1. Chapter 14: Genetic Engineering -Modification of the DNA of an organism to produce new genes with new characteristics

2. Biotechnology
Use of organisms to benefit humanity

3. Recombinant DNA technology
DNA from different organisms is spliced together
Allows scientists to make many copies of any DNA segment (clone)
Can introduce foreign DNA into cells of microorganisms

4. Recombinant DNA technology
Restriction enzymes – cut DNA
Bacteria produce for defense against viruses
Vector – transports DNA into a cell
Ex: bacteriophage
Plasmid – separate, smaller circular DNA that maybe be present and able to replicate inside bacteria
Transformation – uptake of foreign DNA by cells
How plasmids can get into bacteria

5. Bacterial Conjugation and Recombination

6. Recombinant DNA technology
Palindromic sequences – reads the same as complement, in opposite direction
AAGCTT
TTCGAA
Many restriction enzymes cut these sequences
Restriction enzymes cut on a stagger  sticky ends (can pair with complementary single-stranded end of other DNA cut with same enzyme)
DNA Ligase – links 2 fragments  recombinant DNA

7. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination 3
DNA ligase seals strands.
Recombinant DNA molecule

8. Restriction Enzymes

9. Steps of Creating a Recombinant DNA Plasmid (Basic)
1. Plasmids and desired DNA cut by same restriction enzyme
2. Mix 2 types of DNA so sticky ends pair
3. DNA ligase forms bonds between fragments

11. Figure 20.2 A preview of gene cloning and some uses of cloned genes
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2
Plasmid put into bacterial cell
Recombinant bacterium 3
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Copies of gene
Protein harvested
Figure 20.2 A preview of gene cloning and some uses of cloned genes 4
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth

12. Cloning a Gene

13. Fig
TECHNIQUE
Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
Bacteria carrying plasmids
Figure 20.4 Cloning genes in bacterial plasmids
RESULTS
Colony carrying non- recombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
One of many bacterial
clones

14. Cloning DNA Genome – total DNA per cell
Genomic library – collection of DNA fragments more or less representative of all DNA in genome
Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules

15. – Then we would synthesize this probe
A probe can be synthesized that is complementary to the gene of interest
For example, if the desired gene is
– Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5

16. DNA Probe

17. Using a DNA probe

18. Polymerase Chain Reaction (PCR)
Can amplify a small sample of DNA quickly
DNA replication in vitro
2 strands separated by heating so special heat-resistant DNA polymerase called Taq polymerase used (thermophile)
MAJOR BONUS: Only specific sequences can be replicated
Study: crime scenes, archaeological remains

19. PCR

20. Gel Electrophoresis
Separates fragments like DNA, RNA or polypeptides (they carry charge and can migrate in an electrical field
RNA and DNA (-) --- so they move to (+) pole
Smaller fragments go further
Compare sample to standard
Usually “blot” - transfer DNA from gel to nitrocellulose filter for further analysis
DNA Fingerprinting

21. Gel Electrophoresis

22. Mixture of DNA mol- ecules of different sizes
Fig. 20-9a
TECHNIQUE
Power source
Mixture of DNA mol- ecules of different sizes –
Cathode
Anode +
Gel 1
Power source
Figure 20.9 Gel electrophoresis – +
Longer molecules 2
Shorter molecules

23. Fig. 20-9b
RESULTS
Figure 20.9 Gel electrophoresis

27. DNA Fingerprint

28. Transgenic Organisms
Plants and animals in which foreign genes have been incorporated
Animals
Inject DNA into nucleus of egg or stem cell
Eggs implanted in uterus; stem cells injected into blastocysts + then implanted into foster mother
Plants
Disease resistance
Pesticide resistance

29. Transgenics

30. From bone marrow in this example
Fig
Embryonic stem cells
Adult stem cells
Early human embryo at blastocyst stage (mammalian equiva- lent of blastula)
From bone marrow in this example
Cells generating all embryonic cell types
Cells generating some cell types
Cultured stem cells
Different culture conditions
Figure Working with stem cells
Different types of differentiated cells
Liver cells
Nerve cells
Blood cells

31. TECHNIQUE RESULTS Mammary cell donor Egg cell donor
Fig
TECHNIQUE
Mammary cell donor
Egg cell donor 1 2
Egg cell from ovary
Nucleus removed
Cultured mammary cells 3
Cells fused 3
Nucleus from mammary cell 4
Grown in culture
Early embryo
Figure Reproductive cloning of a mammal by nuclear transplantation
For the Discovery Video Cloning, go to Animation and Video Files. 5
Implanted in uterus of a third sheep
Surrogate mother 6
Embryonic development
Lamb (“Dolly”) genetically identical to mammary cell donor
RESULTS

32. Fig
Figure CC, the first cloned cat, and her single parent

33. Cloning Video

34. GE Plants

35. Application of GE Human proteins Human treatments for disease Vaccines
Insulin
Hormones - HGH
Human treatments for disease
Multiple sclerosis, certain cancers, heart attacks, forms of anemia
Vaccines

36. Fig
Figure Goats as “pharm” animals

37. Fig
(a)
This photo shows Earl Washington just before his release in 2001, after 17 years in prison.
Source of sample
STR marker 1
STR marker 2
STR marker 3
Figure STR analysis used to release an innocent man from prison
For the Discovery Video DNA Forensics, go to Animation and Video Files.
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b)
These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

38. Forensics