Amplifying DNA: PCR & cell-based DNA cloning. The importance of DNA cloning: Current DNA technology is based on two different approaches:
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a. Specific amplification (DNA cloning) which involves cell-based DNA cloning (involving a vector/replicon and a suitable host cell) andin vitro DNA cloning (PCR)
b. Molecular hybridization where the DNA fragment of interest is specifically detected using a mixture of different sequences
>3 bp should be avoided.
a.short sizes of amplified products (<5 kb). This is solved by doing Long-range PCR (up to tens of Kb long) which uses a mixture of two heat stable polymerases that provide optimal levels of DNA synthesis as well as a 3’ -> 5’ exonuclease activity.
b.low yields of amplifications which is resolved by cloning the PCR amplified DNA fragment in a vector then propagating the vector in a cell based system (clone by A/T cloning or by using anchored PCR primers).
PCR has 3 major advantages:
- robust (possible to amplify DNA from damaged tissues or degraded DNA)
- Hot-start PCR
- Nested PCR
- Touch-down PCR
- Construction of recombinant DNA molecules. Involves the use of endonuclease restriction enzymes, ligation, and a replicon (vector).
- Transformation in appropriate host cells.
- Selective propagation of cell clones. This step takes advantage of selectable markers.
- Isolation of recombinant DNA from cell clones followed by molecular characterization (such as restriction enzyme analysis).
Two types of libraries are known:
- Genomic library. To be representative of the entire genome, the library should be >4GE. A genome equivalent (GE) is genome size/average insert size. In humans, for a GE=1, you need 3000Mb genome size)/40 kb (insert size) = 75,000 independent clones.
- cDNA library. Takes advantage of reverse transcriptase. Usually much smaller than a genomic library.
- screening by vector molecules which includes antibiotic resistance genes or β-galactosidase gene complementation
- Generalized recombinant screening by insertional inactivation. This can be achieved by β-galactosidase screens or suppressor t-RNA-based screens.
- Directed recombinant screening. This can be achieved by hybridization-based screening by using labeled probes or by using PCR-based screening.
4. Cloning systems for different sized DNA fragments: appropriate selectable marker system.
Three types of λ derived cloning vectors:
a. Replacement λ vectors: removal of central section of the genome and replacing by a foreign DNA fragment (up to 23 kb inserts)
b. Insertion λ vectors: modifications to allow insertional cloning of cDNA fragments into the cI gene (up to 5 kb)
c. Cosmid vectors: cos sequences of λ are inserted into a small plasmid generating a cosmid. Can take 33 – 44 kb inserts.
- centromere sequences (CEN)
- Telomere sequences (TEL)
- Autonomous replicating sequences (ARS) for replication in the yeast nucleus.
- Ampicillin resistance for propagation in E. coli
- Three markers including a suppressor tRNA gene, TRP1, and URA3 genes for selection by complementation in the appropriate yeast host cell.
5. Cloning systems for producing mutagenized DNA: 0.2 – 2.0 Megabases. YACs are propagated in yeast as a linear chromosome which becomes part of the genome and is distributed by the mitotic machinery. YACs must include:
- 5’ adds-on mutagenesis which adds specific sequences at the 5’ of the amplified product. Such sequences may include a phage promoter to drive gene expression.
- Site-directed mutagenesis which results in an amplified product with a specific base substitution to introduce a specific amino acid substitution at the protein level.
- The use of pET-3 bacterial vectors containing T7 promoter in combination with host cells carrying the gene for T7 RNA polymersae expressed under the control of the lacZ promoter (i.e. inducible by IPTG).
- The vector is designed so that the recombinant protein is fused to an endogenous protein (fusion proteins).
- Use an affinity tag so that the recombinant fusion protein be purified by affinity chromatography. Two affinity tagging systems are GST-glutathione affinity and polyhistidine-nickel ion affinity.