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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Tools of Genetic Engineering

 
     
 

Examples of Enzymes

SI Nuclease
It degrades the single stranded DNA or single strand of double stranded DNA with cohesive ends. As a result of action of SI nuclease cohesive ends are converted into blunt ends.

DNA Ligases
Mertz and Davis (1972) for the first time demonstrated that cohesive termini of cleaved DNA molecules could be covalently sealed with E.coli DNA ligase and were able to produce recombinant DNA molecules. DNA ligase seals, single strand nicks in DNA which has   5'3' - OH (hydroxyl) termini. There are two enzymes which are extensively used for covalently joining restriction fragments : the ligase from E. coli and that encoded by T4 phage. The main source of DNA ligase is T4 phage, hence, the enzyme is known as T4 DNA ligase.

For the joining reactions, the E. coli DNA ligase uses nicotinamide adenine dinucleotide (NAD+) as a cofactor, while T4 DNA ligase requires ATP for the same. Both the enzymes contain a -NH2 group on lysine residue. In both cases, cofactor breaks into AMP (adenosine monophosphate) (Fig. 3.2.) which in turn adenylate the enzyme (E) to form enzyme - AMP complex (EAC). EAC binds to nick containing 3' -OH and 5' - PO4 ends on a double stranded DNA molecule. The 5' - phosphoryl terminus of the nick is adenylated by the EAC with 3'-OH terminus resulting in formation of phosphodiester and liberation of AMP (Lehman, 1974). After formation of phosphodiester nick is sealed (Fig. 3.2.). T4 enzyme has the ability to join the blunt ends of DNA fragments, whereas E. coli DNA ligase joins the cohesive ends produced by restriction enzymes. Additional advantage with T4 enzymes is that it can quickly join and produce the full base pairs but it would be difficult to retrieve the inserted DNA from vector. However, cohesive end ligation proceeds about 100 times faster than the blunt end ligation.

 

Content

Enzymes

 

Exonucleases

 

Endonucleases

 

Restriction endonucleases

 

 

Nomenclature

Example of some enzymes

 

SI nuclease

 

DNA ligases

 

Alkaline phosphatase

 

Reverse transcriptase

 

DNA Polymerase

Foreign DNA

Cloning vectors

 

Plasmids

 

Bacteriophages

 

 

Insertion vector

 

 

Replacement vector

 

Cosmids

 

Phasmids

cDNA Clone bank

Gene bank (Genomic Library)

Electrophoresis


Alkaline Phosphates
When plasmid vector, for joining a foreign DNA fragment, is treated with restriction enzymes, the major difficulty arises at the same time. Because the cohesive ends of broken plasmids, instead of joining with foreign DNA join the cohesive end of the same DNA molecules and get recircularized. To overcome this problem, the restricted plasmid (i.e. plasmid treated with restriction enzymes) is treated with an enzyme, alkaline phosphatase, that digests the terminal 5' phosphoryl group (Fig. 3.3). The restriction fragments of the foreign DNA to be cloned are not treated with alkaline phosphatase. Therefore, the 5' end of foreign DNA fragment can covalently join to 3' end of the plasmid. The hybrid or recombinant DNA obtained has a nick with 3' and 5' hydroxy ends. Ligase will only join 3' and 5' ends of recombinant DNA together if the 5' end is phosphorylated. Thus, alkaline phosphatase and ligase prevent recircularization of the vector and increase the frequency of production of recombinant DNA molecules. The nicks between two 3' ends of DNA fragment and vector DNA are repaired inside the bacterial host cells during the transformation.

  Mechanism of DNA ligase enzyme - AMP complex formation and sealing of nick on a double stranded DNA molecule; A, E. coli DNA ligase; B, T4 DNA ligase; A, adenine; P, PO4; R, ribose; N, nicotinamide; NMN, nicotinamide mononucleotide
 

Fig 3.2. Mechanism of DNA ligase enzyme - AMP complex formation and sealing of nick on a double stranded DNA molecule; A, E. coli DNA ligase; B, T4 DNA ligase; A, adenine; P, PO4; R, ribose; N, nicotinamide; NMN, nicotinamide mononucleotide

Reverse Transcriptase
In addition to these enzymes, reverse transcriptase is used to synthesize the copy DNA or complementary DNA (cDNA) by using mRNA as a template. Reverse transcriptase isvery useful in the synthesis of cDNA and construction of cDNA clone bank.


Inhibition of recircularization by alkaline phosphates (to increase recombinant plasmids)  

Until recently, it was known that the genetic informations of DNA pass to protein through mRNA. During 1960s, Temin and co-workers postulated that in certain cancer causing animal viruses which contain RNA as genetic material, transcription of cancerous genes (on RNA into DNA) takes places most probably by DNA poly­merase directed by virus RNA. Then DNA is used as template for synthesis of many copies of viral RNA in a cell. In 1970, S.Mizutani, H.M. Temin and D. Baltimore discovered that informations can also pass back from RNA to DNA. They found

that retroviruses (possessing RNA) contain RNA dependent DNA polymerase which is also called as reverse transcriptase. This produces single stranded DNA, which in turn functions as template for complementary long chain of DNA.

DNA Polymerase

This enzyme polymerizes the DNA synthesis on DNA template (or cDNA template) and also catalyses a 5'3' and 3'5' exonucleolytic degradation of DNA (Kornberg, 1974). The DNA polymerase, investigated by A. Komberg and coworkers in E. coli in 1956 is now known as DNA polymerase I (DNA pol I ). The other two enzymes are DNA polymerase II (DNA pol II) and DNA polymerase III (DNA pol III). These have almost similar catalytic activity. DNA pol I (mol wt. 109,000) has a single polypeptide chain of about 1,000 amino acid residues. The addition of mononucleotide to the free -OH end of a DNA chain is catalyzed by this enzyme. Also it catalyses the other two reaction i.e.. 3'5' exonuclease activity (hydrolyzing single nucleotide residues from 5' - terminus) and 5'3' exonuclease activity (hydrolyzing single nucleotide residues from 5'- terminus). Function of DNA pol II (mol wt 120,000) is little understood. However, it catalyses 3'5' exonuclease activity. DNA pol III (mol wt about 140,000) is about several time more active than the other two. It is a dimer of DNA pol III. It requires an auxiliary protein DNA copolymerase III and after combination, yields a DNA pol III - copol III complex. Where there is preformed DNA template it produces a parallel strand in the presence of ATP.

Fig. 3.3.   Inhibition of recircularization by alkaline phosphates (to increase recombinant plasmids)

 
 
 
     
 
 
     



     
 
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