Recombinant DNA Cloning Introduction
Recombinant DNA cloning technology overview
Plasmid vector design
DNA / Gene Cloning procedure
Recombinant DNA (rDNA) is the artificially recombined DNA sequences or Chimera sequences that don’t exist naturally. It is normally produced by cloning of DNA sequence into plasmids. Recombinant DNA cloning technique was first proposed by Peter Lobban in the early 1970s. He came up with an idea to use G’s / C’s and A’s and T’s for cohesive ends to join two pieces of DNA together. Later, this technique was realized by several papers which discussed the ability to create variety of recombinant DNA by joining two DNA molecules together. In 1974, some scientists signed the Berg letter to address the potential safety issues regarding the recombinant DNA cloning and the moratorium letter entitled "Potential biohazards of recombinant DNA molecules” was published on Science 1974, 185:303 (July 26, 1974). With the discovery and application of restriction endonucleases that make cohesive ends of DNA fragments much easier, the DNA cloning technique eventually became a common molecular biology technique for most biology laboratories.
Plasmids are small circular DNA molecule with a single origin of replication that can propagate in bacterial host. Some plasmids are copied at the same rate as the host chromosome replication and may have single copy of the plasmid in the cell. Some plasmids may replicate at higher rate than host chromosome and therefore produce multiple copies of plasmids in the host cell. To design plasmid vector best suitable for certain applications, one has to choose carefully the DNA sequence, replication origin, selection marker such as antibiotic resistant genes, and bacterial strain.
Few factors to be considered before choosing the vector molecules:
- capacity for long foreign DNA
- copy number of plasmids
- required host range
- availability of vector borne promoter
- selection for insertion
- requirement of ssDNA or RNA
- subcloning, transcription, restriction enzyme mapping and other considerations.
Cleaving and Joining DNA Molecules
The DNA molecule is a long thin thread sufficiently rigid to be broken by shear forces in solution. Such forces include sonication by ultrasound, mechanical stirring. The sheared DNA sequences occur at random position in terms of DNA sequence. DNA can also be cleaved by non-specific endonucleases such as DNAase I. DNAase I cut DNA molecule in an random way. DNA cleaved by both mechanical cleavage and non-specific endonucleases cleavage need to be repaired before the cloning step.
Prior to 1970 no method was available to cut DNA molecule in specified positions until the finding and application of restriction endonucleases (RE). Restriction enzymes are classified in three classes according to their gene organization and the mode of action. General speaking, the specificities of the restriction enzymes are determined by 3 parameters, namely the DNA recognition sequence, the position of the cleavage sites (blunt or cohesive ends), and methylation influency.
After cutting DNA molecules with restriction enzyme, they can be joined by mainly three methods.
1. DNA ligase for cohesive ends joining.
2. T4 DNA ligase for blund ends joining.
3. Terminal deoxynucleotidyl transferase to synthesize homopolymeric 3' ss tails at the ends of the DNA fragments.
Introduction of recombinant DNA in host cells can be achieved by transformation.
General Cloning Strategies
There are several factors need to be considered to chose the cloning methods:
- simplicity and feasibility of DNA manipulation
- need to produe cleavable joints
- influence upon transcriptional or translational read through.
D. A. Jackson, R. H. Symons, and P. Berg, "Biochemical methods for inserting new genetic information into DNA of Simian Virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli," Proceedings of the National Academy of Sciences [PNAS] 1972, 69:2904.
- Molecular Biology