Thursday, June 20, 2013

Uses of Recombinant DNA Technology

  1. Understanding      molecular      basis      of      disease      like      thalassemia,       familial hypercholesterolemia, cancer, diabetes, etc.
  2. Production of human proteins for therapy e.g. insulin, tissue plasminogen activator, etc.
  3. Production of protein for vaccines e.g. hepatitis B and diagnostic testing e.g. AIDS test.
  4. Predict the risk of disease and monitor pharmacological effects
  5. Use In forensic medicine.
  6. Gene therapy e.g. ADA deficiency, sickle cell disease, etc.
  7. Isolation and manipulation of DNA, including end to end joining of sequence from different sources to make chimeric molecule is the essence of recombinant DNA technology. This involves several techniques and reagents.

1. Cutting the target DNA sequence

Endonucleases cuts DNA at specific sequence producing unique and short pieces. These are a key tool. These enzymes were called restriction enzymes because they can restrict the growth of bacteriophage in bacteria by digesting and degrading the bacteriophage DNA without harming host DNA. Since host DNA is methylated by site specific DNA methylase which is present along with endonuclease, renders host DNA unsuitable for digestion.

Restriction enzymes are named after bacterial from which they are isolated. E.g. EcoRI is from

E. coli, and BamH is from Bacillus amyloliquefaciens, etc. First 3 letters include first letter of genus and following 2 letter are of species, it is followed by strain and roman numeral indicate order of discovery (EcorRI means, E. coli strain R and first discovered).

Each restriction enzyme cleaves specific ds-DNA sequence 4-7 bp long producing blunt ends (e.g. HpaI) or overlapping (sticky or cohesive) ends (e.g. BamHI). Most of the  recognition sequences are pallindromic having two fold symmetry.

Fig. Sticky and Blunt Ends

There are 3 types of restriction endonucleases, I, II and III. Type I and III contain both endonuclease and methylase activities (Cytosine and Adenine are methylated). Type I cleave DNA at random sites that can be more than  1000 base pairs from recognition site. Type III cleaves DNA about 25 bp from recognition sequence. Both T-I and III requires ATP for moving in DNA.

Type II require not ATP, and cleave DNA within the recognition sequence itself.

              Fig. Restriction Enzymes 
(Reproduced from Harper's Illustrated Biochemistry)

2. Ligation of cut DNA ends producing chimeric DNA molecules:

Sticky ends can easily ligate themselves if they encounter complementary bases, but this also creates non specific ligation. To ligate sticky ends, new ends are added using enzyme terminal transferase or synthetic sticky ends are added. E.g. if poly d(G) is added to 3’ ends of vector and poly d(C) is added to 3’ end of foreign DNA using terminal transferase, two molecules can only anneal which prevents non specific annealing. This is called homopolymer tailing. Alternatively, synthetic blunt ended duplex oligonucleotide linkers containing the recognition sequences for a given restriction enzyme are ligated to the blunt ended DNA. Direct blunt end ligation is done by using bacteriophage T4 DNA ligase. These DNA ligase use ATP to form phosphodiester bond.

Various synthetic polylinker DNA fragments having multiple sites for restriction endonuclease are created so that they become useful later for inserting additional DNA by cleavage and ligation.

Sticky ends are used in construction of hybrid or chimeric DNA. For each segment in DNA there are 4 possibilities (A T C and G). So a nuclease recognizing 4-bp sequence cuts on average, once every 256 bp (46) and that recognizes 6 bp cuts, on average, once every 4096 bp (46). Since each enzyme recognizes specific sequence in linear array of DNA. A restriction map can be constructed when the recognition sequence and the sequence in genome is known. Since the ends of each DNA fragment cleaved by given endonuclease are same, these fragments can be separated by electrophoresis and can be used for cloning.

 Fig. Plasmid cloning vector cleavage with EcoR1
Various prokaryotic and eukaryotic recombinases are used in conjunction with endonucleases to catalyze specific incorporation of two DNA fragments that carry appropriate recognition site. These enzymes catalyze homologous recombination between the relevant recognition sites.

2.  Cloning for amplification:

A clone is a large population of identical molecule, bacteria, or cells that arise from a common ancestor. So, large number of identical DNA molecules can be produced by molecular cloning.

                                           Fig. Recombinant DNA Technology

This can be achieved by inserting the chimeric or hybrid DNA construct in cloning vectors (bacterial plasmids, phages or cosmids) which will replicate in host cell under their own control system. Now the chimeric DNA is amplified.

The recombinant DNA molecule (plasmid) is inserted into bacterial cell (by transformation, in eukaryotes the process is called transfection), the plasmid DNA replicates independently, replicating the insert. Thus the insert is amplified many fold. Since the sequence recognized by original restriction enzyme is retained in this sticky end, the cloned DNA insert can be cleanly cut back out of recombinant plasmid circle with this endonuclease. In this may the single DNA segment can be amplified.

(Source: Harper's Illustrated Biochemistry and lehninger's Textbook of Biochemistry)

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