BMB 462 Lecture 20 Outline of Last Lecture I Function of DNA and RNA II DNA Carries the Cell s Genetic Information III Primary DNA Structure IV The History of DNA Determining Secondary Structure Outline of Current Lecture I Strand and Base Properties II Base Conformation III Summary of DNA Structure IV Comparing Types of DNA V Denaturation VI Techniques Utilizing Denaturation VII Other Secondary Structures VIII Mutations in DNA Current Lecture Concepts to remembers from previous courses lectures I Strand and Base Properties a Base Pairing i 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 structure b Strand Properties i 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 transmitted ii The cell replicates the information by copying the strands II Base Conformation a Glycosidic Bond Rotation i 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 conformation iii 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 structure a The hydrophobic core in DNA is what makes it so stable Each base pair can make hydrophobic 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 DNA ii The proximity to the bases that the major groove brings the transcription factor allows the factor to recognize specific nucleotide sequences c 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 structure d Purines are in syn conformation so the bases are pointing the other way IV Comparing Types of DNA a 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 ray i It is essentially the B form but dehydrated and therefor scrunched together the major groove is compressed so the molecule is shorter Bform is 34 angstrom the a form is shorter c Both B and A form are right handed helices d 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 Denaturation a 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 strands remained 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 microarrays d Denaturation Curves i To plot denaturation curves you measure the amount of double stranded DNA 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 Denaturation i Organic solvents reduces the hydrophobic interactions between the bases dissolving DNA from the core 1 This decreases tm ii Salt Concentration increases the repulsion between the phosphatesugar 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 strands iii Length The longer the DNA is the more energy it would take to denature it because there are more bases to dissociate 1 Increased length increases tm iv pH changes in pH cause tautomerization changing the h bonding ability 1 When pH deviates from the optimal range tm decreases v GC content GC forms 3 H bonds vs the 2 that AT pairing forms so more energy is required to separate them 1 The higher the GC the higher the tm f Denaturation Mapping i Partial denaturation results in separation of the 2 strands at areas that are AT rich since they are less stable than the GC areas 1 Single strand DNA tends to be AT rich 2 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 Denaturation a Hybridization i 2 strands of DNA could anneal if their sequences have enough in common despite some mutations mismatches 1 This is useful for mapping DNA ii Microbiological Techniques using Hybridization 1 Southern Blot A short oligonucleotide probe can hybridize to a specific DNA sequence a Developed by Edward Southern 2 Northern Blot The probe hybridizes to a specific mRNA sequence a 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 chip a Each spot on the chip has a specific oligonucleotide sequence b In your sample you label the sequences using fluorescent dyes i e the desired target
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