Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization
Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization Complementary strands from any sources Reversible reaction DNA/DNA or DNA/RNA or RNA/RNA
Denaturation Denature: helix separation Hydrogen bonds broken / Strands unwind Double strands to Single strands
Denaturation
Denaturation Heat ~ 100 C for a short period )completely denatured at 90 C( 97 C + salt Alkaline: p. H > 11. 3 (0. 3 N Na. OH(
Denaturation Organic solvent : Urea and formamide directly reacting with bases inhibit normal base pairing reversible reaction
Denaturation Organic solvent : formaldehyde irreversible denaturation form covalent bond with NH 2 group
Renaturation / Hybridization / Reassociation Base-pairing reaction of complementary strands Slow cooling at 65 C Fast cooling (100 --> 0 C): stay separated
Renaturation Two steps of base-pairing reaction I Nucleation step Quite slow Random reaction of 2 strands collide by incidence Rate-limiting step Short stretches of H bonds
Renaturation Two steps of base-pairing reaction II Zippering / Annealing step Fast Extend base-pairing reaction over the whole strands
Hybridization AT vs CG regions
Hybridization Factors affecting hybridization Rate of reaction Criterion or helix stability
Hybridization Rate Factors affecting rate I Concentration of momovalent ion eg. sodium salt (Na(+ higher Conc : higher Rate Conc higher than 0. 4 M Hybrid destabilize
Hybridization Rate Factors affecting rate II Temperature Melting temperature Salt, GC content, Organics Maximum rate = Tm - 25
Melting Temperature Melting curve at 260 nm Temperature at which DNA is half folded
Melting Temperature Tm (AT-rich) < Tm (GC-rich(
Hybridization Rate Factors affecting rate III Fragment Length optimal length at 450 nt Too short: easy mismatch Too long: very slow rate
Hybridization Rate Factors affecting rate IV Organic solvent concentration Denaturing agent higher Conc : slightly lower Rate V Solvent Viscosity higher Viscosity : lower Rate
Hybridization Rate Factors affecting rate VI GC composition higher GC content : slightly higher Rate VII p. H 5 -9 no effect (13) 11 <denature
Criterion / Thermostability Factors affecting criterion I Temperature Incubation temperature or Ti lower Ti by 1 C : higher mismatch by 1% higher mismatch : lower Tm : lower criterion optimal Ti : Tm - 15
Criterion / Thermostability Factors affecting criterion II Concentration of monovalent ion higher salt : higher rate : lower criterion III Fragment length higher length : higher Tm : higher criterion
Criterion / Thermostability Factors affecting criterion IV Concentration of organic solvent higher conc : lower Tm : lower criterion V GC composition higher GC content : higher Tm : higher criterion
Hybridization Hybrid formation Considered rate and criterion Hybrid specificity Considered hybridization stringency
Stringency Conditions for hybridization Effect of degree of mismatch High stringency : best match Low stringency : some mismatch
Stringency
Stringency Factors High stringency Low stringency Temperature Salt Organic solvent
Renaturation analysis Evaluation of degree of genetic similarity between organisms Evaluation of genome complexity
Renaturation analysis DNA with high amounts of satellite DNA Renature much faster When compared to DNA with Mainly single sequences Regardless of genome size
Renaturation analysis Multiple-copy sequence of Genome eg. repetitive sequence Easy nucleation step Quick hybridization
Renaturation analysis Complex genome High amounts of single sequences Long reaction period
Renaturation analysis Eukaryote: 4 DNA groups Foldback DNA Highly repetitive DNA Moderately repetitive DNA Unique / Single copy DNA
Hybridization reaction Fundamental tool in molecular study Hybridization partners ss. Probe : known sequence and labeled ss. Target: related sequence under study Form ds if complementary (to hybridize(
Nucleic acid probe Sequence with known molecular identity Homologous probe: same source Heterologous probe: different source
Nucleic acid probe DNA: genomic DNA (by cloning or PCR( complementary DNA RNA: transcription of DNA inserted in plasmid Synthetic oligonucleotide: specific to target sequence sometimes as a set of degenerate probes
Nucleic acid probe
Probe labeling ds or ss nucleic acid probe to be labeled Working probe: single strands Labeled by incorporating: labeled d. NTPs to new DNA strands labeled NTPs to new RNA strands 32 P (or others) to terminal nucleotides
Probe labeling Nick Translation
Probe labeling Random Primed Labeling
Probe labeling Kinase end labeling
Probe labeling Fill in labeling by Klenow
Probe labeling Riboprobe / RNA probe
Types of Label Isotopic label Commonly used: 32 P, 33 P, 35 S or 3 H Non-isotopic label Direct label: Fluorescene dye Indirect label: Digoxygenin Biotin-Strepavidin
Choices of Label Sensitivity Resolution Probe stability Safety Ease of Use
Radioactive Label Type Half Life 32 P 14. 3 d 25. 5 d 87. 4 d 60 d 12. 35 y 33 P 35 S 125 I 3 H Maximum Energy of Emission (Me. V( 1. 71 0. 248 0. 167 0. 035 0. 018
Radioactive Label Radio-labeled nucleotide Autoradiographic detection Radiation intensity --> signal 32 P: Highly sensitive / Low resolution
Non-Radioactive Label Safe / Easy / High resolution / Low sensitivity Direct Label: Fluorescene dye / Fluorophore Indirect Label: Biotin-Strepavidin Digoxigenin Required conjugated marker
Non-Radioactive Label Detection Fluorescence Colorimetric assay Alkaline phosphatase + NBT + BCIP Chemiluminescence assay HRP + H 2 O 2 + luminol
Fluorophores
Indirect Label
Indirect Label
Nucleic Acid Hybridization Identification of closely related molecules Probe: homogeneous population of identified molecules Target: heterogeneous population of nucleic acid
Nucleic Acid Hybridization Liquid / Solution hybridization slow reassociation of single copy in complex genome Solid / Filter hybridization immobilized target to increase reassociation rate Reverse hybridization: unlabeled immobilized probe In situ hybridization: target in tissue
Nucleic Acid Hybridization Denaturation of double strands : by heating by alkaline treatment Annealing of complementary strands Formation of Homo or Heteroduplex
Nucleic Acid Hybridization
Nucleic acid stability Factors on energy required for strand separation Strand length: negligible if exceed 500 bp Base composition: GC / AT content Chemical environment: monovalent cation formamide or urea
Melting Temperature Tm as a measure for duplex stability Hybridization at Ti lower than Tm to promote heteroduplex formation
Calculation of Tm Hybrids DNA-DNA DNA-RNA or RNA-RNA oligo-DNA or oligo-RNAd Tm (°C( 81. 5 + 16. 6 (log 10[Na+]a ( %) 0. 41 +GCb) - 500/Lc 79. 8 + 18. 5 (log 10[Na+]a) + 0. 58 (%GCb( + 11. 8 (%GCb)2 - 820/Lc For <20 nucleotides: 2 (ln ( For 20 -35 nucleotides: 22 + 1. 46 (ln (
Blotting Transfer of Nucleic acid onto solid support Membrane filter: Nylon / Nitrocellulose By capillary force, vacuum or electroblot
Blotting
Hybridization Southern: electrophoresed DNA Northern: electrophoresed RNA Dot blot: unfractionated target Slot blot: big volume / unfractionated target Colony: bacterial genome Plaque: virus genome
Southern/Northern Hybridization
Dot Blot Hybridization
Slot Blot Hybridization
Colony Hybridization
DNA Microarray Large-scale gene screening / expression analysis Whole genome study on single pass Hybridization of high-density DNA array Robotic spotting of DNA clones or oligonucleotides
Microarray VS Northern
Microarray / DNA chip
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