1. DNA ligase
2. Reverse transcriptase
3. Restriction endonuclease
4. Terminal transcriptase
5. Nuclease
6. DNA polymease
7. Ribonuclease-H
8. Alkaline phosphatase
9. Polynucleotide kinase

1. 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 cloning vector.

2. Reverse transcriptase:

• RT is used to synthesize complementary strand (cDNA) from mRNA template.
• It is also known as RNA dependent DNA polymerase
• It is isolated from retrovirus

3. Restriction endonuclease:

• Restriction endonuclease enzyme recognize and cut DNA strand at specific sequence called restriction site.
• These enzyme is isolated from wide variety of microorganisms. Endonuclease enzyme degrades foreign genome when enter inside microbial cell but the host cell own DNA is protected from its endonuclease by methylation of bases at restriction site.
• There are 3 types of restriction endonuclease:

Type I Restriction endonuclease:

• It has both methylation and endonuclease activity.
• It require ATP to cut the DNA
• It cuts DNA about 1000bp away from its restriction site
• eg. EcoKI

Type II Restriction endonuclease:

• It does not require ATP to cut DNA
• It cuts DNA at restriction site itself
• eg. EcoRI, Hind III

Type III Restriction endonuclease:

• It requires ATP to cut DNA
•  It cuts DNA about 25bp away from restriction site.
• eg. EcoPI

4. Terminal transferase:

• It is the enzyme that converts blunt end of DNA fragments into sticky end.
• If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. So the enzyme terminal transferase converts bunt end into sticky end.
• Terminal transferase enzyme synthesize short sequence of complementary nucleotide at free ends of DNA, so that blunt end is converted into sticky end.

5. Nuclease:

• The enzyme nucleases hydrolyses the phosphodiester bond on DNA strand creating 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 strand is joined by DNA ligase
• Nucelase are of two types; endonuclease and exonuclease

6.  DNA polymerase:

• DNA polymerase is a complex enzyme which synthesize nucleotide complementary to template strand.
• It adds nucleotide to free 3′ OH end and help in elongation of strand
• It also helps to fill gap 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

7. 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

8. Alkaline phosphatase:

• The enzyme Alkaline phosphatase helps in removal of terminal phosphate group from 5′ end
• It prevents self annealing of vector DNA soon after cut open by restriction endonuclease

9. Polynucleotide kinase:

• It adds phosphate group from ATP molecule to terminal 5’end after dephosphorylation by alkaline phosphatase.

Enzymes used in recombinant DNA technology

The post Enzymes used in recombinant DNA technology appeared first on Online Biology Notes.

• The gene for human growth hormone (hGH) is isolated from human pituitary gland.
• Insertion of whole hGH gene into plasmid vector and cloning into E.coli results into production of biologically inactive hormone because bacteria can translate the region of gene that are not translated in human thereby producing a prehormone containing an extra 26 aminoacids which might be difficult to remove.
• Hence the segment of gene that codes for the first 24 aminoacids of hormone is constructed chemically from blocks of nucleotide.

Step I: Chemical synthesis of gene for first 24 aminoacids:

• From the known aminoacids sequence of hGH, gene for first 24 aminoacids are constructed chemically. These genes are constructed in three small fragments and then they are joined by T4 DNA ligase to get whole gene for first 24 aminoacids.

Step II: Isolation of mRNA for hGH

• In this step mRNA for hGH is isolated from human pituitary gland tissue.

Step III: Reverse transcription

• Using reverse transcriptase enzyme complimentary DNA (cDNA) is synthesized from mRNA.
• The cDNA obtained by reverse transcription process, is the gene for  hGH.
• The full gene is cut with restriction endonuclease enzyme to remove first 24 gene.

Step IV: Joining of synthetic gene and cDNA

• In this step synthetic gene (gene for first 24 aminoacids) and cDNA are joined in order to obtain full gene with its own initiation codon (AUG). T4 DNA ligase join these genes.

Step V: selection of suitable vector and recombination:

• Expression vector phGH407 derived from plasmid vector PBR322 is used as carrier vector.
• HGH gene is ligated into a restriction site just downstream of Lac; promotor/operator region of the expression vector.

Step VI: selection and recombination into suitable host cell

• E. coli is used as suitable host cell.
• The recombinant expression vector is then transformed into E.coli.
• The recombinant E. coli then starts producing hGH.
• The recombinant E. coli are isolated from the culture and mass production by fermentation technology to obtain hGH.

Use of recombinant human growth hormone (hGH)

1. Treatment of children suffering from growth deficiency
2. Treat the patient with Tumer’s syndrome and chronic renal insufficiency
3. To treat patient with renal carcinoma
4. Bovine somatotropin hormone is used to increases milk production in lactating cows and also to increase body mass of cattles

Production of human growth hormone (hGH) by recombinant DNA technology

The post Production of human growth hormone (hGH) by recombinant DNA technology appeared first on Online Biology Notes.