Biol/Chem 473

1. Biol/Chem 473 Schulze lecture 9: methods

2. Southern Analysis • Extract DNA • Fractionate by electrophoresis in an agarose gel • Transfer by capillary action to membrane (blot) • Incubate with nucleic acid probe • Detect & analyze & collect Nobel Prize

3. Gel Treatment: Measurements and photography Agarose gel with genome digest in it Photograph of genome digest under UV

4. Set up the SOUTHERN BLOT transfer (denatured! Ie., single stranded!) DNA from agarose gel to membrane

5. Making a Probe • cDNA, genomic fragment, PCR product etc. • Repeat for RFLP analysis • Copy of mRNA (cDNA) to analyze gene structure • Choose a detectable label • Radioactivity (usually P32) • “bumpy groups” like DIG • Incorporate label into probe • Random prime labeling • Nick translation

6. promoter exon exon exon intragenic DNA transcription processing polyA tail Reverse transcriptase cDNA vector A cDNA is a copy of a mRNA

7. Hybridizing probe to Southern Blot • Prehybridization step: blocking • Block off parts of the membrane not already bound with nucleic acid • Hybridization step: stringency can be varied • High salt solution promotes strong pairing between probe and target • High temperature increases specificity of probe • Posthybridization step: washing off excess probe • Temperature, salt concentration again can be varied for purposes of stringency

8. Application: Transgene integrity and copy number (Reza Imani) HindIII digest indicates whether transgene is intact XhoI digest indicates transgene copy number

9. Northern Analysis • Extract RNA • Fractionate by electrophoresis in a denaturing agarose gel • Transfer by capillary action to membrane (blot) • Incubate with nucleic acid probe • Detect & analyze & collect Nobel Prize

10. Poly(A) RNA (mRNA) Total RNA RNA gels

11. Application: Developmental Northern

12. Microarrays • Just like in Northern and Southern analysis, this technique relies on HYBRIDIZATION between complementary sequences • Analyze gene expression of all the genes in the genome AT ONCE by representing probes for every gene on a small glass chip, and incubating the chip with labeled target RNA

13. “Spotted” arrays: presynthesized single or double stranded sequences (“probes”) are printed onto glass slides Probes are generally fairly long several hundred base pairs or more Each experimental trial requires ONE array chip “oligonucleotide” arrays: sets of oligos (“probes”) representing a particular gene sequence are synthesized directly onto the surface of glass wafers Probes are short – usually about 25 nucleotides long; each gene is represented by a set of 25mers. Each experimental trial requires TWO array chips Two kinds of microarrays

14. Robotic microarray production (“Spotted” arrays) www.bio.davidson.edu/.../ CSU/Introscanners.html

15. Spotted Array:1. RNA isolation

16. Spotted Array: 2. Generate labeled cDNAs

17. Spotted Array: 3.Hybridize both cDNA pools to a single microarray chip Pool labeled cDNAs

18. Spotted Array: 3.Hybridize both cDNA pools to a single microarray chip

19. Spotted Array Assay: 4. scan chip

20. Pat Brown, father of glass slide microarrays Stanford University

21. Oligonucleotide arrays: build up probes DIRECTLY on chip surface

22. Oligo array: hybridization and scanning

23. Expression in sample #1

24. Expression in sample #2

25. In silico: at the computer In vitro: at the bench

26. Microarray Application: tumor expression profiles critical invasive melanoma samples supplied by U. of Iowa lab! patient samples gene names Nature 406: 536-540 (2000)

27. RTPCR (Real-Time PCR or Q (quantitative) PCR) • mRNA is converted into DNA via reverse transcriptase (RT). • The polymerase chain reaction (PCR) uses specific primers to make multiple DNA copies. • Measure amount at end of cycle

28. Application: Microarray + RTPCR

29. Chromatin immunoprecipitation • Used to determine whether a given protein binds to a given DNA sequence in vivo • Like all protein analysis involving antibodies (including westerns) a specific antibody is required • If there is no specific antibody, then epitope tagging can be employed (FLAG, MYC, HIS) • An epitope is a portion of a molecule to which an antibody binds

30. secondary antibody (detects primary antibody) Required in westerns, but not chromatin immunoprecipitation Primary antibody Antibodies as probes

31. Back to the Chromatin Immunoprecipitation Assay (ChIP) 1. Crosslink Protein-DNA complexes in situ 2. Isolate nuclei and fragment DNA (sonication or digestion) 3. Immunoprecipitate with antibody against target nuclear protein and reverse crosslinks 4a. Identify protein components of isolated complexes 4b. Identify DNA sequence by PCR, cloning and sequencing MCS

32. The probes on microarrays can be complementary to the entire genome!

33. The probes on microarrays can be complementary to the entire genome!

34. ChIP on ChipChromatin immunopreciptation on a microarray Chip DNA from binding site Fluorescently label http://www.chiponchip.org/

35. Application: where on the chromosomes does the male-specific lethal-3 protein bind?