DNA Technology

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 • Restriction Enzymes – • 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?