PCR and GENOTYPING

1. PCR and GENOTYPING ShanmugavelRajendran Teacher-Intern Center for Biomolecular Therapeutics Rockville, Maryland Summer 2012

3. Alzheimer’s Disease Symptoms Affects brain function – Memory, behavior, and thought processing Presence of amyloid plaques in the brain and inter- neuronal neurofibrillary tangles. Over-expression of genetic factors namely Presenilin-1 and Amyloid Precursor Protein Increases the amount of Aβneurotoxic peptide in certain regions of the brain.

4. Histopathology of brain sections of wild type mice show no plaque formation but PS/APP mice show elevated levels of plaque formation indicative of Alzheimer’s Disease.

5. AMYLOID PRECURSOR PROTEIN [APP]

6. PRE SENILIN- 1 PROTEIN [ PS-1]

7. Background Information S100 Protein family includes S100A and S100B S100A is highly expressed in Melanoma and S100B is highly expressed in Alzheimer’s disease (AD). When S100B level increases, it down regulates p53 (tumor suppressor gene)

8. Research Focus How to restore p53 function? How to block interaction between S100B and p53? Find the drug that can reduce the expression of S100B and prevent the progression of AD.

9. Mouse Model Must be tested in mouse before clinical use. Higher genetic similarities between human & mouse Shares many characteristics of human AD Easy to manipulate the mouse embryo 3 week gestation period Sexual maturity in2 mo. Small litter size (2-10 pups)

10. Mouse Model PSAPP double transgenic mice are used WT – S100B gene is present KO – S100B gene is replaced by Neomycin resistant gene

11. Research Process The specific phenotype for the mice in the presence/absence of the gene is determined by over-expression of the gene or knockout Knockouts give functionally inactive version of the gene; effect of the gene can be checked; Comparative studies. To know the specific phenotype changes in WT/KO, the genotype has to be characterized. PCR is an effective tool

12. Research Process Three Steps 1. Extraction of genomic DNA 2. Polymerase Chain Reaction 3. DNA gel electrophoresis

13. Extraction of Genomic DNA Cut the mice tails Digest the tissues and lyse the nuclei DNA is precipitated from the solution using ethanol and bound to silica membrane After several washes, contaminants are removed DNA is eluted in TE buffer

14. Extraction of Genomic DNA ATL Buffer + Proteinase AL Buffer + ETOH AW 1 & 2 AE Buffer

15. Polymerase Chain Reaction (PCR) • “Amplify” large quantities of DNA from small quantities or to make many copies of small segments of DNA • Analyze single DNA fragments out of large complex mixture.

16. Components of PCR Reaction • Template DNA • Forward & Reverse Primers • Thermo-stable polymerase • Taq Polymerase • dNTP • (dATP, dTTP, dCTP, dGTP) • PCR Buffer (Mg++) • Thermocyler Thermus aquaticus

17. Denaturation Annealing Extension

18. Temperature Cycling • Denaturation 94° • Annealing 55° • Extension 72° • 2. In every cycle DNA between the primers is duplicated

19. Exponential Amplification At the end of 30 cycles --- 1 billion copies

20. Agarose Gel Electrophoresis

21. Visualization of DNA • Ethidium Bromide (EtBr) intercalates into DNA and fluoresces under ultraviolet light

22. Gel Electrophoresis

23. Gel Electrophoresis Results

24. Litter Genotype

25. Summary Evaluate the type of genotype present in mice. Select the line of mice for further experimentation.

26. PCR Basics Purpose of PCR Overview Components of PCR Reaction Variables Temperature Cycle Times and Numbers Primer Buffer Polymerase Experimental Notes

27. 3 STEPS OF PCR • Denaturation • Annealing • Extension

28. DNA Amplification

29. Selection of Primers • Paired primers (forward and reverse) • Length (17-28bp) • GC content 50-60% • GC Clamp • Tm’s (Melting temp.) between 55-80 • Should not have strings of G’s and C’s at the end. • Primers should not be self complementary • Should not form hairpin or loop or dimers

30. PCR Variables • Temperature • Cycle Times and Temperature • Primer • Buffer • Polymerase

31. Temperature • Denaturation • Trade off between denaturing DNA and not denaturing Taq Polymerase • Taq half-life 40min at 95 °, 10min at 97.5° • 95° • Annealing • Trade off between efficient annealling and specificity • 2-5 ° below Tm • Extension • Temperature optimum for Taq Polymerase • 72 °

32. Cycle Times and Temps Typical PCR Run Step Time/Temp • 3 min at 95° • 30 sec at 95° • 1 min at 55° • 2 min at 72° • Go to step 2 - 29 times • 8 min 72° • 0 min 4° • End

33. Gel Electrophoresis An aliquot of the PCR reaction (amplicon) was run on an agarose gel supplemented with Ethidium Bromide. Ethidium bromide intercalates with DNA. The amount of light emitted is proportional to the degree of intercalation. More DNA = More intercalation = Brighter bands.

34. Gel Electrophoresis Visualize the amplified bands and verify the genotype of the mice. Compare the band size of the samples with the known markers or reference standards.

35. Summary PCR is a powerful tool. Characterize the genotype of the mice by designing specific primers in the target region. Evaluate the type of allele present in the genomic DNA Conclude the type of genotype present in mice.