Using Gel Electrophoresis to analyze DNA, RNA and proteins

1. Using Gel Electrophoresis to analyze DNA, RNA and proteins

2. A little background • Did you know that acids and bases neutralize each other? • An acid is anything that will dissociate a hydrogen ion (the acid is an electron acceptor) • A base is anything that will bind to a hydrogen ion (the base is an electron donor) • Carboxyl groups and phosphate groups are good acids • Amine groups are good bases! • If you put an acid in a basic buffer, the acid will become negatively charged. Draw it! • If you use an acidic buffer, a basic group will become positively charged. Draw it!

3. What is DNA? • DNA stands for deoxyribonucleic acid • 99% of it is found in the nucleus (nDNA). 1% is found in mitochondria (mDNA) • It is a polymer: that means individual units were linked together to create the long strand seen here • The individual units (monomers) are nucleotides Pictures from:http://publications.nigms.nih.gov/thenewgenetics/chapter1.html

4. What are nucleotides? • The DNA code of a gene—the sequence of its individual DNA building blocks, labeled A (adenine), T (thymine), C (cytosine), and G (guanine) and collectively called nucleotides Pictures from:http://publications.nigms.nih.gov/thenewgenetics/chapter1.html

5. Let’s see them up-close • Notice their sugar component (deoxyribose) • Notice their base (a purine or a pyrimidine) • Notice their phosphate groups! When put into a basic buffer, the hydrogen atoms dissociate and the phosphate groups become large, negatively charged groups! They act like an acid! Picture from:http://fig.cox.miami.edu/~cmallery/150/gene/c5x29nucleotides.jpg

6. So the DNA is now negatively charged, now what? • In 1973, Dr. Edwin Southern isolated genomic DNA • Used a restriction enzyme to cut the DNA into smaller fragments of varying sizes • Poured an agarose gel • Loaded the sample in the gel • Applied an electrical current • DNA separates according to size (smallest “runs” the fastest). Image from:http://www.scifair.org/+images/Electrophoresis.gif

7. OK, but gels are slippery and can break, plus it’s difficult to store for long term, what else can we do? A Southern Blot! Image from:http://www.gbiosciences.com/Image/BE315.jpg

8. Now we have a piece of membrane with our DNA stuck to it. But I can’t see anything, now what? • We have to hybridize our membrane with a probe. • A probe is basically a piece of a gene that is labeled (fluorescence or radioisotope). • Anywhere there is a gene on the blot that matches (basepairs) with our probe, they will hybridize • They remain hybridized even after washing to remove unbound probe. This is our probe!

9. Let’s review how we can manipulate DNA Image from:http://openlearn.open.ac.uk/file.php/2645/S377_1_007i.jpg Short term Quick viewing of DNA fragments Long term storage Blot can be reused (re-hybridized to other probes) and stored easily http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

10. But that’s not all! RNA molecules can be manipulated, too! • Did you know that RNA is also a polymer? • It is single stranded • Uracil not thymine! • And, RNA can be manipulated on a gel just like DNA. • This technique is called a “northern blot” • Not capitalized, just a “joke” taken from Dr. Southern’s technique. Negative charges when placed in a basic buffer! http://www.steve.gb.com/science/prokaryotes_and_eukaryotes.html

11. Northern blotting • This technique is used to find out which genes are transcribed! • You don’t need restriction enzymes because mRNA segments are short. • Ex: Different genes transcribed in different tissues • Ex: Different genes transcribed at different times of cell cycle within the same cell

12. Just because you see an RNA... • Doesn’t mean the gene was fully “expressed!” • Remember, ultimately the protein is the end product of gene expression. • Don’t worry! Proteins are polymers too! • Their monomers are amino acids! • When placed in a basic buffer, they have negative charges too! • Guess what? Western blots are used for protein analysis.

13. The 20 Amino Acids Glycine Alanine Valine Leucine Isoleucine Serine Threonine Cysteine Methionine Glutamic acid

14. Aspartic acid Lysine Arginine Asparagine Glutamine Phenylalanine Tyrosine Histidine Tryptophan Proline

15. Zwitterions and pI • The pH at which an amino acid exists in solution as a neutral molecule, a zwitterion. • Since zwitterions are not charged, they will not move • Isoelectric points (pI) are for proteins and reflect when their overall amino acid charges are balanced (electrically neutral). As such, they will not migrate to either side of an electrophoresis chamber. • As a zwitterion the amino acid is electrically neutral and will not migrate to either side of the electrophoresis chamber. • the pH at which an amino acid exists determines its net electrical charge and the type of charge (+ or -).

16. If you increase the pH of a solution of an amino acid by adding hydroxide ions, the hydrogen ion is removed from the -NH3+ group. During electrophoresis, this amino acid would move toward the anode (the positive electrode)

17. If you decrease the pH by adding an acid to a solution of an amino acid, the -COO- part of the zwitterion picks up a hydrogen ion. This time, during electrophoresis, the amino acid would move towards the cathode (the negative electrode).

19. Cellulose serum protein electrophoresis (SPE) • Proteins separate according to their electrical charges • Small amount of serum or urine • Current passed through strip • Proteins stained http://www.labtestsonline.org/understanding/analytes/electrophoresis/test.html

20. Bands of SPE • Albumin • Most obvious band. Single protein • α1- globulins • Almost entirely a1-antitrypsin • α2 -globulins • Mainly α2 -macroglobulin and haptoglobin • β-globulins • β 1-transferrin • β 2-C3 complement • γ-globulins • immunoglobulin

21. Example: normal and abnormal serum protein electrophoresis

22. In contrast to cellulose strips, SDS gels allow separation of proteins according to their size because of the negative detergent binding to the amino acids in the same ratio. http://employees.csbsju.edu/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htm

23. For a western, we use antibodies as a probe.....Why?A DNA probe would have nothing to bind to on a protein. We need to use antibodies. http://employees.csbsju.edu/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htm http://www.biology.arizona.edu/IMMUNOLOGY/activities/western_blot/west1.html

24. Let’s play • We have a family of 5: Dad, mom, Son 1, Son 2, and Son 3. • Who has the blue toenail gene? What test would you perform? • Who expresses the transcript? What test would you perform? • Who actually has blue toenails (no cheating by asking to remove shoes and socks!) What tests could you perform?

25. Here’s the Southern blot: + C -C D M S1 S2 S3 • Questions: • Why have a positive control? • Why have a negative control? • What happened to mom’s BT gene? • What can you say about the sons’ BT genes? • Which parent did they inherit them from?

26. Here’s the northern blot: + C -C D M S1 S2 S3 • Questions: • Who expresses the BT transcript? • Does this necessarily mean they have BTs? • What blot will provide you with proof?

27. Here’s the western blot + C -C D M S1 S2 S3 Who has ?

28. What if you ran out of a basic buffer? • Do you have to cancel your experiments? • What if you had an acidic buffer available? • What charges would your molecules now have? • How would you set up your electrophoresis equipment?

29. Enzyme-linked Immunosorbant Assay (ELISA) • Quicker than a Western • Not as specific as a western: antibodies do go “bad” and could bind aberrantly to an antigen they should not bind. • False positives: in addition to an antibody “going bad” antibodies tend to stick to the positive charges on the plastic dish. Blocking and washing are VERY important! • Pregnancy tests (confirmed with blood test and ultrasound). • HIV diagnosis (must be confirmed with western gel)

30. http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/technique.htmlhttp://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/technique.html Step 4 Step 1 Step 5 Step 2 Step 6 Step 3