BIOM 121 1st Edition Lecture 5Outline of Last Lecture 1. Aromatic Rings2. Absorption of Light3. Chirality4. High energy bonds5. Weak chemical interactionsa. Hydrogen bondsb. Ionic bondsc. Van der Waalsd. Hydrophobicity6. Protonation and deprotonationOutline of Current Lecture 1. Denaturation2. Nucleases3. Restriction Endonucleases4. Uses of restriction enzyme5. Gel electrophoreses6. Ethidium BromideCurrent Lecture1. Denaturation- Denaturation is reversible- Denatured DNA can be cooled down and the strands want to fulfill the base pairs and hydrogen bonds. Cooling down can let the two complementary strands find each other- Highly unlikely that the two particular strands from before find the same one, as long as it finds the one with complementary base pairs- Do the two sequences have to be perfectly complementary? No.o You can take two pieces that are almost similar and you can put them togethero Heteroduplexes (hybrid molecules/hybridization)- Two DNA pieces that are similar can realign with the same one or with another one. It is the complementary base pairs between the rest of the molecule that can hold it together if they are not exactly the same.These 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.- The non-hybrid is more stable. You can alter the conditions where hybrid is not form to make it more stable. You can alter the “stringency”/ make it more stringent so only the similar strands complement. 2. Nucleases- Enzymes that cut nucleic acids by hydrolyzing phosphodiester bonds. They cut and breakcovalent bonds.- DNase- cut DNA. RNase- cut RNA; some do both- Exonuclease: starts at the end. It only recognizes the broken end and cuts from there- Endonuclease: recognize in the middle of the DNA and cuts from there- Products: nucleotides (nucleoside phosphates) due to exonuclease or endonuclease cutsto oligonucleotides (still pieces of DNA)- Most nucleases are nonspecific. 3. Restriction endonucleases: recognize specific sequences in DNA- Many cut specifically at the recognition sequence -> oligonucleotides (2 DNA)- Some restriction enzymes produce products with “sticky ends”- Recognition sites vary in length. There can be short ones that occur frequently- Eco R 1: recognize 5’ GAATTC 3’ This sequence is a PALINDROMEo Cuts DNA at the sites between G and A.  EcoR1 site: has ¼ next place has the letter-> probability is 1 in 46 bp or 1 in4096 bp on average! On average you will find a site at 4 kb.- Sau3A1 has 4 and has the frequency of .25 kb- Restriction enzymes cut where it recognizes but doesn’t cut the same way. - EcoR1 cuts at 5’ phosphate and 3’ hydroxyl group- “Sticky” end: has single strand DNA and wants to base pair with something complementary to it. EcoR1 has 5’ overhang- Hpa1: cuts right through the middle: blunt end- Pst1 leaves a 3’ overhang sticky end- Any piece of DNA cut by EcoR1 can bind with any other DNA cut by EcoR1: can take human and put it with E Coli. DNA -> recombinant DNA (different sources to recombine)4. Uses of restriction Enzyme: - Cloning: any 2 pieces of DNA with compatible ends can be easily put together. - Analysis: relative position of restriction sites provide information about the sequence of the DNA- Molecular cloning: o Plasmid: an autonomously replicating piece of DNAo Replicates independently of DNA. Naturally reoccurring.o Some plasmids can resist antibiotics.o Has an origin of replication!- You can cut the plasmid with ECOR1 and another DNA of interest, heat it up so it annihilate, it gives a ligated fragment. As you cool it down, fragments join together with DNA ligase and give it a recombinant. However, not all plasmids can- Take mixture of ligated plasmids and transform to E. coli; however, some E. Coli do not have it- The transformed cells are plated on agar -> the cells transformed will be able to grow into a colony in Tetracycline- Purify the bacteria: and clone the cell.- Why “restriction endonuclease”o The enzymes restrict foreign DNA from coming in the cellso Different bacteria make different enzymes.o How do you keep from cutting up your own DNA: every restriction enzymes comes with a partner enzyme and will methylate one of the groups at the major groove and then the enzyme can no longer recognize the sequence. The new DNA coming in has unmodified (not methylated yet). Modification. Restriction modification systems because they come in pairs5. Gel electrophoreses- DNA separation by gel electrophoreses- Analytical tool: size/length- Preparative tool: separate them from one another- Run a current in the gel. Since DNA is a negative charge, it will migrate from the negativeto the positive pole. The gel separates the molecules based on size so small travels quicker- Nothing will move if there were no electric field- A gel matrix is use to “sieve” the moleculeso Agarose: loose; broad separation rangeo Acrylamide: tighter; narrower separation range- Movement through the gel is dependent on the CHARGE and SHAPE. NOT ON MOLECULAR WEIGHT!!- The two molecules must have the same charge to mass ratio; if that is true, then the separation is proportional to the molecular weight!- DNA has 2 charges per base pair (2 phosphate) 2 -. All DNA has 2 – per base pair. - All linear double stranded DNA have the same shape.- Therefore, movement is proportional to length6. Ethidium bromide- Has a width almost exactly like a base pair. It has a negative charge and amine groups- Ethidium bromide wants to stack with the other base pair: intercalation. - It is a fluorescent molecule. It absorbs light in a quantized fashion. It kicks the electrons up to another state and loses some energy via heat and the electrons fall into anotherstate and then go back to the ground state. When they go back down, they give off energy: photon of light. It will give off a particular wavelength of light. Fluorescent absorb light at some wavelength (UV), loses some energy, then give off light with less energy (visible)- Ethidium in solution vs intercalation in base pair; intercalation lets the intensity increase about 20 fold. It is a