PCR and Forensics YouTube - CSI Intro

1. PCR and ForensicsYouTube - CSI Intro

2. What we’ll be doing • Lesson 1 DNA chemistry • Lesson 2 Theory of PCR • Lesson 3 Prac 330: PCR using supplied template (set up and run PCRs) • Lesson 4 Run gel of PCRs from Lesson 3, Prac 225 do restriction digests. • Lesson 5 Run restriction digests on gels and produce DNA fingerprints

3. Lesson 1: The chemistry of DNA

4. In a nutshell, PCR: • Produces enough target DNA that it can be analysed, either on a gel or in a machine which measures fluorescence. • DNA (deoxyribonucleic acid) is the chemical which carries the genetic code of life on this planet. YouTube - Polymerase Chain Reaction

5. DNA bases Pratt Ch. 3. page 54, 55

6. The bases are linked to a five-carbon sugar to form nucleosides. In RNA, the sugar is ribose. In DNA, the sugar is 2’ deoxyribose. page 55 Nucleosides.

7. The structure of DNA: The linkage between the nucleotides is called phosphodiester bond. Linked nucleotides form a polymer in which the the backbones are the phosphate-sugar groups and the bases project out. One strand page 56 The structure of DNA.

8. The opposing strands of DNA pair anti-parallel 3’ 5’ 5’ 3’

9. Exercise • Draw a 3 base section of DNA indicating the 5’ and 3’ regions • Look at the next slide and explain, using your knowledge of electronegativity and molecular geometry, explain how/why the hydrogen bonds shown exist • Here’s a really tough one: hydrogen bonding doesn’t have an effect on double strand stability, why not? • The pKA of the phosphoryl groups is about 3. Explain why DNA is negatively charged at pH 7.0

10. DNA contains two strands DNA contains 2 strands (double helix) as their bases pair through hydrogen bonding. Which is stronger?? page 57 DNA base pairs.

11. How is DNA measured? • Size of DNA is measured in base-pairs (bp) or, for example, Kilobase pairs (1000bp=1kb). • Most DNA in cells is thousands to millions of bp

12. Genome Sizes of Model SystemsModel system Size (Million Bases) Genes Escherichia coli (Bacterium)  4.6 3,000 S. cerevisiae (Yeast) 15 Neurospora crassa (Fungus) 39.9 10,000

13. What is the mass of one human genome? Assume: • Genome is 109bp • The average mass of a nucleotide residue is 300g/mole

14. DNA replication issues • DNA polymerase must have a primer (a bit of DNA or RNA on which to elongate the DNA strand) • The raw material used to construct DNA is dNTPs • DNA polymerase can only synthesise 5’ 3’ Draw a diagram illustrating how DNA is synthesised.

15. Lesson 2: The theory of PCR

16. POLYMERASE CHAIN REACTION Polymerase chain reaction (PCR) uses thermostable DNA polymerase and specific oligonucleotide primers to amplify genes. The first thermostable DNA polymerase was Taq polymerase from the bacterium Thermus aquaticus which was isolated from hot springs.

17. Figure 3.08 A DNA melting curve.

18. The rate of renaturation depends on the length of the DNA fragment Short fragment anneal faster than long fragments. Figure 3.09 Renaturation of DNA.

19. POLYMERASE CHAIN REACTION Denature DNA sample to separate DNA strands (94oC, 5 min) Hybridise primers to DNA strands (30-65oC, 30 s) 20-30 cycles Denature to separate new DNA strands (94oC, 30 s) Taq polymerase synthesises new DNA strands (65-75oC, 2-5 min)

20. POLYMERASE CHAIN REACTION Target DNA 3’ 5’ 5’ 3’ Denature DNA 3’ 5’ 5’ 3’ Anneal specific primers 3’ 5’ 5’ 3’

21. POLYMERASE CHAIN REACTION 3’ 5’ 5’ 3’ Extend primers with Taq polymerase 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Denature DNA and anneal specific primers

22. 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Extend primers with Taq polymerase 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Size of PCR fragment Repeat cycles

23. POLYMERASE CHAIN REACTION Cycle number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Target DNA 0 1 2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 Cycle number 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Target DNA 16384 32768 65536 131072 262144 524288 1048576 2097152 4194304 8388608 16777216 33544432 67108864 134217728 268435456

24. The PCR Song and a myth • YouTube - PCR • YouTube - The PCR Song • YouTube - CSI PCR in 60 seconds: BS There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases, too. Denaturing, annealing, and extending. Well it’s amazing what heating and cooling and heating will do. PCR, when you need to detect mutations. PCR, when you need to recombine. PCR, when you need to find out who the daddy is. PCR, when you need to solve a crime.

25. POLYMERASE CHAIN REACTION PCR is now used routinely for: • cloning genes • DNA sequence analysis • site directed mutagenesis • generating DNA probes • random PCR to differentiate individuals • cloning flanking DNA • Producing DNA from unculturable microorganisms

26. Cancer detection Valerie’s normal breast Valerie’s breast tumour Valerie’s peripheral blood control std

27. Calculate the final concentrations of the components in the table below. Typical PCR reaction mixture * The PCR buffer used was made after the recommendations of the manufacturer/vendor (Perkin Elmer Cetus). The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2).It is useful to prepare a larger volume of this buffer (10-15ml), aliquot it and store the vials at -20 C for years.

28. PCR Robot from the early 80’s PCR is a little trendier these days... Things Are a Little Different Now...

29. Now try the PCR/bioinformatics exercise…