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.
- 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
     - 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.

Key Stages in the Process

1. Create the recombinant DNA
- this process has been describe in the DNA/mRNA sub group.

2. 

References

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