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Biochemistry ± DNA Chemistry and Analysis x x x DNA x RNA o Adenosine / Guanine / Cytosine / Thymine o Adenine / Guanine / Cytosine / Uracil o Sugar: 2-deoxyribose o Sugar: -ribose o Phosphate linear chain linkage o Phosphate linear chain linkage Structure of Nucleic Acids o Linkage: mononucleotides are linked via phosphodiester bridges between the ¶DQG¶K\GUR[\OJURXS Bases are NOT attached covalently to one another 6WUXFWXUHVDUHW\SLFDOO\ZULWWHQ/WR5¶WR¶XQOHVVRWKHUZLVHVWDWHG o Nonenzymatic Hydrolysis of Nucleic Acids Strong Acidic Hydrolysis of DNA / RNA results in purines removed from sugar x purine bases / pyrimidine nucleosides / deoxyribose or ribose / P* Strong Basic Hydrolysis x 2I51$SURFHHGVWKURXJKD¶¶-cyclic phosphate intermediate o Products: ¶¶ nucleoside monophosphates x Of DNA DOES NOT OCCUR ± ODFNV¶-hydroxy group o Important for separating RNA (n) / DNA (stable base) DNA Double Helix: both prok and euk cell DNA has a double helix wrapped around the same axis o Right handed, Į-helix o Backbone: phosphodiester bonds and deoxyribose sugar on outside of molecule Polar Groups: x P* project to outside, interacting with (+) charged (@ physiological pH) proteins x (-) P* (@ physiological pH) complexed with cations (Na/K/Ca) to stabilize helix Nonpolar Groups: interior of the molecular structure, water excluded o Bases project to the interior of helix, with planes parallel to one another and stacked perpendicular to long axis of strand Van der Waals forces contribute to stability of the helix Base Pairs: bases of strands form hydrogen bonds with bases of the other-shows complementary relationship x A=T GŁ&3DLULQJVNOT in equal distribution x $ 7ZHDNHUWKDQ*Ł&VRVHFWLRQVWKDWDUH$ 7ULFKFDQEHHDVLHUVHSDUDWHG o Strands are complentary (not the same nt sequence) and antiparallel RQHUXQV¶-¶RQHUXQV¶-¶ o 2 3HULRGLFLWLHVRQHDWǖSDUallel bases separation) and 34 ǖ (one complete turn of the helix) B: 10bp to complete one turn of double helix o Major and Minor GroovesVLWHRIUHJXODWRU\SURWHLQELQGLQJPDMRUZLWKPDMRUODUJHUǖWKDQPLQRUǖ Formed by the angle of glycosyl bonds (>180º = major, <180 º = minor) Strands do not need to be separated for protein to read DNA sequence o Stability: normally a flexible chain, below interactions provide stabilization Hydrophobic & electronic interactions (Van der Waals) btwn stacked bases - shielded from aq environment +\GURJHQERQGVEHWZHHQQLWURJHQEDVHVRIFRPSOHPHQWDU\VWUDQGV$ 7*Ł& Ionic: (-) P* and polar sugar exposed to aq environment x Keep strands from collapsing by keeping (-) charges separated from one another o Melting: DNA in solution and then slowly increase temperature in the lab Hypochromic Effect: when DNA heated to the melting temp, and an increase in UV light absorbance occurs x Stacked bases in dsDNA shield UV absorption x Melting causes UV to increase absorbance by bases ± monitor melting based on absorbance in soln Heating causes H-bonds to disrupt, 2 strands separate x A=T melts at lower temps than GŁ& Annealing: H-bond formation allows dsDNA to form complementary single strand o Confirmations of the Double Helix Same base pairing rules apply, but may function differently in gene regulation / expression x Changes in shape of minor / major grooves affect protein binding B: most common, described by Watson-Crick, 10bp C: QRWIRXQGSK\VLRORJLFDOO\ESWXUQǖ A: ESWXUQǖ1-bases not perpendicular but tilt 20º, forms under low hydration conditions x RNA only forms A in double stranded form ± due to ribose structure Z: 12bp/turn, L-handed helix/ staggered zig-zag backbone/ bases found on the outside x Occurs in vivo in alternating pur-pyr stretches (specifically G-C rich) / favored in regions of methylation of deoxycytidine in GC rich regions x Stim Expression of Genes: proteins bind specifically to Z-DNA & may function in gene regulation B & Z can be present in one segment without separating strands Segments of adenosine in a row can bend helix - 6 in a row can cause a bend of 18º x May be a feature recognized by DNA binding proteins ± expression blocked in regions with a bend x x x Information Storage & Retrieval of Information: Two strands compose DNA o Coding Strand: nt sequence is same as that of RNA the DNA was made from, U substituted by T o Template Strand: complementary to Coding strand, and is what the RNA would bind to during protein production Laboratory Chemistry of Nucleic Acids o Denaturing (melting): separation of 2 strands of DNA due to disruption of binding forces Causes: high temp or pH x Low ionic strength (not sufficient along to denature, used to inc efficiency of other 2) Significance x Necessary for DNA Replication / RNA Synthesis ± requires energy expenditure x Development of experimental approaches for diagnosis of disease o Renaturation (*hybridization*): convert DNA to solid substrate and denature them to single strand so probe can bind Hybridization of labeled probe to DNA molecules allows selection of clone DNA sequence x Probe: defined ssDNA sequence of sequence sought in genome, specific gene you want to examine o Synthesized and labeled to follow probe ± DERXWQW¶VRQO\RFFXUs few times in a genome) o Must use specific melting temperature for that probe, so as not to bind non-specifically Non-specific hybridization occurs at temperatures below melting point o Last bound colonies will be most specific in large pop of DNA ± important concept o Blotting: utilizes hybridization SNOW DROP Southern: DNA electrophoresed (size), denatured, transferred to nitrocellulose gel, hybridized with probe x Separation by size, smaller fragments run faster x Looks for gene structure / complexity Northern: RNA electrophoresed, transferred to membrane & immobilized, hybridized with DNA probe x Measure message levels in genes Western: Proteins electrophoresed, transferred to membrane & immobilized, visualized by immuno. Procedure o Enzymatic Hydrolysis (Nucleases) ± metabolism within the cell RNases ± only RNA DNases ± only DNA Exonuclease: eats DNA/RNA one base at a time starting from a specific end x ¶SUHVHQWHGZLWK¶-hydroxyl on terminal nucleotide to remove form polynucleotide chain x ¶SUHVHQWHGZLWKIUHH¶-OH on terminal nucleotide to remove from polynuclotide chain Endonuclease: eat the interior RIDSRO\QXFOHRWLGHVSHFLILFIRU¶RU¶VLGHRISKRVphodiester bond x Can cut ssDNA or dsDNA depending on nuclease o Restriction Endonuclease: recog specific base sequence in foreign DNA for degradation & cleave both strands Found in bacteria and not in eukaryotes ± restrict growth of bacterial viruses x Host modifies DNA so that they do not attack host DNA x Cuts at defined sequences and then allows for annealing with same enzyme produced ends o Cut at certain sequence and then put it back together Beneficial: chromosome structure / creating recombinant DNA / isolating genes / sequencing DNA molecules Palindrome: reads the same way forward and backward ± many cut at these sequnces x Creates specific ends of DNA and assist cloning & creating specific size DNA fragments Cloning DNA with Restriction Endonucleases o RecombiQDQW'1$OLQNLQJUHVWULFWLRQIUDJPHQWVIURPRQHRUJDQLVPZLWKDQRWKHU¶V'1$ DNA of organism 1 linked to vector (bacterial / viral fragment propagated in host organism) o Procedure DNA fragment from organism of interest obtained from genomic / cDNA library Fragment introduced into plasmid (small circular DNA from bacteria) Recombinant DNA introduced into bacterial cell via transformation Bacteria with engineered plasmid selected for via Ab & isolated Bacteria replicate and DNA cloned o Selection of a Vector ± How can we propagate this DNA fragment? Possess a number of sites cleaved by different restriction enzymes Selectable marker: Genes that encode for resistance to Ab ± allows for selection of bacteria Internal signals (promoters, etc) permit insert to be transcribed & translated into protein o Sticky Ends: H-bonding of complementary extensions to facilitate DNA fragment ligation Ligation = formation of phosphodiester bonds via enzymes SE created from blunt ends which have Linker Sequences added to them Linker Sequences: joined covalently to ends of DNA & contain restriction enzymes ± convert to specific restriction enzyme type o Isolation of DNA to be Cloned ± to isolate specific gene, screen a DNA Library (contains fragments of DNA) Single DNA molecule (pure fragment of DNA) not often used, usually isolate a gene for specific protein x DNA Source: genomic DNA digest (genomic library) or DNA from mRNA (cDNA library) x Library consists of transformed cells bearing different fragments of DNA or cDNA - coding Genomic Library for isolating a specific gene ± has non-coding regions in it x cDNA is just the coding region and will have introns removed to generate protein x Vector: DNA to be inserted is small (6-10kb) o ȜSKDJHYHFWRUV can handle up to 20kb ± infects E.coli o COSmids: can handle up to 50kb Plasmids with DNA sequences (cos sites) allowing plasmid to package into bacteriophage coat protein Allows efficient expression of the library, replicated as a plasmid & insert Cloning: constructed by cloning all DNA of genome of an organism x Larger vectors used for fewer colonies to clone entire genome / genes of interest obtained complete ¶¶flanking sequences) x Insert DNA from partially digesting genomic DNA with restriction endonuclease (Sau3A) x Separated fragments separated by electrophoresis& removed for cloning x 5DQGRPJHQRPLF'1$IUDJPHQWVOLJDWHGWRȜSKDJHYHFWRUV cut with BamHI x ȜEDFWHULRSKDJHJHQRPLFOLEUDU\: random DNA ligated to Ȝ genome arms o infect bacteria and propogate o Screening of library allows you to look at human genome Conducted via hybridization with a probe for sequence of interest x Human Genome Project done with bacterial artificial chromosomes (BACs) which can accommodate up to 300kb Eukaryotic DNA Sequences x Highly repetitive: localized @ centromere & participates in alignment during mitosis o aka satellite DNA and not transcribed o More of a structural role in chromosome formation x Moderately Repetitive: both transcribed and untranscribed sequences o Ex. rRNA rubunits / tRNAs / histone proteins o Randomly repeated with untranscibed spacer regions between them o Several hundred to several thousand per genome required to satisfy need of cell