Chapter 12 DNA technology

12.1 Plasmids are used to customize bacteria Recombinant DNA technology Lab techniques used to combine genes from different sources (species) into a single DNA molecule Use of natures “tools” have filled our recombinant DNA toolbox. Enzymes, plasmids Examples are plentiful Bacteria produces human insulin, glow in dark bacteria, Flavor Saver Tomatoes, Golden Rice, Round-Up Ready Soybeans and Corn…

12.1 Plasmids are used to customize bacteria Introduction to a plasmid Small circular DNA molecules that replicate separately from larger bacterial chromosomes Used for “gene swapping” amongst bacteria Antibiotic Resistance!! Conjugation “bacterial sex”

12.1 Plasmids are used to customize bacteria Plasmids (con’t) After swapping sometimes genes are inserted into host chromosome which makes it a great vector Gene carrier providing a means of integrating “genes” into a host

12.1 Example of plasmids ability to transfer gene into host Plant tumor caused from Agrobacterium tumefaciens

12.2 Enzymes used to cut and paste DNA Restriction Enzyme – Enzymes that have the ability to cut DNA and is therefore used as molecular technology tool Extracted from bacteria as they serve as a protective measure from invading viruses “restricting” them from surviving in cell. Sticky ends – staggered (as opposed to a blunt cut) cuts in DNA exposing un-bonded nitrogen bases. Allows for recombinant DNA technology

12.2 Enzymes used to cut and paste DNA Restriction enzymes produce “sticky ends” which allow scientists to cut and paste genes between organisms. They are like a molecular scissors.

12.1 Example of plasmids ability to transfer gene into host Tumor causing bacteria Agrobacterium tumefaciens Gene to be transferred Cellular DNA Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome Recombinant plasmid Plant cell colonies Transformed bacteria introduce plasmids into plant cells Complete plant is generated from transformed cell http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio40.swf::The%20Ti%20Plasmid Agrobacterium tumefaciens is a bacteria that uses a plasmid to infect plants. Scientists have utilized this plasmid to insert “genes of interest” into plants

12.3 Plasmids can be cloned in recombinant plasmids Recombinant DNA Gene for human growth hormone DNA recombination Human Cell Sticky ends DNA insertion Bacterial Cell Bacterial chromosome Bacterial cell for containing gene for human growth hormone Plasmid animation review

12.4 Cloned genes can be stored in genomic libraries (plasmid) Genomic Library – The entire collection of all cloned DNA fragments from a genome (genome – entire genes from one organism) Uses Storage, sequencing, study

12.4 Cloned genes can be stored in genomic libraries (phages – virus) Phage libraries much less space. 70 petri dishes can hold entire human genome Compared to 300 petri dishes for plasmid library Phage

12.5 Reverse transcriptase helps make genes for cloning. cDNA – Complementary DNA made from expressed mRNA. Made using reverse transcriptase from retroviruses such as HIV. Advantage is that it makes DNA from already edited mRNA so DNA in our libraries won’t have junk in them (saves space!) Advantage of using cDNA Studying specific cells and genes associated with them. If we want to target genes from a brain cell or a cancer cell 2. Smaller to work with.

Genomic Library vs. cDNA library

Genomic Library vs. cDNA library Larger Includes all possible genes from organism Includes all exons and introns cDNA Smaller Includes only expressed genes from specific cell Includes only coding regions (exons) Proteins can be expressed by transformed prokaryotes b/c no introns

12.8 Nucleic acid probes identify clones carrying specific genes How do we find books in the library? Colonies will produce protein. If not… Probing - Nucleic acid probe consists of a small synthesized strand of DNA, labeled with a radioactive isotope, that is complementary to the “gene of interest”

12. 6 Recombinant cells and organisms Bacteria Plasmids make ideal vectors, grown rapidly and cheaply, “secretion” of proteins Prokaryotic organisms not as good at producing eukaryotic proteins. Yeast Eukaryotic so better at producing eukaryotic proteins. Single celled, also has a plasmid Mammals Many proteins are glycoproteins unique to mammal protein synthesis Difficult for secretion (via milk in some cases). Gene of interest is accompanied with a promotor specific to genes in mammary gland. SpiderGoat!

12.7 DNA technology is changing pharmaceutical industry Hormones Insulin and HGH via recombinant E.coli Diagnosis and Treatment of disease Genetic Testing very common Gene Therapy not where they hoped it would be Vaccine – harmless variation of pathogen given to trigger immune response Genetically engineer bacteria to make surface proteins associated with pathogen Alter the gene of the actual pathogen that makes it harmful

12.9 Microarray – measures gene expression Collect Tissue Isolate RNA - density Isolate mRNA – poly A tails Make labeled cDNA – using RT Apply cDNA to microarray (hybridize) Scan microarray Analyze data *Red = expressed in cancer *Green = expressed in healthy Yellow = expressed in both (* genes of interest) Analyze data

Lab Techniques to be familiar with 1. DNA microarray – Can be used to determined which genes in a cell are being expressed. Provides a large scale analysis of gene expression. Animation 2. Gel electrophoresis – Used to separate fragments of DNA (produced by restriction enzymes) to make DNA comparisons. Animation 3. PCR – Polymerase Chain Reaction. Uses heat resistant Taq enzyme to make billions of copies of targeted DNA sequences. Animation 4. CRISPR-Cas9 – Highly specific technique used to “target” specific place on DNA to alter. Animation

12.10 Electrophoresis

12.10 Electrophoresis

12.10 Electrophoresis Whose the father?

12.14 PCR – Polymerase Chain Reaction Use to amplify DNA (game changer) Id of viruses (can determine if HIV has been integrated into host) Amplify indiscriminate amount of DNA at a crime scene Genetic testing Technology Primers – “paper clips” that locate and isolate the region of DNA to be copied (these are programmable – for example there are HIV primers). This is where DNA polymerase binds Polymerase – copies DNA. Must be able to withstand temps required. Thermus aquaticus – bacteria found in thermal vents Thermal cycler – machine that cycle temperatures needed 95 – breaks DNA 50 – allows DNA primers to bind 75 – DNA is copied

animation Polymerase binds to primers and copies DNA