DNA is a polymer of 2-deoxyribonucleotides pGCTA 5 end 3 end C G T A OPO-CH 2
O H 2 N-C
C C
HN
N N
CH
C
O
N
O O O P O CH 2
O O C N N
CH C CH
NH 2
NH 2
C
C N
N N
CH
C
N
HC
O O O P O CH 2
O OH
O O O P O CH 2
O N C
C O HN
CH C O CH 3
1 2 3 4 5 3 RNA is a polymer of ribonucleotides ribose sugars Phosphodiester linkages Directional chain (5 to 3) 4 Bases purines: adenine & guanine pyrimidines: cytosine & uracil pGCUA C G U A 5 end 3 end 1 2 3 4 5 3 OH PO-CH 2
O H 2 N-C
C C
HN
N N
CH
C
O
N
O O O P O CH 2
O O C N N
CH C CH
NH 2
OH O O O P O CH 2
O N CH
C O HN
CH C O OH NH 2
C
C N
N N
CH
C
N
HC
O O O P O CH 2
O O-H
OH RNA is easily hydrolyzed under alkaline conditions The reaction proceeds through a 2,3-cyclic monophosphate intermediate. Enzymatic hydrolysis of RNA by RNase proceeds through a similar intermediate. Because DNA lacks the 2-OH group, it is stable under alkaline conditions. . . . . O P O-CH 2
O O O N OH O O P O CH 2
O O N OH O O P O O ... OH O N OH O O P O O ... HOCH 2 O . . . . O P O-CH 2
O O N O O P O O H + . . . . O P O-CH 2
O O O N OH O O P OH O mixture of 2- and 3- monophosphate derivatives H 2 O shortened RNA RNA The phosphate groups of DNA and RNA are negatively charged A phosphodiester group has a pK a of about 1, and so will always be ionized and negatively charged under physiological conditions (pH ~7). Nucleic acids require counterions such as Mg 2+ , polyamines, histones or other proteins to balance this charge. 5 3 HO-CH 2
O N O O P O CH 2
O O N O O P O CH 2
O O N O O P O CH 2
O O O-PO 3 2 N + M + M + M + M
BENTUK DNA B-form DNA consists of a right-handed double helix with antiparallel strands 34 (10 bp) per turn major groove minor groove major groove minor 3.4 per bp These dimensions are for DNA fibers. In solution, there are ~10.5 base-pairs per turn. 5 3 5 3 Melting and Renaturation of DNA Renaturation driven by homologous base pairing Will also hybridize with a radiolabeled 5-ACGGCTA-3 probe. The two strands of the double helix separate reversibly at high temperatures The temperature at which this denaturation or melting occurs depends on the pH and salt concentration, and increases with the GC content of the DNA. (The curves drawn here are schematic.) 100 80 60 40 20 0 %
D e n a t u r e d 110 100 90 80 70 Temperature / o C 40 50 70% GC 60 If the temperature is lowered, the strands recombine. The rate of reassociation is inversely proportional to the complexity of the DNA. xx Guanine (keto form) H 2 N-C
C C
HN
N N
CH
C
O
N
Thymine (keto form) N C
C O HN
CH C O CH 3
N
N C
C OH CH C O CH 3
Thymine (enol form) N
OH
H 2 N-C
C C
N N
CH
C
N
Guanine (enol form) The ring NH atoms of G and T have pK a values of about 9. At physiological pH, about 99% of the base is in the keto form and 1% in the enol form. Tautomeric forms of G & T can cause mutations due to mispairing during DNA replication
DNase I DNase II
Exonuclease Palindromic* sequences (inverted repeats) in DNA or RNA can form hairpin or cruciform structures inverted repeats in an antiparallel double helix 3 5 5 3 T G C G A T A C T C A T C G C A A C G C T A T G A G T A G C G T hairpin C A C T 3 5 T A G C G T A T C G C A T G A G C A C T cruciform 3 5 5 3 T A G C G T A T C G C A T G C G A T A C G C T A Mirror repeats cannot form these structures. *A palindrome reads the same in either direction (Radar, Madam, Im Adam). STRUKTUR RNA ADA PERTANYAAN ? TERIMA KASIH General Theory of Electrophoresis Analytical or preparative separation of charged molecules due to differential migration through a matrix upon application of an electric field, with net movement towards electrode of opposite charge ElectrophoresisAn Overview Definition: The differential movement for migration of ions by attraction or repulsion in an electric field. Separation of components of a mixture using an electric field v=Eq/f v = velocity of molecule E = electric field q = net charge of molecule f = friction coefficient Electrophoresis- overview cont. Can determine the size, shape, and charge of a molecule Different forms of electrophoresis are used for each of these factors independently or in combination. Types of Electrophoresis Capillary Native Polyacrylimide Gel Electrophoresis (PAGE) SDS-PAGE Slab Paper
Electrophoresis Support/Matrix Solid matrix is most often agarose (larger pore size) or polyacrylamide (smaller pore size). Here, buffer provides ions for current while maintaining constant pH
For capillary electrophoresis, buffer serves as matrix Components of electrophoretic mobility Voltage difference, E = voltage applied/distance between electrodes; generally expressed as volts/cm Charge on molecule, q Frictional component, f, determined by size and shape of molecule, pore size of matrix, viscosity of buffer Velocity of particle, v= Eq/f Mobility of particle, = v/E = q/f Approximate only- accounts for neither interaction of particle with matrix nor shielding of particle by buffer ions Separation is achieved due to differences in either or both major components Size/shape Charge Both size/shape and charge
Electrophoresis can be, but is not always, run to an endpoint- if molecules are detected in the matrix, an empirical endpoint chosen such that all molecules remain in the matrix V = IR Voltage is a function of both current and resistance Resistance decreases during electrophoretic run, therefore current increases if maintaining constant voltage To minimize this, sometimes gels are run at constant current or at constant power (W = I 2 R) Why minimize current increase during run? o As current increases, power increases- much of power is dissipated as heat o Heat affects electrophoretic separation- diffusion increases; samples can be sensitive to heat; buffer viscosity decreases therefore resistance decreases and uneven heating occurs due to greater cooling at gel edges Outline DNA/agarose- horizontal; separation by size/shape RNA/agarose- horizontal; denaturing; separation by size Protein/polyacrylamide- vertical separation by size- SDS-PAGE (denaturing) separation by size and charge-native (non-denaturing) separation by charge (pI)- isoelectric focusing separation by charge, then size- 2D PAGE (denaturing) Capillary Electrophoresis
DNA/Agarose Gel Electrophoresis Horizontal submarine electrophoresis most common; separation is by size Agarose concentration 0.3-3% Buffer most often Tris-Borate-EDTA (TBE) at 1X or 0.5X; sometimes Tris-Acetate-EDTA (TAE) at 1X (recipes- Maniatis, Current Protocols) Detection of DNA is generally by ethidium bromide intercalation (dye in gel, in buffer, in sample, or in immersion solution after run) or by other dyes (e.g., Sypro) Agarose Good separation of all but smallest DNAs (<100); mid- range to large pore size provides sieving effect; relatively inert Linear polysaccharide purified from seaweed extract is composed of repeating units of agarobiose When dissolved by heat in aqueous solution, then cooled, agarose solution gels due to formation of inter- and intra-chain H bonds => The higher the concentration, the smaller the pore size Preparing and running an agarose gel Suspend agarose in running buffer (NOT H 2 O) to desired concentration Heat to boiling; once dissolved, cool to ~65 o C; add EtBr if desired to 1 g/ml; pour into gel tray with comb to form wells; let set completely Prepare DNA samples- add loading dye to 1X (provides high density to allow sample to sink, and provides dye for monitoring migration) Remove comb from gel, set up in tank and submerge in buffer Load samples by pipetting slowly through buffer into wells Attach leads to tank and power supply; set V so I < 60 mA DAYA PISAH ELEKTROFORESIS GEL Agarosa % Fragmen kb 0,5 1-30 0,7 0,8-12 1,0 0,5-10 1,2 0,4-7 1,5 0,2-3 3 0,01-0,5 Akrilamid% Fragmen kb 3,5 100 Running an agarose gel- agarose concentration Agarose concentration needed is determined by sizes of DNA fragments to be separated o for fragments between 0.3 and 3 kb, 0.7-1% are good standard concentrations o to separate small fragments (0.2-0.7 kb) from one another, use 1.5-3% (smaller pore size) o for separation of larger fragments (>3 kb and less than 30 kb) use 0.3-0.5% Running an agarose gel- agarose concentration At low agarose concentrations, very little frictional resistance for small fragments => mobility is similar between different sizes; for high agarose concentrations, very little separation of large DNA fragments of different size because high frictional resistance for all Separation of fragments above 20 kb is not ideal using normal electrophoresis setup Estimating molecular size by determining electrophoretic mobility Dyes provide both the means of determining endpoint of run and the basis of determining mobility (M r )
and estimated size (DNA in kb)
Bromophenol blue (BPB) and xylene cyanol are the most common agarose gel tracking dyes. Both dyes run with characteristic mobility that varies with gel percentage; in general, BPB runs at 0.3-0.6 kb and xylene cyanol at 3-6 kb Estimating molecular size by determining electrophoretic mobility Mobility, M r = distance migrated by band of interest/distance migrated by dye front
M r is related to logMW or log of molecular size in bp in a linear fashion, therefore if plot standards on graph, one can determine masses of unknowns Estimating molecular size by determining electrophoretic mobility Distance from origin in cm, or M r
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