Recombinant DNA Technology

Introduction

Recombinant DNA molecules are new artificial DNA strands that are produced by combining two unrelated (non-homologous) genes, for example: hybrid of E. coli plasmid with human insulin gene. It is possible to join two unrelated genes from different species because all organisms in the world share the same DNA makeup (nitrogen bases, sugar, and phosphate backbone) and only differ in the sequence  . So one strand of DNA can complement the other strand according to Chargaff's rules.

Molecular Tools for making Recombinant DNA

There are severals Biological Tools required to make the Recombinant DNA:

1. Enzyme

• Restriction Endonuclease: act as molecular scissor, to cleave DNA at specific sequence. Most common type is Endonuclease type II, it recognises 4-8 palindromic sequences. Different Endonuclease will have different way of cleaving the DNA, there are two types: Asymetrical Clevage which leaves either 5' sticky end or 3' sticky end, other type is Symetrical clevage, it leaves blunt end.
• DNA Ligase: this enzyme responsible for joining fragments of DNA together by reforming the Sugar Phosphate backbone.
• Taq Polymerase: is an enzyme that is used during PCR to amplify copies of gene. It is stable at the high temperatures required for PCR to take place.
• Reverse Transcriptase: enzyme that is used to convert mRNA back to cDNA (DNA without intron)

2. Vectors:

DNA that act as vechicle to transport the Recombinant DNA into host cells.

A. General requirements for vector:

• Contain unique restriction sites, that act as an attachment site for new DNA.
• Contain efficient origin of replication.
• Can be introduced easily to the host cells.
• Contain genes that allow for selection, such as: antibiotic resistance.
• May contain Expression factors.

B. Most commonly used vectors:

• Plasmids
• Plasmids Cosmids - hybrid of Plasmid and Bacteriophage.
• Bacteriophage

3. DNA/mRNA

We can use either of the molecules as source for the gene of interests.
A. DNA as source:

• the DNA is isolated from a lysed cells.
• dsDNA is then seperated and partially cleave.
• lastly, being refer to Genomic Library

B. mRNA as source:

• mRNA molecule is transcribed back to DNA using reverse transcriptase.
• the cDNA is then being refer to cDNA library.the advantages of using cDNA is that there is no longer any intron in the DNA, so we won't produced truncated proteins.

C. We could also use PCR to amplify particular genes of interest.

4. Cells

• Is used for amplification or sometimes expressed the product of the recombinant gene.
• Type of cells depend on the purpose of the experiment, but most common cell type:
  • Bacteria
  • Yeast
  • Insect
  • Mammalian

Certain types of cells are preferred as expression systems due to characteristics they have. For example yeast, insect, and mammalian cells all perform post translation modifications required when producing human proteins. These cell types would be preferred over bacterial cells that are unable to conduct these modifications.

Key Stages in the Process

1. Create the recombinant DNA

• The DNA of interest is the cut using restriction endonuclease, the same type of restriction endonuclease is also use to cut the vector, in this case plasmid.
• The DNA is then ligated into the vector

2. Cloning of recombinant DNA

• Recombinant plasmid is then inserted into host cell, but the host cell have to be in a state of competent.
• The host cell will then grow and divide, so does the recombinant plasmid.

3. Selection

• Not all bacteria are transformed. Therefore we have to select those bacteria that contain plasmid only. This can be done by aplying antibiotic on the agar plate, so only the bacteria that contain the plasmid survive.
• Not all Recombinant DNA successfully ligate to the plasmid. Therefore we have to select bacteria that contain the recombinant DNA, by a technique called Blue or White Selection.
• Other selection methods to choose specific Recombinant DNA from Genomic/cDNA library are:
  • Hybridisation to ssDNA, which will complementary bind to the sequence of interest.
  • Using Primers that specifically bind to specific sequence.
  • Screen for the expression of the product of recombinant DNA.

4. Using the Recombinant DNA     

   A. Express the Protein require (expression vector)

      - To produce protein for purification.
      - To learn the function of particular protein in a cell/organism that don't usually produce them.
   B. Modifiy
      - To change the properties of a protein.
      - Study the Structure of protein in detail.

Application of the Technique

Recombinant DNA is now widely used in biotechnology, medicine, research and also farming. 
Below are several application of DNA recombinant Technology:

- Insulin Production

Recombinant DNA corresponding to the A chain of human insulin is prepared and inserted into plasmids that are used to trasform Escherichia coli cells. The bacteria then synthesises the Insulin chain, which is purified. A similar process is used to obtain B chains. The A and B chains are then mixed and allowed to fold and form disulphide bonds, producing active insulin molecules. 
- Golden Rice Production
- Insect-resistance Crops
- Herbicide-resistance Crops
- Recombinant blood clotting factor VIII for males suffering from haemophilia A