1. Chapter 20 – DNA Technology and Genomics

2. Recombinant DNA
Def: DNA in which genes/nucleotide sequences from 2 different sources are combined in vitro into the same DNA molecule
Genetic engineering: direct manipulation of genes for practical purposes
Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

3. DNA Cloning
Permits the production of multiple copies of a specific gene or DNA segment
Most practical applications utilize bacteria and bacterial plasmids

4. Overview of gene cloning with a bacterial plasmid

5. Creating Recombinant DNA
Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific (flash movie)
Restriction site: recognition sequence for a particular restriction enzyme
Restriction fragments: segments of DNA cut by restriction enzymes in a reproducible way
Sticky end: short extensions of restriction fragments
DNA ligase: enzyme that can join the sticky ends of DNA fragments
Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids

6. Restriction Enzymes

7. Cloning Cloning of human DNA using a bacterial plasmid
Same technique used to create plasmid for our transformation lab

8. Cloning a Gene

9. Identifying Clone Cells
How identify cells?
Insert ampicillin resistance gene (ampr) into plasmid, culture cells on ampicillin, if live, have incorporated plasmid
Nucleic acid hybridization, uses radioactive nucleic acid probe

10. Genomic Libraries
Can store these genes in bacteria or viruses to create a genomic library
a cDNA or complimentary DNA library is made in vitro by reverse transcription of all the mRNA produced by a particular cell
cDNA is used to create plasmids

11. Rainbow (donor) and CC (clone)
Organismal Cloning
Producing one or more organisms that are genetically identical to the parent that donated the single cell
Important because can generate stem cells – unspecialized cells that can reproduce indefinitely and differentiate into specialized cells
Stem cells have great potential for regenerating damaged tissues

12. Cloning Technique
Technique used to create/clone Dolly, the first mammal ever clonned.

13. Stem Cells
Difference between adult and embryonic stem cells

14. DNA Anaylsis and Genomics
Gel Electrophoresis
Restriction Fragment Length Polymorphism or RFLP analysis
Southern Blot
Polymerase Chain Reaction or PCR
DNA Sequencing
DNA microarray of gene expression

15. Gel Electrophoresis
Used to separate nucleic acids or proteins that differ in size or electrical charge
Nucleic acids carry a negative charge (why?) so they will move towards the positive end of the porous gel
1. samples loaded in gel at negative end
2. an electrical current “pulls” the fragments towards the positive end, smaller fragments move further than larger ones
DNA binding dye used to visualize the fragments

16. Restriction Fragment Length Analysis
Useful in comparing two different DNA molecules, like 2 alleles of a gene
If have a small change in bp sequence, restriction enzymes will cut at different places producing different fragments
Example: β-globin allele (sickle-cell)

17. Restriction Fragment Length Analysis (cont.)
What if when comparing different genomic DNA there are too many bands for analysis? Use southern blotting
Southern Blot – combines gel electrophoresis and nucleic acid hybridization (next slide)
RFLP (restriction length polymorphism) – differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns, found in noncoding regions of the DNA; because these are inherited they can be used as genetic markers for making linkage maps

18. RFLP and Southern Blotting

19. PCR Polymerase Chain Reaction
Method of amplifying samples of DNA in vitro
Requires: dsDNA, heat-resistant DNA polymerase, RNA primers, DNA nuclotides
After 20+ cycles target DNA vast outnumbers all other DNA sequences

20. Genome Mapping Human Genome Project – completed in 2003
3 step approach
Genetic (linkage) mapping – uses recombination frequencies, the ordering of genetic markers like RFLPs
Physical mapping – cutting the DNA with restriction enzymes and putting them in order, make fragments overlap and use probes
DNA sequencing

21. DNA Sequencing Dideoxy chain-termination method
ssDNA incubated with tagged nucleotides
DNA synthesis is stopped with tagged nucleotide
Labeled strands separated through a gel and analyzed with a fluorescence detector

23. Gene Expression Levels
DNA microassay – can test thousands of genes simultaneously to determine which are expressed at any one time (tissue type, environmental conditions, stages of development, etc…)

24. DNA micro assay cont.

25. Applications of DNA Technology
Medical
Diagnosis of diseases
Human gene therapy
Pharmaceutical Products
Forensic Evidence
Environmental Cleanup
Agricultural
Animal husbandry
Genetic engineering in plants