Techniques Reading Handout of Chapters 3 (phosphorylation and ligation) and 10 from

Blackburn & Gait Describe UV absorption by nucleic acids Absorbance of free nucleotides >> single stranded nts >> double stranded nts Hypochromic effect Recognize a melting curve and understand denaturation Know that the Tm is affected by base composition Understand the use of PAGE to separate nucleic acids Be able to describe when nondenaturing vs. denaturing gels would be used Know the characteristics of agarose vs. polyacrylamide gels Know the DNA modification reactions of: Phosphorylation by T4 polynucleotide kinase Ligation by T4 DNA ligase Cleavage of DNA by restriction endonucleases (Types I, II, III) Understand process of PCR Understand why you would use Northern and Southern blotting

DNA & RNA IsolationDNA very stable and long; easy to inactivate deoxyribonucleasesRNA more subject to degradation by RNases

General steps:Isolate cells, Lyse cells, Remove proteins, Isolate pure nucleic acid

DNA - during isolation exclude free Mg2+ (cofactor for DNases and can be excluded by adding 10 mM EDTA as a chelator and by use of SDS) and avoid rough treatment (rigorous pipetting, vortexing should be avoided if DNA > 10,000 bp)

1. Isolate cells by centrifugation2. Lyse cells by adding SDS for euks or lysozyme + SDS for proks

also French press and sonication3. Remove protein by adding protease (proteinase K) and extracting with phenol (denatures proteases and other proteins); Nucleic acids are precipitated by adding NaOAc and ethanol (spool genomic DNA or centrifuge smaller DNA)4. Isolate pure DNA by equilibrium density gradient ultracentrifugation which uses the fact that DNA has a characteristic density (1.7 g/mL); density gradient of CsCl, ultracentrifuge, ethidium bromide added

RNA - need to get rid of RNases with phenol extraction and presence of divalent metal ions is helpful• Hard to isolate RNA from tissues b/c does not homogenize so can use guanidinium isothiocyanate (dissolves tissue)• Separate tRNA from rRNA b/c different solubilities, rRNA ppt’d and tRNA purified by anion exchange chromatography• purify mRNA with poly A tail by affinity chromatograpy (oligo-dT cellulose chrom)

UV spectroscopyDNA and RNA nucleotides have strong UV absorption at max = 260 nm Absorption due to complex electronic transitions in Pu/Py ringsFree nucleotides have highest absorptionSingle stranded nucleic acids have medium absorptionDouble stranded nucleic acids have low absorptionHypo- and hyperchromicityMelting temperature (Tm) depends on base composition, [salt] and counterions

Nucleotides & Nucleic AcidsChemistry - denaturation

High temp/pHNo covalent bonds broken

Normal temp/neutral pHPartially denatured (12 bp)

SLOW

QUICK

Nucleotides & Nucleic AcidsChemistry - denaturation

Lowest absorption of UV light

Lower absorption of UV light than free nucleotides

HYPOCHROMIC EFFECT(can monitor transition from ds to ss)

Nucleotides & Nucleic AcidsChemistry - heat denaturation

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Nucleotides & Nucleic AcidsChemistry - denaturationElectron microscopy

FluorescenceEmission of radiation given out as molecule returns to its ground state from an excited electronic stateCan add fluorophore to DNA/RNA and use FRET FRET Fluorescence Resonance Energy Transfer energy passed over a distance donor molecule is a fluorophore - excite just it and it

transfers the energy of an absorbed photon to an acceptor molecule can look at topology

Structure DeterminationCD - circular dichroismIR - infrared spectroscopyNMR - nuclear magnetic resonance spectroscopyX-ray crystallographyNAIM - nucleotide analog interference suppression

SeparationCentrifugationSeparate by differences in size, viscosity or densityEquilibrium centrifugation (buoyant density) - use CsClDifferential - separates by differences in size Sucrose density gradient

Low speed

Tissue homogenization

DIFFERENTIAL CENTRIFUGATION

Supe to medium speed

Tissue homogenate

Supe to high speed

Pellet of whole cells, nuclei, cytoskeleton, plasma membrane

Pellet of mitochondria, lysosomes, peroxisomes

Pellet of micorsomes (fragments of ER), small vesicles

Pellet of ribosomes, large macromolecules

Supe to very high speed

Supernatant contains soluble proteins

ISOPYCNIC (SUCROSE-DENSITY)CENTRIFUGATION

centrifugation

Sample

Sucrose gradient

Less dense

More dense

Fractionation

SeparationElectrophoresisDNA/RNA molecules move under the influence of electric fieldMigration based on size, charge, shape

(nondenaturing vs. denaturing)

Protein Purification and Analysis

ElectrophoresisUse gels made of crosslinked polymer polyacrylamide Proteins/DNA/RNA migrate based on their charge-to-mass ratio (denaturing) Proteins/DNA/RNA migrate based on their charge-to-mass ratio and their shape (nondenaturing)

Purification of RNA polymeraseSteps 1 2 3 4 5 6

Protein Purification and Analysis

Denaturing Gel Electrophoresis

Used to estimate purity and molecular weightDenature protein or nucleic acid by adding SDS/formamide/urea (separate by size only)

Electrophoresis of denaturing polyacrylamide gelVisualize DNA/RNA by UV shadowing

Xylene Cyanol (28-mer)

Bromphenol blue (8-mer)

20% denaturing acrylamide

SeparationElectrophoresis

Agarose PAGE (polyacrylamide)Separate large mcs (2000 kbp) separate smaller mcs (< 1 kbp)Solidify crosslinkNondenaturing denaturing vs, nondenaturingEthidium bromide EtBr, radioactivity, fluores

Mobility observed based on migration of dyesAgarose gel - can detect difference between supercoiled, relaxed and linear DNA

PAGE - can detect bent DNA since it migrates slower than straight

DNA shapePulsed field electrophoresis

efficient resolution of very large DNA (> 1000 kbp)alternating polarity field + pulse longer

Modeling3D visualization of structure

Microscopy look at structure of large systems

Separation

Chromatography

1. Column is packed with material (resin) that can absorb molecules based on some property (charge, size, binding affinity, etc.)2. Molecules washed through the column with buffer3. Fractions are taken, at some point your molecule will elute4. May have to change buffer to get elute tightly bound molecules

DNA/RNA modification5’-phosphorylation

enzyme T4 polynucleotide kinasetransfers -phosphate of ATP to 5’-OH terminus of DNA/RNA (ss or ds)

T4 polynucleotide kinase

Phosphatasehydrolyzes monoesters to produce Pi and alcoholmost are nonspecificalkaline phosphatases found in bacteria, fungi, and higher animals (not plants) and remove phosphates from polynucleotides, carbs, phospholipidscatalytic sequence Asp-Ser-Ala (serine proteases)

DNA/RNA modificationLigasescatalyze formation of a phosphodiester linkage between 2 chains Need 5’-phosphate and 3’-OH and in reaction get PPi hydrolysis

DNA/RNA modificationRestriction endonucleasesType I - trinucleotide and tetranucleotide sequence separated by 6 nt,

cleavage < 7000 bp awayType II - palindromic, cleavage site within or real close Type III - asymmetric pentanucleotide sequence, cleavage, < 25 bp

DNA/RNA modificationNucleases

PCR

PCR

After cycle 30, > 1 billion identical molecules (230 = 1.07 x 109)

Northern and Southern Blotting

Detection of specific nucleic acid sequencesProcess: 1. if genomic DNA - cut into workable pieces with restriction endonucleases2. Separate by electrophoresis3. Blot onto nitrocellulose4. Gene of interest detected on filter by hybridizing a complementary nucleic acid strand labeled either with radioactivity or an affinity label such as biotin

Southern - transfer DNA onto filterNorthern - transfer RNA onto filter (have to keep denatured so use formaldehyde)

In situ hybridization - cells and organisms smaller than 1 mm are fixed using formaldehyde; larger organisms are sliced into thin sectionsSpecimens are probed with radioactive nucleic acid