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SectionNotes1 - Nucleotide units: Sugar, base, and...

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Unformatted text preview: Nucleotide units: Sugar, base, and phosphate! Notes for lectures 1 and 2 DNA and RNA structure base! Nomenclature: Important terms: nucleotide vs nucleoside: phosphate! (mono, di, or tri )! sugar! RNA: 2’ OH! DNA! Reactive oxygen thus RNA is not stable 1! (wikipedia) numbering carbons around a ring: C1’ is always the one that bases are attached. Duplex/double strand: The most stable conformation of DNA is a structure containing two Structure and ftis featuresdoublea tabilityor ucleotOften written as! dsDNA. strands, rtDNA 2-stranded or ofto s polyn a duplex. i de chain Imposo anactors contributing stranded : antiparallel:! A term used to describe the opposite orientations of the two strands of a DNA double helix; the 5' end of one strand aligns with the 3' end of the other strand. annealing/hybridizing: When one strand of DNA or RNA hydrogen bonds (or base pairs) with another strand to form a duplex. 5’ PO4- backbone has! bases have hydrogen bon d! cognate charge! or complementary strand: a strand ceptors that hasna rs! negative ac of DNA an d do o sequence that can base paired with another strand. the the complementary sequence for 5ʼ CGAT 3ʼ that can base pair to it would be 5ʼ ATCG 3ʼ. Important features of polynucleotide chain: 3’ OH chemically reactive! phospho diester linkage &! glycosidic bon d! the chain has polarity: ! 5’-3’! 3! Chargaff’s rule: # of A = # of T • -1 charge/nucleotide • • chemically reactive bonds become big players in DNA synthesis as well as repair #of G = # of C This forms the assumption that bases are paired chemical directionality: 5ʼ phosphate to 3ʼ hydroxyl  ­ ­>Attractive forces between bases in a pair are hydrogen bonds (horizontal with respect to the 5’ ­3’ polarity) Important features of polynucleotide duplex: • • • each base has functional groups that can hydrogen bond stably to only one of 3 other bases. The base A pairs with T, and G pairs with C. The base pair formed is predominantly planar and the distance from A to T in a base pair is roughly the same as the distance from G to C in a base pair. A common geometry for ! Watson-Crick (WC) base pairs (bps)! GC have 3 H ­bonds, comparing to 2 H ­bonds formed by AT  ­ ­> G and A are purines with two rings; T and C (and U in RNA) are pyramidines with 1 ring. These rings facilitate a base pair to interact with bp above and below it: base stacking (vertical with respect to the 5’ ­3’ polarity, think rungs on a ladder). This is accounted for by dipole ­dipole/van der waals intearactions as well as the pi orbitals in the rings. GC pair stacks better with AT pair above or below it in the duplex than TA because G and A have two rings to interact, and C and T have 1 ring to interact  ­ ­> The negative charges on the backbone want to repel each other as much as possible resulting in twist, this is also contributed by the need for duplexes to bury the more hydrophobic bases inside of the duplex but leave hydrophilic backbones toward the outside. If you store your DNA/RNA molecules in solutions with some salt (i.e. positively charged ions), you’ll be able to shield the negative charges from each other, and resulting in a more stable duplex. Aqueous or polar environments favor duplex and hydrophobic or non ­aqueous environment tend to break the duplex Stabalizing Interactions H-bonds between bases (since GC base pairs have 3 H-bonds and AT have 2, sequences enriched in GC content will be more stable) base stacking (Van der Waals interactions/ dipole-dipole interactions, and Pi orbital interactions from the aromatic bases) Destabalizing Interactions charge repulsion between the (-) phosphates on the backbone loss of translational/rotational degrees of freedom, ie effects entropy (donʼt need to know this one by heart) Hydrophobic effect (putting hydrophillic backbone on the outside to interact with water and hiding the more hydrophobic bases on the inside of the structure away from water. Also known as exclusion of water molecules from around the bases) the stabalizing interactions win Nucleic acids as information out and s DNA exists as a double helix. But can cause the carrierthus duplex to fall apart if you disrupt the stabalizing interactions: chemically or thermally by heating it up. Major grooves present different functional groups the helix ydrogen, methyl groups, Salt and length dependencies of such as h to coil transition H ­Melting curves for dsDNA for proteins to recognize and bind to nucleic acids, bond donors/acceptors, whereas minor grooves have degenerate information, i.e. GC will be read the same way as CG base pairs, AT same as TA. A versus B form duplex: Reiter et al. 2008  ­ ­> more space between BPs  ­ ­> shorter distance among BPs vertically in the vertical direction thus better base stacking  ­ ­> shorter diameter if looking  ­ ­> larger diameter thus better down a cross section of helix electrostatic repulsions of neg charge *Therefore, A ­form dsRNA is more stable  ­ ­> shallow major grooves for access  ­ ­> major grooves are buried too deep *Therefore, B form dsDNA is better info carrier Endonucleases: endo = cuts inside of a polynucleotide These enzymes evolved in bacteria to protect own genome against foreign genetic material, where its own genome is modified somehow, e.g. methylated3 types of ends: 5’overhang, 3’overhang, or blunt (cutting at the same base pair on both strands) Useful in cloning: If you are trying to introduce a piece of DNA into a bacteria plasmid and have the bacteria amplify this DNA for you, you’ll need endonucleases. 0 EcoRI 200 NotI 300 BamHI 350 insert- 1500bp total Plasmid 4kB EcoRI 200 EcoRI 800 In this case, you can’t use EcoRI to cute your plasmid and pop back in an insert that’s also cut with EcoRI, because that’ll give you three pieces of the insert But you can use NotI and BamHI sites. You would have to introduce those recognition sites on either end of the insert (use both to specify direction, if you only add NotI sites on both end, there’s 50% chance that your insert will be ligated into the plasmid in the reverse direction) After cutting both plasmid and insert with NotI and BamHI, ligation of the now compatible ends, you’ll get a plasmid that looks like the left figure. Insert + plasmid plasmid only 0 EcoRI 200 4.6 kB NotI 300 Insert 1500 Plasmid 4kB 600bp BamHI 1800 Linearized 4kB 300bp Gel after EcoRI digest; smaller fragments run faster Other techniques: Southern blots are for DNA, Northern blots are for RNA. Both involves cutting nucleic acids to be examined with restriction enzymes, run a gel, blot the gel onto membrane. After denaturing the blotted nucleic acids into single stranded pieces, you add a radiolabeled or fluorescently labeled probes (also denatured). Your probes should be specific to the region/sequence of interest. Then you can either test for radioactivity of certain bands on a blot or image the fluorescence Fluorophores and many other compounds such as ethidium bromide used to image DNA contain aromatic rings, so they can intercalate into the duplex and mimic base stacking interactions. ...
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This document was uploaded on 09/12/2011.

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