Molecular Cloning Protocols

Molecular cloning protocols encompass the procedures used for defining, isolating and replicating a DNA sequence. Traditional restriction and ligation cloning protocols initiate DNA fragmentation with restricted endonucleases (enzymes that cut DNA strands at restriction sites), DNA fragment ligation (the repair of discontinuities in DNA molecules), transfection (the introduction of nucleic acid into a cell body) and selection (the choosing of individual genomes for replication).

Isolation

• Protocols for the isolation of a DNA fragment, the first step in molecular cloning, often incorporate a polymerase chain reaction (PMR), which uses cycles of heating and cooling to amplify pieces of DNA. To reach the goal sequence size, other protocols include DNA sonication, reaction enzyme digestion and the use of chemically synthesized oligonucleotides. These protocols vary depending on the magnitude and amount of isolated DNA needed.

Ligation

• Protocols for ligation involve using an enzyme, DNA ligase, to join DNA molecules together with a covalent bond. Protocol dictates combining DNA fragments, the cloning vector, a ligation buffer, the DNA ligase and sterilized water in a microcentrifuge tube and incubating overnight at 4 degrees Celsius.

Transfection

• Protocols for transfection, or infusing DNA into a cell using a non-viral means, often involve injecting DNA directly into the cell cytoplasm. Other methods include using chemical reagents, such as calcium phosphate and lipids, to deliver the transfection complex through the cell membrane. This method tests the effects of gene modification on the functioning of specific genes.

Selection

• Selection, or screening, protocols determine which cells successfully held the DNA insert and indicate which cells need isolation. A centrifuge harvests cells that contain the transfected DNA, which are then incubated in a lysozyme (a naturally occurring enzyme) buffer and treated with an alkaline detergent, giving the proteins and membranes solubility. Using acetate to precipitate the proteins, a centrifuge and cheesecloth filter the DNA-containing supernatant (the soluble liquid remaining from a centrifuged compound). The supernatant is precipitated with polyethylene glycol, centrifuged, and suspended in a cesium chloride and ethidium bromide buffer. The ethidium bromide stains the DNA according to density, and using a long-wave UV light, the lower-band DNA is extracted with a five cc syringe. An equilibrated ion exchange column separates the DNA from the ethidium bromide and cesium chloride, and the final DNA pellet is suspended in a buffer solution and detected on agarose gel electrophoresis.