Polymerase Chain Reaction (PCR)

1. Polymerase Chain Reaction (PCR) • Polymerase chain reaction: Starting with VERY SMALL AMOUNTS OF DNA (sometimes a few molecules), one can amplify the DNA enough to detect it by electrophoresis.

2. 5’ 3’ 3’ 5’ Denaturation 5’ 5’ 3’ 3’ 3’ 5’ Annealing Cycle 1 3’ 5’ Extension 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ Cycle 2 3’ 5’ 5’ 3’ Cycle 3 3’ 5’

3. Rate of PCR 2n InitialDNA 1 2 4 8 Number of DNA molecules

4. Copies of DNA=2N

5. RT-PCR • Polymerase chain reaction amplification of cDNA can also be used to detect specific transcripts in a RNA sample. • In this procedure, known as RT-PCR, reverse transcriptase is used to copy all of the mRNAs in an RNA sample into cDNA. • Usually, oligo dT molecules, that anneal to the poly A tails of the mRNA, are used as primers. • This single stranded cDNA can then be amplified by PCR using primers that anneal to a specific transcript sequence.

6. AAA(A)n AAA(A)n RT-PCR 5‘-Cap mRNA AAA(A)n (dT)12~18 primer anneal 3‘ 5‘ Reverse transcriptase dNTP 5‘ Regular PCR cDNA:mRNA hybrid

7. The amplified DNA fragments that are produced can by analyzed by agarose gel electrophoresis. • The amount of amplified fragment produced is proportional to the amount of target mRNA in the original RNA sample. • Although less quantitative than Northern blots, RT-PCR is extremely sensitive and can be used to detect very rare mRNA species.

9. How do we accurately quantify the amount of DNA? Real-time PCR

10. Real Time PCR

11. 3. intensifier 5. ccd detector 350,000 pixels 1. halogen tungsten lamp 2b. emission filters 2a. excitation filters 4. sample plate

12. Level off/ plateau Log phase 2 4 8 16 32 Amplification Plot of real-time PCR DNA copy number (log) PCR cycle (Ct)

13. DNA sequencing • Sequencing is used to determine the precise order of nucleotides in a DNA molecule. • The Sanger di-deoxy method involves the synthesis of DNA by a DNA polymerase. • DNA synthesis is terminated at specific nucleotides by the incorporation of di-deoxy nucleotides that are missing the 3’ OH.

23. Sequence analysis • Four different reactions produce DNA fragments that are terminated (randomly) at each of the four nucleotides. • These samples are resolved by electrophoresis. • The shortest fragments, those terminated closest to the primer, run faster than the longer fragments.

25. A C G T • DNA sequencing reactions can be radioactively labeled. • Bands detected by X-ray film exposure. • Sequence can be read in the 5’ to 3’ direction from the bottom of the image towards the top. A A T C T A A C G

26. This is great but… Wouldn’t it be great to run everything in one lane? Saves space and time, more efficient Fluorescently label the ddNTPs so that they each have a different color…

27. 07_03.jpg

28. Automated DNA sequencers use dideoxy terminators labeled with four different fluorescent dyes. • All four reactions can be carried out simultaneously in a single reaction.

29. Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer. • The DNA sequence is read automatically.

30. Beckman CEQ 2000, 8 capillary ABI Prism 3730, 96 capillary

31. Affymetrix Gene Chip

32. Affymetrix Expression Arrays http://www.affymetrix.com/technology/ge_analysis/index.affx

35. Analysis of microarrays • Microarrays allow for the simultaneous analysis of the expression of thousands of mRNAs. • Useful for determining changes in gene expression patterns from one sample tissue to another. • For example, microarrays have been used to study differences in gene expression in different tumor tissues.

36. Genes with similar expression patterns are clustered together. Gene expression patterns can be associated with different disease patterns.

37. Microarray example #1: Biomarker identification - lung cancer Samples Genes Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.

38. Data partitioning clinically important: Patient survival for lung cancer subgroups 1 Cum. Survival (Group 1) .8 Cum. Survival (Group 2) Cum. Survival (Group 3) .6 Cum. Survival .4 .2 0 0 10 20 30 40 50 60 Time (months) p = 0.002 for Gr. 1 vs. Gr. 3 Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.

39. Another microarray example: understanding malaria IDC Transcriptomes for three P.falciparum strains Llinas, M. et al. Nucl. Acids Res. 2006 34:1166-1173; doi:10.1093/nar/gkj517

40. Yeast 2 Hybrid Allows the geneticdetection of physical interactions between proteins

41. Yeast Two Hybrid Assay The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions. If two proteins interact, then a reporter gene is transcriptionally activated. e.g.gal1-lacZ - the beta-galactosidase gene A colour reaction can be seen on specific media. You can use this to Study the interaction between two proteins which you expect to interact Find proteins (prey) which interact with a protein you have already (bait).

42. GAL4 has two domains: one binds DNA (UAS) , the other activates transcription . GAL1 { UAS GAL4 { { DNA-binding Activating GAL1 GAL1 POL POL

43. If protein X and Protein Y interact, they lead to expression of the reporter gene.

44. Two-hybrid assay SNF4 1. B SNF1 A 3. 2. GAL4-DBD Transcription activation domain UASG 4. Fields S. Song O. Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163 GAL1 Allows growth on galactose

45. Two-Hybrid Selection UAS-Reporter GAL4-dbd/BAIT GAL4-ad/X Library No expression UAS-Reporter GAL4-dbd/BAIT GAL4-ad/X Expression