A description of enzymes commonly used in the field of Genetic Engineering.
Reverse Transcriptase is an enzyme that makes a DNA strand using mRNA that makes a DNA strand using mRNA as a template strand. It is also known as RNA dependent DNA synthetase.
It is isolated from retroviruses (Rous Sarcoma Virus (RSV), Mouse Mammary Tumour Virus (MMTV). Avian Myeloblastosis Virus (AMV) and Murine Leukaemia Virus (MuLV). This enzyme is bound with RNA core of the viruses. It is 70,000 daltons in molecular weight. It adds complementary deoxyribonucleotides onto the mRNA template to synthesise a complementary DNA strand.
This enzyme requires a free 3'-OH group to add the first complementary base to the mRNA. The 3'- OH group is provided by a primer.
Reverse transcriptase is used to make cDNAs from mRNAs.
Ribonuclease- H (RNase-H)
Ribonuclease- H is a nuclease enzyme that selectively hydrolyses the mRNA in a RNA-DNA hybrid.It is isolated from retroviruses such as RSV, AMV, MMTV, MuLV, etc. It is found associated with reverse transcriptase enzyme in the RNA core of the viruses. The molecular weight of this enzyme is 92,000 daltons.
This enzyme hydrolyses the mRNA in RNA-DNA hybrid, after the full development of cDNA on the mRNA. Thus it releases the cDNA to synthesize the second DNA strand to form a duplex DNA. This enzyme however does not degrade free RNAs.
RNase- H is used to hydrolyse the mRNA present in RNA-DNA for releasing the cDNA to synthesise the second strand.
Klenow Enzyme or Klenow Fragment
Klenow enzyme is a product of enzymatic breakdown of DNA polymerase I from E. coli. The molecular weight of this enzyme is 76000 daltons. It has 5'-3' polymerase activity and 3'-5' exonuclease activity. It has the ability to do DNA polymerization under in vitro condition. A 3'- OH group is required for this polymerization reaction. The enzyme moves one nucleotide further on the template to add the next complementary nucleotide.
Klenow enzyme is used to synthesise a second DNA strand on cDNA strand to form a duplex DNA, (cDNA clone).
It is also used in PCR for the amplification of the DNA.
SI nuclease is an enzyme that selectively cuts and degrades single-stranded portions of DNA. It is a glycoprotein consisting of 82% protein and 18% carbohydrate units. The molecular weight of the enzyme is 38000 daltons.
This enzyme breaks the phophodiester bond between two nucleotides in single stranded portion of DNA and then degrades single stranded extensions. It does not degrade double- stranded portions of DNA and RNAs.
SI nucleases used to degrade the hairpin loop formed while making a duplex DNA from complementary DNA strand (cDNA).
It is used to remove unwanted tail sequences from DNA fragments to make blunt ends.
It is used to remove the extra adenine base from DNAs fragments by polymerase chain reaction.
It can also be used to determine the degree of complementary base pairing between DNA strands during hybridization .
Taq DNA Polymerase
Taq DNA polymerase is isolated from the gram negative, rod shaped, thermophilic bacterium, Thermus aquaticus. It consists of a single polypeptide chain having the molecular weight 95000 daltons. It is a thermostable enzyme that can withstand heating to 95ºC or more. The polymerization activity however is maximum at around 75ºC. This enzyme is resistant to pH9.
It adds complementary nucleotides onto the template DNA strand to form a complementary DNA strand. This enzyme has an unusual property that, it adds an extra adenine base to the 3' end of the growing strand.
Taq DNA polymerase is used to make numerous identical copies of a desired DNA segment by polymerase chain reaction. The DNAs thus amplified are used for-
Gene manipulation by genetic engineering.
DNA sequencing by Maxam-Gilbert method.
Producing DNA probes for clinical diagnosis and DNA fingerprinting.
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