Current LectureMCB 250 1st Edition Lecture 6Outline of Last Lecture1. Denaturation2. Nucleases3. Restriction Endonucleases4. Uses of restriction enzyme5. Gel electrophoreses6. Ethidium BromideOutline of Current Lecture 1. Gel electrophoresis2. Southern Blot3. Probe 4. Colony Blot5. Protein6. Properties7. Disulfide Bridges8. Nomenclature9. Folding10. Secondary StructureCurrent Lecture1. Gel electrophoresis:- Size: Distance migrated is inversely proportional to the logMW (log # basepairs)- Quantity: How much DNA is present in each band. Because the amount of ethidium that binds is dependent on how much DNA is there.o Twice as many BP, would be twice as bright. Ethidium gets intercollated within the base pairs- A marker has a known size and quantity. Compare unknown band to known band. Brightness can be similar but the size isn’t the same but the total amount of DNA is thesame.- The size can be from how much molecules there are, but the brightness is how much total DNA are in a bandThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Molecular weight (total in base pairs) and How much DNA is within the band. The2 pieces of information are INDEPENDENT from one another- Does the presence of contaminating protein interfere with gel electrophoresis? NO: Protein may be positive or negatively charged. The negative will move towards the positive pole, uncharged would stay in the well (some DNA can be trapped), some protein are positive and went the other way. The protein doesn’t bind to ETHIDIUM BROMIDE: therefore you cannot see the protein. - Is this as accurate as measuring the absorbance? No, using the UV spectrophotometer will be more accurate but depends on pure DNA. This is relatively accurate by needing to know that it is just 100 ng not 103 or anything.- If the sample has DNA binding proteins, it can just shift the band. - The density of the matrix affects the mobility of the DNA. You can alter the density to maximize the fragment of DNA you want to resolve. o Gives a better resolution - The distance traveled is inversely proportional to the molecular weight but the relationship drops off a bit at top and bottom of gel.2. Southern Blots- Ethidium bromide stains all DNA because it is nonspecific. If you want to identify a part of a DNA gene ->- Take E. Coli (4 mil bp) and cut is with ecoRi1: 4600kb/4kb (how often it cuts) = We expect 1150 fragments. Cant possibly resolve all fragments- Do electrophoreses and stain (it’ll be smeared)- Southern Blotting: combine gel electrophoresis with hybridization/ probing a particular piece of DNA- Take gel and put it on transfer paper. Run electricity another way to have bands transfer into a piece of paper. Change conditions of buffer to denature the DNA. Add aprobe to find a particular piece of DNA.3. Probe- If you know a little about gene of interest, you make a probe: a single piece of DNA with a specific sequence. You would label that DNA with radioactivity. (P-32). DNA on paper is mix of total DNA. One of the bands has the sequence that is complementary to the probe. Add the probe and it will hybridize with the DNA of interest, wash off the extras and put it in a piece of x- ray film and it will be recognized as the radioactivity decays and expose the x-ray film: Radio-otography- Have a complex mixture of DNA -> what particular band has the sequence of interest by probing. Using hybridization to pull out the sequence you’re interested in instead of using ethidium bromide.4. Colony Blot- If you want to clone a gene into a plasmid. Cut plasmid and DNA of interest, mix, ligate, and transform -> colonies. The mixture at ligation reaction is complex. Only a small fraction of the plasmid may have the gene of interest. Use colony blot to find which colony has plasmid of interest.- Transform DNA and get individual E. Coli colonies with different plasmid. Which colony has the one with interest? Transfer the CELLS to paper and treat it to open E. Coli and crosslink DNA and probe it with a probe of known sequence to put it on X-ray film. - Using hybridization to find a piece of DNA5. PROTEINS- Proteins are made up of amino acids. Amino acid has an Alpha carbon with an Hor side group with carboxylic acid and amino group. - They are all L-Amino acids (except glycine isn’t a chiral carbon (no L or P)- Amino acids are connected with a DEHYDRATION REACTION (remove water) between carboxyl group and amino group of another -> make PEPTIDE BOND. This is not a spontaneous reaction. There is a mechanism that does this.- NOTE: the charge of both carboxyl and amino group went away after this reaction. Dipeptide (2 amino acids) but the N terminus of the protein still has a charge (amino) and the C terminus (carboxy group) still has a charge. - Always write proteins N terminus to C terminus!!!!- The peptide bond is a resonant structure. The electrons are being shared between the oxygen and nitrogen-> similar to double bond- The peptide bond is FLAT and does not rotate. The only rotation is around the alpha carbon.6. Properties of side groups of the amino acids!!!!- Hydrophobic amino acids can’t form dipole moment.. They do have charges,- Acidic amino acids are around 4: its not exact because it depends on the local environment- Hydrophilic: can be active group cysteine - Basic side groups: histidine is closer to neutral because it is the protonated form: only occurs of the pka is below that.- Aromatic: Tyrosine has hydroxyl group that can be active site*need to know side chains properties and structure7. Disulfide Bonds- 2 cysteine side groups can form a disulfide bond. 2 Cysteine = CYSTINE.- Can have effects on protein structure: 2 cysteine can form a COVALENT linkage (it can be intramolecular in the same protein or intermolecular between 2 different proteins)- It can occur spontaneously. But we hardly see disulfide bonds inside the cells. This is because cells control the redusive potential so di-sulfide bonds don’t form as much. Don’t see generally inside cytoplasm, rather see more disulfide bonds outside. E. coli, protein secretes out the cell, when secrete, outside makes disulfide bonds. Enzyme mediation can create or break disulfide bonds. - Forces affecting protein structure:- H bonds, ionic bonds, Van der Waals bonds, disulfide bond, hydrophobic cluster,stacked rings, negatively charged amino acids.8. Nomenclature - Primary