Purifying DNA from Living Cells

Genetic engineers require 3 types of DNA. The total cell DNA, plasmid DNA, and phage DNA. M13 is the exception, where plasmids rather than phage techniques are used.

To extract DNA, we need to follow the following steps:

1. Harvest bacteria/viruses
2. Cell is broken to release their contents
3. Cell extract is treated to remove all components except the DNA
4. The resulting DNA solution is concentrated

Manipulating DNA with enzymes

Once pure sample are prepared, we need to construct recombinant DNA molecules. To prepare recombinant DNA, vector DNA is cut at specific places, desired DNA is inserted at then the DNA is joined. Cutting and joining are done by restriction enzymes. DNA manipulative enzymes can be grouped into 5 classes depending on the type of reaction they catalyze:

1. Nucleases: are enzymes that cut, shorten, or degrade nucleic acid molecules
a. Exonucleases: remove nucleotides one at a time from the end of a DNA molecule - Exonuclease III
b. Endonucleases: are able to break internal phosphodiester bonds within a DNA molecule - Bal31
2. Ligases: join nucleic acid molecules together
3. Polymerases: make copies of molecules
4. Modifying enzymes: remove or add chemical groups
5. Topoisomerases: introduce or remove supercoils from covalently closed circular DNA - not used in genetic engineering

Polymerases
DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template. There are 4 types of polymerases used in genetic engineering:

1. Basic reaction: a new DNA strand is synthesized 5' to 3'
2. DNA polymerase I: fills in the nicks (open spaces) and continues replacing existing nucleotides - Taq DNA polymerase
3. Klenow Fragment: only fills in the nicks
4. Reverse transcriptase: uses a template of RNA to create DNA. Useful in cDNA techniques

Type II restriction endonuclease
Each type II restriction endonuclease cuts DNA and specific nucleotide sequences and nowhere else. This is why these enzymes are very important in gene cloning. Some make blunt cuts while others make sticky (cohesive) ends. Examples: EcoRI, BamHI, HindIII, etc.