1. AP Biology Chapter 20
DNA Technology

2. DNA Technology
Recombinant DNA: genes from 2 different sources, can be 2 dif. species
Genetic engineering: direct manipulation of genes for practical purposes
Biotechnology: manipulation of organisms or their components for practical tasks (ancient idea)
Gene cloning vs. cell cloning and cloning of whole organisms – including mammals
Lots of copies of a gene to be used in research
Cloning of bacteria, mice, pigs, etc for their products
Making new organisms…..?

4. Bacterial Tools Restriction Enzymes 1960’s Cut foreign DNA
Recognize specific sequences
Leave sticky ends
Cuts are reproducible because they cut at specific sequences
Fusions occur with DNA ligase
Plasmids
Multiple copies
“clones” of the gene
Genes that are frequently expressed
Can be transferred to other cells
DNA is universal, so bacteria will produce plasmids even if DNA is not theirs originally
Used as a “vector”

5. Problems and Solutions
Expression vectors are used to get prok promoter “ON”
Make cure inserted gene is ‘downstream’ from this type of promoter
Must remove introns for prok to read mRNA
Start with mRNA from cells that have already created the edited version and make something called cDNA (complementary)
Use cells that aren’t bacteria/prok
Yeast and single celled fungi and plants
Also helps with modification that occurs to proteins after translation in euk cells
Use artificial chromosomes
Origin, centromere and telomeres with foreign DNA
Force uptake
Euk cells exposed to pulses of current can allow DNA through plasma membrane….
Inject DNA
Using “shot gun approach”

6. Genomic Libraries
Genomic library is a collection of all the recombinant plasmids in a particular colony of bacteria
cDNA libraries contain all the gene that there are cDNA for

7. Polymerase chain reactions, etc.
Gel electrophoresis employs electricity to separate strands of DNA based on their size
Bands of DNA can be identified using Southern blotting – a small sequence is radioactively tagged, then put with DNA and every where it pairs up with complementary bases it can be read
Restriction fragment length polymorphisms (RFLP’s)
Differences in Southern blot bands between homologous chromosomes, especially in noncoding areas of genome
PCR is a technology that amplifies the DNA so there are billions of copies
DNA from woolly mammoth
DNA from embryonic cells
DNA from crime scenes for DNA fingerprinting

8. Diagrams Be sure to read information on the following:
Gel electrophoresis pg. 374
RFLP’s and Southern Blotting pg. 375
Sanger method of sequencing pg. 378
Your lab manual – Lab 6 A and 6B

9. Human Genome Project
Computer systems worked internationally for almost 15 years to list all the human genes, including the noncoding regions.
3 billion base pairs long
100,000 genes
Also mapped for research and comparison
E. coli (and other bacteria)
Saccharomyces cerevisiae (yeast)
Caenorhabditis elegans (nematode/worm)
Drosophilia melanogaster (fruit fly)
Mus musculus (mouse)
Danio rerio (zebra fish)

10. Terms
Genetic mapping: linkage map determined by recombination frequencies
Currently have 500 markers on human genome
Physical mapping: map units
DNA sequencing: list of bases for all 3million nucleotides pairs
Genome analysis
find genes by looking for sequences, that when transcribed have a start and stop codon
Compare sequences found in one organism and look for similar sequence in other organsims
Microarray assay: microscope slide with known genes in wells – mRNA from a cell is obtained, reacted with cDNA, if bases match they will pair up and when hybrid DNA is placed on slide it will glow in the well if its for the gene in that well
Genomics: the study of genomes and genes

11. Human Health Applications
1. Diagnosis of disease by showing a marker for the gene
2. Human Gene Therapy when there is a known allele
Enhance immune system
Fight cancer
Stem cell products
3. Pharmaceuticals
Manufacture proteins that are required
Design vaccines that are specific and harmless

12. Environment, Agriculture and Forensics
Forensics uses RFLP, southern blot and gel electrophoresis to create a DNA fingerprint
Genetically engineered microbes can ‘feast’ on oil spills and heavy metal contaminants in water and soil
Transgenic organisms are being developed as a source of organs and as a source of medically useful proteins
Genetically engineered plants are created to have specific useful features, as well as being pest resistant and potentially able take up N without fertilizers

13. Safety and Ethics Currently regulated by the following US agencies
USDA, United States Department of Agriculture
FDA, Food and Drug Administration
EPA, Environmental Protection Agency
NIH – National Institute of Health Recombinant DNA Advisory Committee
And potentially , voters like you….