1. DNA Technology and Genomics
Chapter 20
DNA Technology and Genomics

2. Making multiple copies of the gene sized DNA
I. Gene Cloning
Making multiple copies of the gene sized DNA

3. 1. Restriction Enzymes
Enzymes that cut DNA at a specific location called a restriction site
Cut DNA in a reproducible way / restriction fragments
Makes sticky ends which overhang and DNA ligase fuses these pieces permanently to cloning vector’s plasmid

5. 2. Gene Cloning
Cloning Vector – original plasmid / bacteria are the host cell
Isolation of Vector and DNA to be cloned
Insertion of DNA into vector / use a restriction enzyme and ligase
Introduce cloning vector to cell
Grow Cells
Identify cell clones

8. 3. Problems Cloning Eukaryotic Genes
Clone normal way you have 2 types of cells (pro and eu) / Use an expression vector which has a prokaryotic promotor
Introns / use reverse transcriptase to make complementary DNA (cDNA) from mRNA
Use eukaryotic cells (Yeast, YAC) instead of bacteria
Use electroporation where they shock eukaryotic cells and this allows DNA in through temporary holes
Use needles and particle guns

9. 4. Genomic Library A complete set of thousands of plasmid clones
Sometimes use cDNA when making them to remove the introns / cDNA library

10. 5. Polymerase Chain Reaction (PCR)
DNA is amplified without using a cell
Use heat resistant polymerase, primers, and nucleotides
Denature (heat), Primers attach (cooling), Extending/Annealing (cooling)

11. Best Video
Ever!

12. II. DNA Analysis and Genomics
Genomics – sequencing entire genomes in hopes of studying genes and their interactions
Gel Electrophoresis – separates nucleic acids or proteins based on size, charge, etc

14. 1. Restriction Fragment Analysis
Cut DNA w/ restriction enzymes and look at the bands
Label certain DNA for observation with radioactive labels
Southern Blotting – electrophoresis is used then this technique is used to identify certain bands w/ the genes you are looking for Video

16. 1. Restriction Fragment Analysis
RFLP / Restriction Fragment Length Polymorphisms – differences in DNA sequence on homologous chromosomes that can result in different restriction fragment patterns / found in non-coding DNA
Passed down Mendelian style
Can be used to identify differences in DNA between people.

18. 2. Mapping Entire Genomes
1980, Botstein said RFLPs could be used to map the genome
Human Genome Project, 1990
Whole Genome Sequencing – Genetic map (genes, RFLPs, etc) / Physical Map / Figure out nucleotides
Venter, 1992 – started with last step and was very successful

21. 3. So We Have the Genome, Now What?
Surprisingly few genes / 20,000 – 25,000
Lots of non-coding DNA
Human genes subjected to a lot of splicing
Reinforces our evolutionary connections
Research how genes work together / DNA Microarray Assay – technique used to see what genes work under certain conditions

23. 3. Continued
How do we discover gene function? Turn off the genes and see the result.
- In vitro mutagenesis – insert changed sequence
- RNAi - ds RNA is used to break down mRNA
Proteomics – study of a full set of proteins encoded by the genome
Most human genome differences are in SNPs (single nucleotide polymorphisms) differences in single bp variations

24. III. Applications of DNA Technology

25. 1. Diagnosis of Disease PCR can be used to diagnose HIV
Identify carriers of harmful recessive alleles by using RFLPs / link certain genes to certain markers / Huntington’s disease

26. Gene Therapy Altering the genes of an afflicted person
Change bone marrow cells
Not real effective, yet
May lead to eugenics – deliberate effort to control the human populations genetic makeup

27. 3. Pharmaceutical Products
Insulin and Human Growth Hormone
Can be expensive
Make attenuate viruses for vaccines / better response

28. 4. Forensic Uses
Test blood type or tissue type / determine paternity issues, and “who dunnit”
Use RFLPs and Southern Blotting to identify a persons DNA fingerprint
- can also use microsatellites
- simple tandem repeats (STR) – small units (10-100) that repeat in different amounts for everyone

30. 5. Environmental Uses Bacteria may be made to clean up heavy metals
Bacteria may be able to mine
Sewer treatment

31. 6. Agricultural Uses
Transgenic organisms – genomes carry genes from other organisms
Ex. Cows w/ clotting factor gene secreted in the milk
How are they made? Get an egg / Clone desired gene / Inject in egg / Hope it integrates / Implant eggs
Plants are easier to work with because any cell may develop into an adult
Use Ti plasmid / only works w/ dicots
Herbicide resistance / “yellow” rice / plants that fix their own nitrogen

32. Should we be doing this?