Recombinant DNA technology

I. Restriction fragment length polymorphism's (RFLP's) and other nucleotide polymorphism's

A. Happens in regions between genes mainly

1. Restriction enzymes recognize different palandromic base sequences

2. No obvious relation to reproductive success - neutral

3. Are heritable

B. Use electrophoresis to detect

C. Applications

1. DNA fingerprinting

a. Uses RFLP's or other nucleotide polymorphism's

b. Often uses VNTR's variable length tandem repeats = minisatellites

2. Human population studies/ Forensics: note comments in text p 324-325

3. Creation of gene libraries/gene mapping - positional cloning

a. Use RFLP markers with pedigrees to do linkage studies

b. note tactic to exclude RFLP markers not associated with the disorder: exclusion map

c. Clone RFLP's linked to the gene and hunt for protein coding amino acid sequence

d. CF case study and genes identified by positional cloning pp 317-318

4. Mapping at the chromosomal level Fluorescence in situ hybridization (Fish)

a. uses cloned gene as probe to bind to chromosome

b. analogous to southern blot

5. Prenatal and reimplantation testing

6. DNA chips: silicon wafers with probes for different genes

a. Used to study gene expression: Functional genomics

b. Rapid assays and biosensors

c. Example of use

D. Human genome project

1. Size of the human genome

2. Objective of the project

a. Produce detailed genetic maps of the chromosomes

b. Physical mapping in terms of bp distances

c. Creating gene libraries for the genes

d. Sequencing the genome

E. Legal and ethical issues

1. Privacy issues

2. Ownership issues: who owns genetic information? Patent rights?

F. Recombinant DNA technology and bioengineering

1. Transgenic organisms: gene cloned from one species used to alter genotype of another species

a. Bacillus thuringiensis gene for delta-endotoxin proteins

b. Germ line transformation: injection of DNA vector into germ line nucleus;embryonic stem cells

c. Some plasmids from plant bacteria capable of joining plant genome and serving as vectors

2. Examples of genetically engineered organisms

a. Giant salmon with active promoter for growth hormone

b. Nutritionally engineered rice: add genes for B carotene synthesis enzymes

c. Production of useful proteins: GH, insulin, blood clotting factors

3. Genetic engineering with retroviruses

a. review retroviruses

b. use in gene therapy...still problematical

pdg 11/29/01