BMB 462 Lecture 20 Outline of Last Lecture I. Function of DNA and RNAII. DNA Carries the Cell’s Genetic InformationIII. Primary DNA StructureIV. The History of DNA – Determining Secondary StructureOutline of Current Lecture I. Strand and Base PropertiesII. Base ConformationIII. Summary of DNA StructureIV. Comparing Types of DNAV. DenaturationVI. Techniques Utilizing DenaturationVII. Other Secondary StructuresVIII. Mutations in DNACurrent LectureConcepts to remembers from previous courses/lectures:-I. Strand and Base Propertiesa. Base Pairingi. Watson realized that you get base pairing when the bases are close enough together b/c the atoms need to be a certain distance apart for the hydrogen atoms to interact and cause hydrogen bonding.1. He noticed you could form the same overall structure with an AT base pair as a GC base pair (when you place them on top of each other, they take up the same amount of space - the distance between one C1' and the other in an AT base pair is about the same as in a GC base pair) which allows both pairs to fit in the core of the helix.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.ii. The base pairing also allowed replication by helping determine the positioning of each base in the structureb. Strand Propertiesi. The two strands had to run in opposite directions and complementary means one strand has all the info for the second strand, which allows replication. This gave enough information to understand the model of how DNA information is transmittedii. The cell replicates the information by copying the strands.II. Base Conformationa. Glycosidic Bond Rotationi. Rotation of the glycosidic bond can occur, changing the nucleotides between syn- and anti-conformations.ii. This is particularly important for the pyrimidines because when they are rotated to syn- position, the oxygen on the base would clash with the oxygen in the sugar. (i.e. cytidine)1. Pyrimidines are always in the anti- conformationiii. Purines can be found in syn- or anti- conformation. They are most often found in the anti- conformation in B-DNA (which is the most common type of DNA in the cell)III. Summary of DNA structurea. The hydrophobic core in DNA is what makes it so stable. Each base pair can makehydrophobic reactions with the pairs above and below it, increasing stability.b. The helix also has a major groove and a minor groove. The major groove is key in transcription and replication for binding transcription factors.i. Most transcription factors have an alpha-helix known as a recognition helix that binds to the major groove and allows the factor to recognize DNAii. The proximity to the bases that the major groove brings the transcription factor allows the factor to recognize specific nucleotide sequencesc. The H-bonds give DNA its specificity and also give stability. The hydrophobicity also gives stability.i. The Phosphates have hydrophilic reactions that also allow for greater stability of the structured. Purines are in syn- conformation so the bases are pointing the other wayIV. Comparing Types of DNAa. B Form - Standard, most common form in cells.i. It has a wide major groove that the alpha-helix can fit in.b. A Form - The form Franklin had seen in x-rayi. It is essentially the B-form but dehydrated and therefor scrunched together (the major groove is compressed so the molecule is shorter (B-form is 34 angstrom, the a-form is shorter)c. Both B and A form are right handed helicesd. Z Form - A third type of DNA is Z-form, which has a left-handed orientation and it is unknown whether or not they exist in humans. It is also missing its major groove.V. Denaturationa. By heating up DNA, you can denature it, or cause the double helix to melt. This results in the separation of the strands.b. The reverse is annealing, which allows the strands to come back together. i. This can either be a very fast process (if at least a small part of the strandsremained in contact) or a very slow process (if the two strands became fully dissociated, since the two strands would have to spend time to get properly oriented)c. This ability is used in many procedures, i.e. PCR or microarraysd. Denaturation Curvesi. To plot denaturation curves, you measure the amount of double strandedDNA vs. single stranded DNA present in solution. 1. This is measured via UV absorbance or by using differential dyes that can only be incorporated into dsDNA or ssDNA (i.e. SYBR green)ii. It’s also possible to measure the melting temperature and the tm (the temperature where half the DNA is melted)e. Factors altering tm of Denaturationi. Organic solvents - reduces the hydrophobic interactions between the bases, dissolving DNA from the core1. This decreases tmii. Salt Concentration - increases the repulsion between the phosphate-sugar backbones (the positive ions in solution increase repulsion of the negative charges in the backbones)1. Increasing salt increase tm because you are reducing the repulsion of the 2 strandsiii. Length - The longer the DNA is, the more energy it would take to denature it because there are more bases to dissociate1. Increased length increases tmiv. pH - changes in pH cause tautomerization, changing the h-bonding ability1. When pH deviates from the optimal range, tm decreasesv. GC content - GC forms 3 H-bonds, vs. the 2 that AT pairing forms, so moreenergy is required to separate them.1. The higher the GC, the higher the tmf. Denaturation Mappingi. Partial denaturation results in separation of the 2 strands at areas that areAT rich, since they are less stable than the GC areas.1. Single strand DNA tends to be AT rich2. Promoter regions and origins of replication are also AT rich; these regions have to be melted to allow either transcription or replication.VI. Techniques Utilizing Denaturationa. Hybridizationi. 2 strands of DNA could anneal if their sequences have enough in common, despite some mutations/mismatches1. This is useful for mapping DNAii. Microbiological Techniques using Hybridization1. Southern Blot - A short oligonucleotide probe can hybridize to a specific DNA sequence.a. Developed by Edward Southern2. Northern Blot - The probe hybridizes to a specific mRNA sequencea. This used to be the only way to see if a certain gene was present.3. Microarray – Short DNA oligonucleotide sequences are attached to the surface of the microarray chipa. Each spot on the chip has a
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