BIOL 200 - DNA Cloning

Overview of recombination DNA

Vector + DNA → Recombinant DNA → Replication of recombinant DNA within host cells → Isolation, sequencing, and manipulation of pure DNA fragments

Plasmids characteristics

• Most common types of vectors

• Replicate independently of chromosomes

• Most are found in bacteria (E.coli)

Restriction enzymes role

Cut phosphodiester bond in symmetrical fashion in both strands, creating sticky ends of DNA

DNA Ligase Role

Joins the two sources of DNA together, creating recombinant DNA

Plasmid structure

• Circular structure

• Contain origin of replication

• Ampr - Which codes for antibiotic resistance

• Polylinker - 6 nucleotides in length, giving us a desired sequence once every 4^6

How are E.coli plasmids created?

Mixed and fused together with the presence of CaCl2 and heat

How are recombinant bacteria cultured?

Placed on nutrient agar plates containing ampicillin. Only cells containing the plasmid will undertake ampicillin and therefore survive.

Types of DNA libraries

1. Genomic libraries (consist of chromosomal DNA)

   1. cDNA libraries (which represent mRNA in a given sample)

Reverse Transcriptase use

Used in reverse transcription, in which a RNA molecule becomes a cDNA (complementary DNA molecule)

Recombinant DNA uses

1. Microarray

2. In situ hybridization

3. Expression of exogenous genes

4. Cluster analysis to identify coordinately regulated proteins

5. Production of eukaryotic proteins in E.Coli with lac promoter

6. Cloned genes are also expressed in cultured animal cells

Microarray

Measures levels of expression for multiple RNAs simultaneously using fluorescent labels

In Situ Hybridization

Reveals the location of multiple RNAs within a tissue using fluorescent labels

Transient transfection

Uses a plasmid with a viral origin of replication in order to express cloned genes in animal cells. Plasmid eventually leaves the cell

Stable transfection (transformation)

cDNA in plasmid is placed directly in the chromosome of the animal cell.