DNA Technology and Genomics Chapter 20 DNA Technology and Genomics

Making multiple copies of the gene sized DNA I. Gene Cloning Making multiple copies of the gene sized DNA

1. Restriction Enzymes Enzymes that cut DNA at a specific location called a restriction site Cut DNA in a reproducible way / restriction fragments Makes sticky ends which overhang and DNA ligase fuses these pieces permanently to cloning vector’s plasmid

2. Gene Cloning Cloning Vector – original plasmid / bacteria are the host cell Isolation of Vector and DNA to be cloned Insertion of DNA into vector / use a restriction enzyme and ligase Introduce cloning vector to cell Grow Cells Identify cell clones

3. Problems Cloning Eukaryotic Genes Clone normal way you have 2 types of cells (pro and eu) / Use an expression vector which has a prokaryotic promotor Introns / use reverse transcriptase to make complementary DNA (cDNA) from mRNA Use eukaryotic cells (Yeast, YAC) instead of bacteria Use electroporation where they shock eukaryotic cells and this allows DNA in through temporary holes Use needles and particle guns

4. Genomic Library A complete set of thousands of plasmid clones Sometimes use cDNA when making them to remove the introns / cDNA library

5. Polymerase Chain Reaction (PCR) DNA is amplified without using a cell Use heat resistant polymerase, primers, and nucleotides Denature (heat), Primers attach (cooling), Extending/Annealing (cooling)

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II. DNA Analysis and Genomics Genomics – sequencing entire genomes in hopes of studying genes and their interactions Gel Electrophoresis – separates nucleic acids or proteins based on size, charge, etc

1. Restriction Fragment Analysis Cut DNA w/ restriction enzymes and look at the bands Label certain DNA for observation with radioactive labels Southern Blotting – electrophoresis is used then this technique is used to identify certain bands w/ the genes you are looking for Video

1. Restriction Fragment Analysis RFLP / Restriction Fragment Length Polymorphisms – differences in DNA sequence on homologous chromosomes that can result in different restriction fragment patterns / found in non-coding DNA Passed down Mendelian style Can be used to identify differences in DNA between people.

2. Mapping Entire Genomes 1980, Botstein said RFLPs could be used to map the genome Human Genome Project, 1990 Whole Genome Sequencing – Genetic map (genes, RFLPs, etc) / Physical Map / Figure out nucleotides Venter, 1992 – started with last step and was very successful

3. So We Have the Genome, Now What? Surprisingly few genes / 20,000 – 25,000 Lots of non-coding DNA Human genes subjected to a lot of splicing Reinforces our evolutionary connections Research how genes work together / DNA Microarray Assay – technique used to see what genes work under certain conditions

3. Continued How do we discover gene function? Turn off the genes and see the result. - In vitro mutagenesis – insert changed sequence - RNAi - ds RNA is used to break down mRNA Proteomics – study of a full set of proteins encoded by the genome Most human genome differences are in SNPs (single nucleotide polymorphisms) differences in single bp variations

III. Applications of DNA Technology

1. Diagnosis of Disease PCR can be used to diagnose HIV Identify carriers of harmful recessive alleles by using RFLPs / link certain genes to certain markers / Huntington’s disease

Gene Therapy Altering the genes of an afflicted person Change bone marrow cells Not real effective, yet May lead to eugenics – deliberate effort to control the human populations genetic makeup

3. Pharmaceutical Products Insulin and Human Growth Hormone Can be expensive Make attenuate viruses for vaccines / better response

4. Forensic Uses Test blood type or tissue type / determine paternity issues, and “who dunnit” Use RFLPs and Southern Blotting to identify a persons DNA fingerprint - can also use microsatellites - simple tandem repeats (STR) – small units (10-100) that repeat in different amounts for everyone

5. Environmental Uses Bacteria may be made to clean up heavy metals Bacteria may be able to mine Sewer treatment

6. Agricultural Uses Transgenic organisms – genomes carry genes from other organisms Ex. Cows w/ clotting factor gene secreted in the milk How are they made? Get an egg / Clone desired gene / Inject in egg / Hope it integrates / Implant eggs Plants are easier to work with because any cell may develop into an adult Use Ti plasmid / only works w/ dicots Herbicide resistance / “yellow” rice / plants that fix their own nitrogen

Should we be doing this?