What is recombinant gene technology?
Recombinant DNA technology or genetic engineering is a technology that allows manipulation and expression of DNA. It has at its core the process of gene cloning.
The key technology that allowed gene cloning was the discovery and use of restriction endonucleases. These enzymes recognize specific base sequences in the DNA and will cut the DNA in a precise and predictable manner. Many of these enzymes leave unpaired bases at the ends of the cuts which can initiate the reassociation of complementary ends preparatory to the addition of DNA ligase. DNA ligase reforms the phosphodiester bonds.
Handout on Restriction Enzymes. Review terminology.
What is gene cloning?
Gene cloning is the process of construction, amplification, and identification of a recombinant DNA molecule.
Fig. 1.3 Cloning, transformation and plating allows the separation of individual clones.
The greatest challenge in gene cloning identification of the gene of interest.
A genomic library is a set of clones that represents "all" of the genome.
A cDNA library is a set of clones that represents "all" of the expressed mRNA.
How many clones from a bacterial genomic library would you need to look at to find a clone of interest?
N = ln(1 - P) / ln[1 - (Insert / Genome)]
988 = ln(1 - 0.99) / ln[1 - (20,000 / 4,300,000)] in bacteria
(where 529,592 clones would be needed to find a gene in the human genome)
where
N = the number of independent clones to screen.
P = the probability that a clone will be represented in the library.
Insert = the size of the average cloned fragment in bp.
Genome = the size of the target genome in bp.
Polymerase Chain Reaction (PCR Movie) allows us to amplify a small portion of the genome and selectively clone that region.
Fig. 1.2 The basic steps in the polymerase chain reaction.
Amplification is accomplished by cycling between three temperatures: Denaturing (94°C), Annealing (40°C), and Extension (72°C). The themostable Taq polymerase is most functional at 72°C and stable for a few minutes even at 94°C.
The table below shows the amplification of the first copy, which has a primer at one end of the fragment, vs the discrete fragment, which has primer define ends at both sides of the fragment. Note that amplification usually results in a 10,000 fold increase of discrete fragments which should ideally be attainable in about 14 cycles. Even with more cycles, the limited number of Taq polymerase molecules prevents further exponential increase.
Figure 1.5 PCR allows the selective amplification of a portion of the genome.
Figure 9.13 PCR fragments can be cloned into either TA cloning vectors or vectors with specific restriction enzyme sites.
There are limitations when working with PCR:
1- Taq polymerase does not proofread and therefore is more prone to introduce errors.
2- You need to know DNA sequence of primer areas in order to design specific primers.
3- You are limited to about 40,000 bp during the amplification (10,000 usual high end).