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

Heat ~ 100 C for a short period (completely denatured at 90 C) 97 C + salt Alkaline: pH > 11.3 (0.3 N NaOH)

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 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

Temperature at which DNA is half folded Melting Temperature Melting curve at 260 nm

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 pH 5-9 no effect > 11 (13) 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

Factors High stringencyLow stringency Temperature Salt Organic solvent

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 ssProbe : known sequence and labeled ssTarget: 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 dNTPs to new DNA strands labeled NTPs to new RNA strands 32 P (or others) to terminal nucleotides

Nick Translation Probe labeling

Random Primed Labeling Probe labeling

Kinase end labeling Probe labeling

Fill in labeling by Klenow Probe labeling

Riboprobe / RNA probe Probe labeling

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 TypeHalf Life Maximum Energy of Emission (MeV) 32 P14.3 d P25.5 d S87.4 d I 60 d H12.35 y0.018

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

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 Strand length: negligible if exceed 500 bp Base composition: GC / AT content Chemical environment: monovalent cation formamide or urea Factors on energy required for strand separation

Melting Temperature Tm as a measure for duplex stability Hybridization at Ti lower than Tm to promote heteroduplex formation

Calculation of Tm Hybrids T m (°C) DNA-DNA (log 10 [Na + ] a ) (%GC b ) - 500/L c DNA-RNA or (log 10 [Na + ] a ) (%GC b ) RNA-RNA (%GC b ) /L c oligo-DNA or For <20 nucleotides: 2 (l n ) oligo-RNA d For nucleotides: (l n )

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