DNA cloning

Why DNA clone?

• LArge scale protein production

• Derive gene function and characteristics

• Elucidate if mutation change gene function

• Genetic fingerprinting

How do you DNA clone?

• Start with DNA sequence of the gene of interest

• Design PCR primers to clone the gene

• Ligation

• Transformation of competent cells with plasmid

• Selection of bacterial clones

What is origin of replication?

allows the plasmid to replicate inside the bacterium

What is antibiotic resistance gene?

Confers resistance to the bacteria containing the plasmid. This allows you to select only for the bacteria that you have the successfully introduced the plasmid into

What is the selectable marker?

can often have another that allows for selection of the plasmid, eg another antibiotic resistance gene

What is the promoter?

Chosen to produce high expression of the inserted gene - often preferable to using the promoter that was part of the cloned gene

What are 5’ and 3’ primer sites?

• Defined vector-based primers you can used to confirm the size of the inserted gene - no inserted gene = v.small products

What are restriction sites?

Specific DNA sequences cut by specific restriction nucleases to allow introduction of foreign DNA

What are useful rules for primers?

• Aim for primers to be 15-30 bp long

• Optical G-C content between 40-60%

• Take care with palindromes, di-nucleotide repeats and runs of more than 4

What is competence?

Ability of a cell to alter its genetics by taking up extracellular DNA from its environment in the process called transformation.

DH5-alpha cells are treated to make them transiently permeable to DNA

What are considerations for primers?

Optimal melting temperatures (Tm) for primers range between 52-58°C (can be expanded to 45-65°C)

• Final Tm for both primers should differ by no more than 5°C.

• The 3' end of primers should contain a G or C in order to clamp the primer and prevent "breathing" of ends, increasing priming efficiency.

• 3' ends of a primer set, should not be complementary to each other (primer dimers)

• 3' end of a single primer should not be complementary to other sequences in the primer (hairpin loop structure)

What is calcium chloride transormation?

• The addition of calcium chloride to a E. coli promotes the binding of plasmid DNA to lipopolysaccharides (LPS)

• Positively charged calcium ions attract both the negatively charged

• DNA backbone and the negatively charged groups in the LPS inner core

• The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (4oC) are heated to a higher temperature (42oC) for a short time

What are the controls?

• Uncut plasmid control

• Cell only control (what if the cells are Ab resistant?)

• Transformation control: circular empty vector to measure

transformation efficiency

• Agar plate only (sterility)

• Sequence your PCR product (correct gene, correct orientation,no Taq-induced mistakes...) and/or your isolated clone construct?

• If controls and sequence correct: can still optimise ligation/transformation or repeat from step 1

What is next with successful clones?

• Sequence

• Store your successful clones in glycerol

• Functional studies

• Bulk up the clone to perform protein extraction, purification DNA analysis.