1. Chapter 9 Molecular Genetic Techniques and Genomics
Presented by:
Troy Coke

2. What and Why?
What?: A gene of interest is inserted into another organism, enabling it to be cloned, and thus studied more effectively
Why?: Detailed studies of the structure and function of a gene at the molecular level require large quantities of the individual gene in pure form

3. Terms to Know
Vector: an autonomously replicating genetic element used to carry DNA fragments into a host, typically E. coli, for the purpose of gene cloning
Plasmid vector
Bacteriophage gamma vector
Recombinant DNA: any DNA molecule composed of sequences derived from different sources

4. Cleavage
Restriction enzymes: endonucleases produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction sites, and then cleave both DNA strands at this site

6. Inserting DNA Fragments
DNA fragments are inserted into vector DNA with the aid of DNA ligases
Ligases catalyze the end-to-end joining of DNA fragments
Blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligase than sticky ends

8. Use of E. Coli Plasmid Vectors
Plasmids are circular, double stranded DNA molecules separate from a cell’s chromosomal DNA
Suitable for cloning isolated DNA fragments
Used for fragments ranging from a few base pairs up to ~ 20 kb
Polylinkers increase a vector’s versatility

10. Bacteriophage Gamma Vectors
Approximately 1000 times more efficient than plasmid vectors
Permit efficient construction of large DNA libraries
Can take up DNA fragments up to ~ 25 kb

12. Summary
Plasmid vectors are relatively inefficient, but suitable for cloning isolated fragments
Bacteriophage vectors are used to construct DNA libraries
mRNA is used in higher eukaryotes

13. Thank You! Questions?