Introduction

DNA recombination is a technique that enables genetic engineering, hence the design of genetically modified organisms (GMOs) and cloning. Recombination refers to the action of cutting open the plasmid (restriction) and then inserting or replacing a gene into the plasmid (ligation). A recombinant plasmid has components coming from two or more. Usually, a plasmid is used as a vector of the insert. The insert (gene of interest) is obtained from DNA of other organism or chemically synthesised.

General Terms

• Insert
• A DNA fragment that contains the gene of interest, i.e. the gene that encodes the targeted protein of desired trait
• Vector
• Gene carrier. Usually a plasmid.
• Donor cell
• The cell which the gene of interest originally comes from
• Recombinant plasmid
• Plasmid that has components coming from two or more sources
• Host cell
• The cell that accept the recombinant plasmid and express the desired traits
• Selective marker
• It is an antibiotics resistance gene. It will be used to select cells successfully transformed with recombinant plasmid in transformation. (Transformation and selection will be discussed in details in the coming unit)

Overview of cloning

1. Minipreparation
2. Extract plasmid (vector and/or gene of interest) from cell
3. Restriction digestion
4. Restriction enzymes cut the plasmid DNA at a specific sequence
5. Ligation
6. DNA ligase ligates the DNA inserts to the plasmid backbone
7. The plasmid is now called a recombinant plasmid
8. Transformation
9. Put the recombinant plasmid back to the host cell

Minipreparation

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method. When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. Palleting the mixture. Then run the supernatant through the spin column. The spin column is a tube with a special filter that traps DNA. After spinning the spin column, plasmid DNA is captured by the filter paper. Discard the flow-through and dissolve the plasmid DNA on filter paper.

We can use this technique to extract plasmid containing the gene of interest or plasmid as the vector.

Restriction Digestion

Restriction enzymes cut DNA into fragments. The enzymes were originally isolated from bacteria that produce these enzymes in order to defend themselves from viral attack by destroying the viral DNA.

Restriction enzyme recognizes and cuts DNA at a specific sequence called a restriction site. The restriction site of every enzyme is unique. Many restriction enzymes make staggered cuts in the double stranded DNA, creating fragments that have extensions of single-stranded DNA. these single-stranded ends are called sticky ends because they are capable of base-pairing complementarity with other sticky ends produced by the same restriction enzyme. Other restriction enzymes cut both DNA strands in the same place, producing blunt ends.