1. DNA Technology

2. Genetic Engineering
Genetic engineering is the direct manipulation of genes for practical purposes
Manufacturing protein products
Biotechnology is the manipulation of organisms or their components to make useful products
Selective breeding of cattle
Making wine and cheese
Genetic engineering/biotechnology are major areas of research in biology today

3. DNA Cloning Preparing identical copies of a specific gene
Can be used to produce a protein product (gene  protein)
Copies of a gene can be used for:
Basic research
Endowing an organism with a new metabolic capability
pGLO

4. Practical Applications of Gene Cloning
Gene for pest resistance inserted into plants
Genes used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
Others??

5. Restriction Enzymes & Recombinant DNA
enzymes that cut DNA molecules at specific locations
Look for specific sequences:
Ie: ATTACCGTA
Recombinant DNA –
DNA in which nucleotide sequences from 2 different sources (often different species) are combined into the same DNA molecule

6. Recombinant DNA

7. Recombinant DNA Sticky Ends:
Single-stranded ends of a restriction fragment (piece of DNA)
Sticky ends of restriction fragments can form hydrogen-bonded base pairs with other sticky ends
These bonds are sealed with DNA ligase

8. Plasmids
A plasmid is a DNA molecule that is not part of the chromosome in bacteria
Often give bacteria an “advantage” such as antibiotic resistance
Plasmids are often used as cloning vectors
Take foreign DNA into a cell and replicate there

10. Polymerase Chain Reaction (PCR)
PCR is a process used to copy a segment of one or many DNA molecules quickly in vitro (in a test tube)
PCR can make BILLIONS of copies of a target segment of DNA in a few hours

11. Polymerase Chain Reaction (PCR)
Denaturation
Mixture is heated to separate the DNA strands
2. Annealing
Mixture is cooled to allow primers to form (beginning of DNA replication)
3. Extension
DNA polymerase adds nucleotides to 3’ end of primer

12. Polymerase Chain Reaction (PCR)

13. Applications of Polymerase Chain Reaction (PCR)
Used to amplify (copy) DNA from a variety of sources
Fragments of DNA from a 40,000-year-old woolly mammoth
DNA from fingerprints or tiny amounts of blood, tissue, semen at crime scenes…
If there is not much DNA available for analysis, PCR can produce enough DNA for other applications (electrophoresis, etc.)

14. Restriction Fragment Analysis
Restriction fragment analysis detects certain differences in the nucleotide sequences of DNA molecules

15. Restriction Fragment Length Polymorphisms (RFLPs)
RFLPs are differences in restriction sites on homologous chromosomes that result in different restriction fragment patterns
Everyone has a different DNA sequence, so the restriction enzymes will cut the DNA at different places
The length of the restriction fragments can be analyzed using electrophoresis

16. Gel Electrophoresis
The DNA fragments produced by restriction enzymes are sorted by gel electrophoresis
Sorted by rate of movement through a gel in an electric field
Distance is inversely proportional to the length of the fragment –
Long fragments don’t go far, short ones do
The banding pattern (where the fragments stop) is used to analyze/identify DNA

17. Gel Electrophoresis

18. The Human Genome Project
Largely completed in 2003
Essentially, a detailed map of the entire human genome
Genetic mapping –
Relative order of genes (1st, 2nd, 3rd, etc.)
Physical mapping –
Definitive distance between genes (2457 bp, etc.)
DNA sequencing –
complete nucleotide sequence (ATCCG…)

19. Applications of DNA Technology
Medical Applications
Diagnosing genetic diseases
Amplifying (cloning) genes and analyzing them
Gene therapy
Altering the genes of an afflicted individual
Replacing a “defective” allele with a “normal” allele
To be effective, it must be done in cells that multiply throughout life
Bone marrow cells (somatic cells)
Gametic cells (sperm/egg)
Lots of ethical issues involved here!

20. Applications of DNA Technology
Pharmaceutical Products
Using DNA cloning, genes that code for the production of specific proteins can be made in vitro
Human insulin (diabetes)
Human growth hormone (HGH)
Vaccines

21. Forensic Evidence
DNA fingerprinting (RFLP analysis and electrophoresis) is used to solve crimes
The likelihood that 2 people would have the same DNA fingerprint (restriction enzymes would cut their DNA at all the exact same places and therefore have the same banding pattern) is between 1 in 100,000 and 1 in 1,000,000,000
Also used in paternity testing

22. Applications of DNA Technology
Environmental Cleanup
Many bacteria can extract heavy metals (copper, lead, nickel) from their environments
These genes could be added to other organisms to clean up mining wastes
Bacterial strains can degrade some of the chemicals released during oil spills

23. Ethics & DNA Technology
The progress in genetic technology has raised new ethical questions:
Who should have the right to examine someone else’s genes?
How should that information be used?
Should a person’s genome be a factor in suitability for a job or eligibility for insurance?