Amplifying DNA: PCR & cell-based DNA cloning

1. The importance of DNA cloning:

• Current DNA technology is based on two different approaches:a. Specific amplification (DNA cloning) which involves cell-based DNA cloning (involving a vector/replicon and a suitable host cell) and in vitro DNA cloning (PCR)

b. Molecular hybridization where the DNA fragment of interest is specifically detected using a mixture of different sequences

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• 2. Polymerase Chain Reaction (PCR): features & Applications:

• Template DNA: DNA (linear or circular) or cDNA (complementary DNA produced from produced mRNA by reverse transcriptase)

• Primers: pairs of oligonucleotides each 18-25 nucleotides long; 40%-60% GC content; melting temp of both should not differ by >5oC; 3’ terminal sequences of any primer should not be to any sequences of the other primer in the pair; self-complimentary sequences (inverted repeats) of

>3 bp should be avoided.

• Cycling nature & exponential amplification: denaturation; primer annealing; and DNA synthesis (extension).

• Regular Taq DNA polymerase lacks 3’ -> 5’ exonuclease activity needed to provide proof-reading function.

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• PCR has two limitations: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).

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• General Applications of PCR:

PCR has 3 major advantages:

- rapid

- sensitive

- robust (possible to amplify DNA from damaged tissues or degraded DNA)

• Primer specificity is very important in PCR. Several modifications have been developed to reduce nonspecific binding (see Box 5.1):- Hot-start PCR- Nested PCR- Touch-down PCR

• The correct base pairing at the extreme 3’ end of bound primers is a requirement for producing a PCR product. This allowed the use of PCR to distinguish between alleles of the same gene that differ in a single nucleotide (allele-specific PCR). This method is known as ARMS (amplification refractory mutation system).

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• Degenerate oligonucleotide primed PCR (DOP-PCR) allow the amplification of a different but closely related genes (novel genes) at the same time.

• Indiscriminate amplification of whole genomes can be performed using linker-primed PCR (ligation adaptor PCR).

• PCR could be used to amplify unknown DNA sequences neighboring a known sequence. Such methods include anchored PCR, inverse PCR, RACE-PCR.

3. Principles of cell-based cloning:• Four steps in cell-based cloning

- 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).

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• Endonuclease restriction enzymes type II (RE), are a powerful tool (molecular scissors) used in restricting target DNA (whole genome or plasmid) into smaller DNA fragments. The restriction of a DNA double helix molecule may result in a blunt end or a cohesive end terminus (sticky end generating a 3’ or 5’ single strand overhang). See Table 5.1

• RE are used to generate recombinant linear molecules (concatemers) or circular molecules (cyclization).

• Simple cloning vectors include bacterial plasmids and bacteriophages.

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• Recombinant DNA molecules are transferred into appropriate host cells (e.g. bacteria) for propagation. Normally a single recombinant DNA exists per cell but sometimes co-transformation may result in two or more recombinant DNA molecules per host cell.

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• DNA libraries are a collection of clones that represent the entire genome of an organism.

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.

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• Selection of recombinant clones necessitates the use of an appropriate selectable marker system.- 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.

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4. Cloning systems for different sized DNA fragments:

• Such cloning systems normally include an antibiotic resistance gene (to enable screening for presence of vector) and a marker gene with a multiple cloning site (to enable screening recombinant clones).

• See Table 5.2 for different cloning vectors and the DNA insert sizes that each could accommodate.

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• Lambda and cosmid vectors are used in cloning moderately large DNA fragments in bacterial cells.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.

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• Bacterial Artificial Chromosome (BAC) vectors are used to clone large fragments (>300 kb). Low copy number (1-2 copies/cell) fertility factor (F-factor) plasmids are used for this purpose.

• Bacteriophage P1 vectors and P1 artificial chromosomes (PACs). Components of the P1 phage are included in a circular plasmid and can accommodate up to 122 Kb DNA fragments.

• Yeast Artificial Chromosomes (YACs) permit the cloning of 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:- 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.

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5. Cloning systems for producing mutagenized DNA:

• Cell-based oligonucleotide mismatch mutagenesis can be used to generate a specific nucleotide substitution in a coding sequence of a gene. This is achieved by using M13 vectors to generate single-stranded recombinant DNA

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• Production of single-stranded DNA for use in sequencing is obtained using M13 vectors or phagemid vectors.

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• PCR-based mutagenesis could be used to achieve two types of changes:- 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.

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6. Cloning systems designed for gene expression:

• Bacterial cells are used as hosts for recombinant expression vectors designed for the production of large amounts of a recombinant protein (fusion proteins or tagged proteins).

• Problems with overexpression in bacteria include toxicity of large amounts of the recombinant protein, lack of posttranslational processing, inability to synthesize very large mammalian proteins, and protein folding and solubility.

• To solve the above mentioned problems:

- 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.

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