Download Biochemistry ± DNA Chemistry and Analysis DNA o Adenosine

Biochemistry ± DNA Chemistry and Analysis
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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
ƒ Bases are NOT attached covalently to one another
ƒ 6WUXFWXUHVDUHW\SLFDOO\ZULWWHQ/WR5¶WR¶XQOHVVRWKHUZLVHVWDWHG
o Nonenzymatic Hydrolysis of Nucleic Acids
ƒ Strong Acidic Hydrolysis of DNA / RNA results in purines removed from sugar
x purine bases / pyrimidine nucleosides / deoxyribose or ribose / P*
ƒ 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
ƒ 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
ƒ 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
ƒ Van der Waals forces contribute to stability of the helix
ƒ 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)
ƒ B: 10bp to complete one turn of double helix
o Major and Minor GroovesVLWHRIUHJXODWRU\SURWHLQELQGLQJPDMRUZLWKPDMRUODUJHUǖWKDQPLQRUǖ
ƒ Formed by the angle of glycosyl bonds (>180º = major, <180 º = minor)
ƒ Strands do not need to be separated for protein to read DNA sequence
o Stability: normally a flexible chain, below interactions provide stabilization
ƒ Hydrophobic & electronic interactions (Van der Waals) btwn stacked bases - shielded from aq environment
ƒ +\GURJHQERQGVEHWZHHQQLWURJHQEDVHVRIFRPSOHPHQWDU\VWUDQGV$ 7*Ł&
ƒ 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
ƒ 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
ƒ Heating causes H-bonds to disrupt, 2 strands separate
x A=T melts at lower temps than GŁ&
ƒ Annealing: H-bond formation allows dsDNA to form complementary single strand
o Confirmations of the Double Helix
ƒ Same base pairing rules apply, but may function differently in gene regulation / expression
x Changes in shape of minor / major grooves affect protein binding
ƒ B: most common, described by Watson-Crick, 10bp
ƒ C: QRWIRXQGSK\VLRORJLFDOO\ESWXUQǖ
ƒ 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
ƒ 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
ƒ B & Z can be present in one segment without separating strands
ƒ 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
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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
ƒ Causes: high temp or pH
x Low ionic strength (not sufficient along to denature, used to inc efficiency of other 2)
ƒ 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
ƒ 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
ƒ 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
ƒ 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
ƒ Northern: RNA electrophoresed, transferred to membrane & immobilized, hybridized with DNA probe
x Measure message levels in genes
ƒ Western: Proteins electrophoresed, transferred to membrane & immobilized, visualized by immuno. Procedure
o Enzymatic Hydrolysis (Nucleases) ± metabolism within the cell
ƒ RNases ± only RNA
ƒ DNases ± only DNA
ƒ 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
ƒ 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
ƒ 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
ƒ Beneficial: chromosome structure / creating recombinant DNA / isolating genes / sequencing DNA molecules
ƒ 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$OLQNLQJUHVWULFWLRQIUDJPHQWVIURPRQHRUJDQLVPZLWKDQRWKHU¶V'1$
ƒ DNA of organism 1 linked to vector (bacterial / viral fragment propagated in host organism)
o Procedure
ƒ DNA fragment from organism of interest obtained from genomic / cDNA library
ƒ Fragment introduced into plasmid (small circular DNA from bacteria)
ƒ Recombinant DNA introduced into bacterial cell via transformation
ƒ Bacteria with engineered plasmid selected for via Ab & isolated
ƒ Bacteria replicate and DNA cloned
o Selection of a Vector ± How can we propagate this DNA fragment?
ƒ Possess a number of sites cleaved by different restriction enzymes
ƒ Selectable marker: Genes that encode for resistance to Ab ± allows for selection of bacteria
ƒ 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
ƒ Ligation = formation of phosphodiester bonds via enzymes
ƒ SE created from blunt ends which have Linker Sequences added to them
ƒ 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)
ƒ 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
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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
ƒ Plasmids with DNA sequences (cos sites) allowing plasmid to package into
bacteriophage coat protein
ƒ 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
ƒ 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