endonuclease - It has both methylation and endonuclease activity. It requires ATP to cut the DNA. It cuts DNA about 1000bp away from its restriction site. For example EcoKI.
- Type II Restriction endonuclease - It does not require ATP to cut DNA. It cuts DNA at the restriction site itself. For example, EcoRI, Hind III
- Type III Restriction endonuclease - It requires ATP to cut DNA. It cuts DNA about 25 bp away from the restriction site. For example, Sty
Terminal transferase
It is the enzyme that converts the blunt end of DNA fragments into a sticky end. If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. So the enzyme terminal transferase converts the blunt end into a sticky end. Terminal transferase enzymes synthesize short sequences of complementary nucleotides at free ends of DNA, so that the blunt end is converted into a sticky end.
Nuclease
The enzyme nucleases hydrolyze the phosphodiester bond on the DNA strand creating the 3’-OH group and 5’-P group. It usually cut DNA on either side of distortion caused by thymine dimers or intercalating agents. The gap is filled by DNA polymerase and the strand is joined by DNA ligase. Nucleases are of two types; endonuclease and exonuclease.
DNA polymerase
DNA polymerase is a complex enzyme that synthesizes nucleotides complementary to template strands. It adds nucleotide to free the 3′ OH end and help in the elongation of the strand. It also helps to fill gaps in double-stranded DNA. DNA polymerase-I isolated from E. coli is commonly used in gene cloning. Taq polymerase isolated from Thermus aquaticus is used in PCR.
Ribonuclease-H (RNase H)
RNase-H removes mRNA from DNA-RNA heteroduplex and that mRNA is used to synthesize cDNA. It is isolated from retrovirus.
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