1. DNA Technology & Genomics
Started in 1970s
The transfer of specific genes (segment of DNA) from one species to another.

2. Synonymous Terminology
Recombinant DNA technology
Genetic modification/manipulation (GM)
Bio technology
Gene splicing

3. Recombinant DNA DNA in which genes from 2 different sources are linked
ANDi

4. Genetic engineering
Direct manipulation of genes for practical purposes

5. Biotechnology
Manipulation of organisms or their components to perform practical tasks or provide useful products

6. How do we get the gene from species A to species B?
A vector is used to carry the gene into the host nucleus.

7. What are vectors commonly used in GE?
Plasmid (of bacteria)
Viruses
Shotgun technique (blindly shoots tiny particles coated with the gene into the host cells)

8. DNA Cloning   Restriction enzymes (endonucleases):
in nature, these enzymes protect bacteria from intruding DNA
they cut up DNA at specific sequences (restriction site)
Ex. EcoR1, BamHI, HindIII
Restriction fragments:
segments created by the cutting of DNA with restriction enzymes
Sticky end:
short extensions of exposed nucleotides from restriction fragments
DNA ligase:
enzyme that can join the sticky ends of DNA fragments

9. Bacterial plasmids in gene cloning

10. Steps for eukaryotic gene cloning
Steps for eukaryotic gene cloning
Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) using the same restriction enzyme
Insertion of gene-source DNA into the cloning vector with DNA ligase
Induce transformation of E. coli
E. coli reproduces rapidly cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest

11. How do I know which bacteria will have the plasmid?
Use a plasmid that codes for antibiotic resistance (R plasmid)
Add specific antibiotics to the agar (growth) culture
All those bacteria which do not have the plasmid will die
Only those with the recombinant plasmids can grow and multiply

12. How do we know the gene made it onto the plasmid?
Insert in the middle of a gene with a known phenotype
If the phenotype of that gene is interrupted then “great success”
Example:
pUC19 plasmid has the lacZ gene that turns X-Gal blue when expressed
Proper insertion = not blue

13. DNA Analysis & Genomics
PCR (polymerase chain reaction)
Gel electrophoresis
Restriction fragment analysis (RFLPs)
Southern blotting
DNA sequencing
Human genome project

14. Polymerase chain reaction (PCR)
Amplification of any piece of DNA without cells (in vitro)
Materials:
Heat
DNA polymerase
Nucleotides
DNA primers
Applications:
Cloning genes
Analysis of ancient DNA
Prenatal diagnosis
Genetic fingerprinting
Forensics
Paternity testing

15. (PCR) Steps
Heating : heating to 94-98°C disrupts the hydrogen bonds between complementary nitrogenous bases, yielding single strands of DNA.
Priming : lowered to 50-65°C allowing annealing of the primers to the single-stranded DNA template.
Copying : Heat stable DNA polymerase added along with supply of all four nucleotides, copies rest of fragment as in DNA replication.
Repeating the cycle : Each cycle doubles amount of DNA after twenty cycles one fragment = more than one million

16.
DNA Analysis
Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length
Small fragments travel further through the gel faster than longer fragments
Creates a DNA Fingerprint

17. Restriction fragment analysis
Restriction fragment analysis
Restriction fragment length polymorphisms (RFLPs)
Each individuals DNA has a unique sequence with specific restriction sites causing unique fragment lengths
DNA Fingerprinting

18. O.J. Simpson capital murder case,1/95-9/95
Odds of blood in Ford Bronco not being R. Goldman’s:
6.5 billion to 1
Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
8.5 billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:
21.5 billion to 1
Number of people on planet earth:
6.1 billion
Odds of being struck by lightning in the U.S.:
2.8 million to 1
Odds of winning the Illinois Big Game lottery:
76 million to 1
Odds of getting killed driving to the gas station to buy a lottery ticket
4.5 million to 1
Odds of seeing 3 albino deer at the same time:
85 million to 1
Odds of having quintuplets:
Odds of being struck by a meteorite:
10 trillion to 1

19. Finding the Gene of Interest
Finding the Gene of Interest
Southern blot technique
Fragments spread apart by electrophoresis
Gel blotted with nitrocellulose, DNA transfers to sheet
Radioactive probe poured onto nitrocellulose sheet
Only fragments with proper gene hybridize with probe

20. DNA Sequencing Determination of nucleotide sequences
DNA Sequencing
Determination of nucleotide sequences
Genomics: the study of genomes based on DNA sequences
Human Genome Project
Sequencing of the entire human genome