1. Lab Techniques: Gel electrophoresis Doug Dluzen Lab Lecture 2 ddluzen@hmc.psu.edu

2. What goes in a Gel? Agarose (1%) – Can range from 0.5% to 2.0% depending on fragment size TAE Buffer – Mixture of Tris base, acetic acid, and EDTA – Commonly made as a 50X Stock and diluted to 1X Stock with Water – At 1x general concentrations are as follows: 40 mM Tris 20 mM acetic acid 1 mM EDTA Water Ethidium Bromide

3. Hazardous!!! – Possible carcinogenic properties – Handle with care Used as a fluorescent tag to visualize DNA and RNA – Expose gel with UV light and DNA bands will glow and can be visualized – Incorporate into DNA can induce up to 20-fold intensity of flourescence – Intercalates between DNA and RNA base pairs http://course1.winona.edu/sberg/ILLUST/eth-br.JPG https://web3.unt.edu/riskman/Images/EtBr_CAS_1239-45-8.bmp

4. How to Make a Gel 1. Dilute 50x stock reagent TAE buffer into a 1x using water Add 1% weight to volume dry agarose – i.e. 0.5 grams agarose into 50 mL 1x TAE buffer solution Heat up in microwave until all agarose dissolved Cool down enough to add EtBr (generally 4-5 uL) Pour gel (watch for air bubbles!) and allow to solidify (~20 minutes) Add DNA/RNA samples into lanes – Mixed with Loading Dye to visualize lanes – Be sure to include controls and DNA size ladder

5. Gel Electrophoresis One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties A current is applied that causes charged molecules to move through the gel Molecules are sorted into “bands” by their size Adopted from Dr. Sairam Lecture Slides

6. Pulsed field gel electrophoresis Adopted from Dr. Sairam Lecture Slides http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/dsDNA.jpg

7. Mixture of DNA mol- ecules of different sizes Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE RESULTS 1 2     Adopted from Dr. Sairam Lecture Slides

8. Analyzing DNA

9. Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.” Adopted from Dr. Sairam Lecture Slides

10. Restriction Digest Sites Adopted from Dr. Sairam Lecture Slides

11. DNA Ligase Sticky ends can bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments Adopted from Dr. Sairam Lecture Slides

12. Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5 5 5 5 5 5 5 5 55 5 5 55 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 231 Sticky end GAATTC CTTAAG CTTAA G AATTC G G G CTTAA G G G G AATT C C TTAA Adopted from Dr. Sairam Lecture Slides

13. Using Restriction Enzymes In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Restriction fragment analysis can be used to compare two different DNA molecules, such as two alleles for a gene if the nucleotide difference alters a restriction site Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms) Adopted from Dr. Sairam Lecture Slides

14. Normal  -globin allele Sickle-cell mutant  -globin allele Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of the  -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp Large fragment Dde I RFLP Analysis Adopted from Dr. Sairam Lecture Slides

15. Restriction Digests Need to check if PCR fragment, generally cloned into a useful reporter, has the correct orientation – DNA inserts can ligate into a plasmid in two directions

16. Detection of Specific DNA and Protein Sequences - Blotting Whether you are analyzing DNA, RNA, or protein the underlying principle is the same Use of a probe that signals presence/absence of a gene or protein of interest Labeled (chemically, radioactively, etc.)probe is used to visualize your target

17. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel Southern Blotting Adopted from Dr. Sairam Lecture Slides

18. Southern Blotting Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

19. Southern Blotting A southern blot can distinguish: 1.The presence of a particular gene of interest 2.Number of copies of that gene 3.Genomic rearrangements 4.Mutations of restriction digest sites -Southern blots are very sensitive Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

20. Northern Blotting Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane Identification of mRNA at a particular developmental stage suggests protein function at that stage Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns Adopted from Dr. Sairam Lecture Slides

21. Northern Blotting Same principle as southern blotting, except RNA is measured as opposed to DNA – RNA can also bind to nitrocellulose membrane – Uses formaldehyde as a denaturing reagent Used to identify tissue and temporal expression of a particular gene – Sensitive – Used to measure expression levels of particular mRNA

22. Western Blotting Powerful tool to detect presence and expression levels of a particular protein – Use of an antibody – specific protein molecule will bind to specific protein sequence on the protein of interest This specific protein sequence is called an epitope As with northern and southern blotting, proteins are sorted by molecular weight, transferred to a membrane, and probed – Protein presence, expression, and quantity can be measured

23. Western Blotting - Principles Adopted from GE Healthcare: Western Blotting Principles

24. Western Blotting Methods 1. Electrophoresis – Denaturing Gel Adopted from GE Healthcare: Western Blotting Principles

25. Western Blot Methods 2. Transfer from gel to membrane Adopted from GE Healthcare: Western Blotting Principles

26. Western Blot Methods Once protein transferred to membrane – Incubate in protein buffer (generally 5% milk solution) to bind all regions of blot not bound by transferred protein Incubate with primary and secondary antibodies Visualize!

27. And now for something completely different…

28. Browsing the Genome Browsers NCBI - http://www.ncbi.nlm.nih.gov/ Ensembl - http://useast.ensembl.org/index.html University of California Santa Cruz - http://genome.ucsc.edu/ Pubmed - http://www.ncbi.nlm.nih.gov/pubmed/

29. NCBI

30. BLAST!

31. Ensembl

32. Ensembl – THE MANY USES

33. I see U see UCSC

34. UCSC

35. Other Useful Odds and Ends HapMap Project – encyclopedia of human and mouse SNP variation and genotype frequencies www.hapmap.org TargetScan – microRNA prediction Pubmed – uses NCBI database for literature searches, protein and nucleotide sequences

36. Thank you! Questions? ddluzen@hmc.psu.edu