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

2. DNA Technology
Genetic Engineering – direct manipulation of genes for practical purposes
Scientists can make recombinant DNA and then introduce it into cultured cells that replicate the DNA and may express its genes, yielding a desired protein.
Often, E. coli is used as the “host”
Biotechnology – manipulations of organisms to make useful products.
Recombinant DNA – genes from two different sources are combined in vitro (outside the living body in a lab).

3. Techniques for Gene Cloning

4. Techniques for Gene Cloning
Plasmid – circular DNA that replicates within bacterial cells (separate from bacterial chromosomes)
Isolation of plasmid
Gene insertion into plasmid
Plasmid put into bacterial cell
Cell cloned
Identification of desired clone
Copies of the gene/Copies of the protein
Used in gene therapy (HGH) and agriculture (resistance to pests)

5. Restriction Enzymes
Restriction enzymes cut up foreign DNA – recognize short nucleotide sequences
Restriction site – area recognized by restriction enzyme (5’ – 3’)
Restriction fragments – nucleotide sequence left after DNA has been cut.
Sticky end – single-stranded DNA fragment; DNA ligase can permanently fuse fragments together

6. Restriction Enzymes

7. Transgenic organism
An organism that contains genes from other species.
Results from the formation of recombinant DNA.
Ex: Fruit fly w/firefly gene for luciferase

8. Bacterial Transformation
Another method used to form transgenic organisms
Plasmids inserted to change the proteins produced by cells
Lab 6A
pGLO

9. DNA Cloning

10. DNA Cloning Cloning Vector – the original bacterial plasmid
Procedure: Cloning a Eukaryotic Gene in a Bacterial Plasmid
The E. coli plasmid has two useful genes: ampR (resistant to ampicillian) and lacZ (catalyses hydrolysis of lactose)
Isolation of vector (bacterial plasmid) and gene-source DNA (human tissue)

11. DNA Cloning Insertion of DNA into the vector
Restriction enzyme cuts plasmid DNA at single restriction site
Cuts human DNA making thousands of fragments
One of these fragments carries the gene we want
The restriction enzyme creates compatible sticky ends on both the human DNA fragments and the plasmid DNA
The DNA and plasmids are combined – DNA ligase “glues” the pieces together

12. DNA Cloning Introduction of cloning vector into cells Cloning of cells
Transformation – uptake of naked DNA from surrounding solution
Some acquire the desired DNA; others take up other DNA
Cloning of cells
Put bacterial cells on a plate containing medium with ampicillin and X-gal
Bacteria with recombinant plasmids carrying foreign DNA with form white colonies on medium containing ampicillin and X-gal

13. DNA Cloning
Identification of cell colonies carrying the gene of interest
Nucleic acid hybridization – base pairing between the gene and a complementary sequence using a nucleic acid probe
The probe can be labeled with a radioactive isotope or a fluorescent tag
Then the strand of DNA is separated by denaturing it with heat or chemicals
The desired DNA can then be isolated an grown in large amounts

14. Cloned Genes Are Stored in DNA Libraries
A complete set of thousands of recombinant plasmid clones
Can be stored as a plasmid library (bacterial cells) or phage libraries (viruses)

15. Polymerase Chain Reaction (PCR) Clones DNA In Vitro
Any piece of DNA can be quickly copied many times without using a host cell
DNA is placed in a test tube with:
Special DNA polymerases (first isolated from bacteria growing in hot springs – the enzyme does not denature with heat)
Supply of nucleotides
Synthetic single-stranded DNA that serves as primers
Can be used to amplify a specific gene prior to cloning

16. PCR can be used to quickly amplify DNA from a 40,000 year old woolly mammoth; tiny amounts of blood, tissue, or semen from a crime scene; and DNA of viral genes from HIV-infected cells

17. DNA Analysis and Genomics
Suppose we have cloned a DNA segment carrying a human gene of interest – now we begin to ask some far-reaching questions:
Do genes differ in different people, and are certain alleles associated with a hereditary disorder?
Where and when is it expressed?
What is its location within the genome?
Evolutionary questions - Species to species differentiation?

18. DNA Analysis and Genomics
Genomics – the study of whole sets of genes and their interactions
Gel Electrophoresis – method of sorting DNA molecules into bands, each consisting of molecules of the same length
Separation on the basis of size, electrical charge, and other physical properties
Restriction fragments – detect DNA differences that affect restriction sites
Gel Electrophoresis used to sort DNA fragments by size after being treated with restriction enzymes

19. Gel Electrophoresis
Digest two different DNA samples with same restriction enzyme (the samples should differ in one or more restriction site)
Use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest (usually used in combination with Southern blotting)

20. Gel Electrophoresis

21. Gel Electrophoresis

22. Gel Electrophoresis
The DNA fingerprints below represent four different individuals.
Which of the following statements is consistent with the results?
B is the child of A and C.
C is the child of A and B
Which of the following are probably siblings?
A and D
C and D

23. Entire Genomes Can Be Mapped at the DNA Level
Human Genome Project – begun in 1990 to map all the human genes
Surprisingly, there are few genes in the Human Genome

24. Practical Application of DNA Technology
Diagnosis of Diseases
PCR can be used to amplify and detect HIV DNA in blood or tissue samples
Scientists can diagnose hundreds of human genetic disorders before the onset of symptoms, even before birth
We can identify symptomless carriers of potentially harmful recessive alleles
Genes have been cloned for many human diseases, including hemophilia, cystic fibrosis, and Duchenne muscular dystrophy

25. Practical Application of DNA Technology
Human Gene Therapy – the alteration of an afflicted individual’s genes
Theoretically, it is possible to replace or supplement the defective gene with a normal allele
The new allele could be inserted into the somatic cells of the tissue affected by the disorder
The cells that receive the normal allele must be ones that will continue to reproduce during the patient’s life – one example is stem cells from bone marrow that give rise to all blood and immune cells

26. Practical Application of DNA Technology
Use of stem cells to promote healthy bone marrow

27. Practical Application of DNA Technology
Pharmaceutical Products
Mostly proteins
Human insulin
Human growth hormone (HGH)
Tissue plasminogen activator (TPA) – protein that helps dissolve blood clots (very expensive)
Drugs that mimic a receptor protein that HIV binds to in entering white blood cells – the HIV binds to the drugs and fails to enter the blood cell
Vaccines

28. Practical Application of DNA Technology
Reproductive Cloning
Producing complete, genetically identical animals
Usually talked about in the press
DOLLY the SHEEP.

29. Practical Application of DNA Technology
Forensic Uses
DNA fingerprinting
Environment Uses
Sewage treatment plants rely on the ability of microbes (bacteria and protists, mainly) to degrade many organic compounds into nontoxic forms
Agricultural Uses
Animal Husbandry and “Pharm” Animals
Transgenic organisms – their genomes carry genes from another specie

30. Practical Application of DNA Technology
Agricultural Uses (continued)
Genetic engineering in plants (editable cotton seed – 2006)
Delayed ripening
Resistance to spoilage and disease
Increase nutritional value (“golden rice”)

31. WITH EVOLUTION IN THIS WAY?
SO THE BIG QUESTION IS:
SHOULD WE INTERFERE
WITH EVOLUTION IN THIS WAY?