PCR & electrophoreisis

I. PCR A) Polymerase Chain Reaction B) Amplifies DNA…..amplifies means… (makes lots of copies of) C) add: sample DNA, Taq polymerase, nuclotides, primer D) 3 steps –each repeated 20-40 times

1) Denaturation a) heat used…no enzymes b) breaks H-bonds to separate DNA strands

2) Annealing a) no RNA polymerase so b) add synthetic RNA primers c) primers match DNA code on both sides d) cooling temp anneals them to the DNA

3) Extension a) Add Taq polymerase b) Taq adds nucleotides to primers making DNA complementary to original strand c) Taq = Thermus aquaticus enzyme i) heat resistant does not denature ii) T. aquaticus from hot springs @95˚C

Review on line : http://passel.unl.edu/pages/animation.php?a=PCR.swf http://www.dnalc.org/ddnalc/resources/pcr.html Review on line : http://passel.unl.edu/pages/animation.php?a=PCR.swf

II. Electrophoresis A. Separates DNA fragments based on size B. Agarose gel – DNA in wells drawn through gel C. Electric field draws DNA frags through gel 1. DNA is negative so moves to cathode 2. buffer in gel and tray have ions to create electric field

D. Speed of DNA fragments varies by size 1. large fragments move slow…don’t get as far down the gel as small fragments 2. size of band on gel has NOTHING to do with the length of the DNA fragment!!!

E. Speed altered by 1. Concentration of gel…. a. high concentration slows large fragments b. pores in gel too small 2. Higher voltage = faster travel

F. Gel analysis 1. DNA standard marker of known length are in lane 1 2. Measure distance each segment moved 3.Graph distance vs size 4. Best fit line Estimate unknown size

G. DNA Fingerprinting 1. Human DNA 99% the same 2. Differences are in non-coding DNA between genes 3. Tandem Repeats = 2 bases repeated 4. ATATATAT # repeats varies so 5. Different people have different lengths of DNA between genes

H. DNA Fingerprinting Uses 1. Crime scene investigations 2. Paternity tests 3.Cut all samples with same enzymes 4.Do PCR on Samples to amplify DNA 5.Do electrophoresis to sort DNA by size 6.Compare results to crime scene or potential parents 7. 1 in 3,000,000,000,000 chance of unrelated people sharing same DNA fingerprint

8. Child gets all DNA from Mom or Dad 9. Any fragment found in child must be in either Mom’s DNA or Dad’s DNA

I. DNA Sequencing 1. Alter nucleotides so they will stop replication 2. Add modified A nucleotides to one sample 3. T to second, G to third, C to fourth 4. Run PCR then electrophoresis 5. Order of segment sizes indicates order of bases in sequence

J. Sanger Method (di-deoxy method) 1. Uses Di-deoxynucleotide to stop replication 2. named after Frederick Sanger, won the 1980 Nobel prize in chemistry http://www.dnalc.org/resources/animations/sangerseq.html

K. Fluorescence DNA Sequencing 3. Label Nucleotides with fluorescent dye each base different color 4. Alter labeled nucleotides so they stop DNA replication 5. Add both labeled nucleotides and normal nucleotides to sample DNA 6. Run PCR and electrophoresis

7. Segments separated by size 8. Base that fluoresces is always the last base in line

III. Gene Cloning :using bacteria to amplify DNA A. create recombinant plasmids (DNA of 2 species) 1. restriction enzymes a. evolved as bacterial immune system b. cut double stranded DNA a specific sites called restriction sites c. cut up virus DNA that invades host

d. Restriction site DNA is symmetrical i. read same way ii. palindromic sequences iii. Cut can be blunt or make sticky ends v. sticky ends bond to any other DNA cut by same restriction enzyme

2. Bacterial plasmid and human gene cut with same restriction enzyme a. sticky ends will fuse human gene into the plasmid b. makes recombinant DNA plasmid C. Transformation = bacteria take up recombinant plasmid from the environment D. Cloning... As bacteria divide they copy the plasmid…amplifying that gene

E. Gene Cloning Uses 1. Amplify Genes of interest that are harvested from bacteria and isolated by electrophoresis http://www.ydae.purdue.edu/Apple_genomics/flash/movie2.swf 2. Create populations of bacteria that produce proteins needed medically (insulin) http://www.dnalc.org/view/15476-Mechanism-of-Recombination-3D-animation-with-with-basic-narration.html

4. provides genes for gene therapy 5. transgenic plants/animals 3. oil eating bacteria 4. provides genes for gene therapy 5. transgenic plants/animals a. have gene incorporated into egg/ zygote/ embryo so all cells have the gene b. genes are passed on to offspring http://www.bioteach.ubc.ca/TeachingResources/Applications/GMOpkgJKloseGLampard2.swf