Nucleic Acid Hybridization

Nucleic acids Complementary bases

Hybridization Complementary strands fromany 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 9 0 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 NH2

group

Renaturation

Renaturation / Hybridization / Reassociation

- Base pairing reaction of complementary strands

Slowcoolingat6 5 C

-- Fast cooling (1 0 0 > 0 C): stay separated

Renaturation

TT T TTTTT TT TTTT-TTTTTTT TTTTTTTT I Nucleation step

Quite slow Random reaction of 2 strands

collide by incidence- Rate limiting step Short stretches of H bonds

Renaturation

TT T TTTTT TT TTTT-TTTTTTT TTTTTTTT II Zippering / Annealing step

TTTTTTTTTT TTTT-TTTTTTT TTTTTTTT

over the whole strands

Hybridization

AT vs CGregions

Hybridization

Factors affecting hybridization

Rate of reaction

T TTTTTTTT TT TTTTT TTTTTTTTT

Hybridization Rate

TTTTTTT TTTTTTTTT TTTT I Concentration of momovalent ion

eg. sodiumsalt (Na+) higher Conc : higher Rate

Conc higher than 0.4 MT TTTTT TTTTTTTTTTT

Hybridization RateTTTTTTT TTTTTTTTT TTTT

II TemperatureT TTTTTT TTT TTTTTTTT

Salt, GC content, Organics - Maximum rate = Tm 25

Temperature at which DNA is half folded

Melting Temperature

Melting curve at 2 6 0 nm

Melting Temperature

- -Tm (AT rich) < Tm (GC rich)

Hybridization RateTTTTTTT TTTTTTTTT TTTT

III TTTTT TTT TTTTTT 450optimal length at nt

Too short: TTTT T TTT TTTT Too long: very slow rate

Hybridization RateTTTTTTT TTTTTTTTT TTTTIV T TTTTTT TTTTTTT TTTTTTTTTTTTT

Denaturing agent higher Conc : slightly lower Rate

V Solvent Viscosity higher Viscosity : lower Rate

Hybridization RateTTTTTTT TTTTTTTTT TTTT

VI GC compositionTTTTTT T T TTTTTTT T TTTTTTTT TTTTTT TTTT

VII pH- 5 9 no effect

1113> ( ) denature

Criterion / Thermostability Factors affecting criterion

I Temperature Incubation temperature or Ti

1 1lower Ti by C : higher mismatch by % higher mismatch : lower Tm : lower criterion

- 15optimal Ti : Tm

Criterion / Thermostability Factors affecting criterion

II Concentration of monovalent ion highersalt: hi gher r at e : l ower cr i t er i on

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 higherGC content : higherTm :higher criterion

Hybridization

Hybrid formation Consi der ed r at e and cr i t er i on

Hybrid specificity Considered hybridization stringency

Stringency

Conditions for hybridization Effect of degree of mismatch

High stringency : best match Low stringency : some mismatch

Stringency

Stringency

Fact or s High stringency Low stringencyTemperature Sal t

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

T TTT TTT TTTTT TT T T T T TTT 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

Longreactionperiod

Renaturation analysis

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

T T T T genomic DNA (by cloning or PCR) complementary DNA

RNA: transcription of DNA inserted in plasmid Synthetic oligonucleotide:

specific to targetsequence 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

T TTTTT TTT TTTTTTTT

Probe labeling

Fill in labeling by Klenow

Probe labeling

Riboprobe / RNA probe

Probe labeling

Types of Label

Isotopic label Commonly used: 32TT 33TT 35 S or 3H

- Non isotopic label Direct label: Fluorescene dye

Indirect label: Digoxygenin -Biotin Strepavidin

Choices of Label

SensitivityResolution

Probe stabilitySafety

Ease of Use

Radioactive LabelType Half Life Maximum Energy

of Emission (MeV)32T 143. d 171

33T T2 5 .5 0.24835T T8 7 .4 0.167125I 6 0 d 0.0353H 1235. y 0.018

Radioactive Label

- Radio labeled nucleotide Autoradiographic detection

-- Radiation intensity > signal32 P:Highly sensitive / Lowresolution

- Non Radioactive Label

Safe / Easy / High resolution / Low sensitivity Direct Label: Fluorescene dye / Fluorophore

Indirect Label: -Biotin StrepavidinDigoxigenin

Required conjugated marker

- Non Radioactive Label

DetectionFluorescence

Colorimetric assay Alkaline phosphatase + NBT + BCIP

Chemiluminescence assay HRP + H

2O2

T TTT TTTT

Fluorophores

Indirect Label

Indirect Label

Nucleic Acid Hybridization

Identification of closely related molecules Probe: homogeneouspopulation

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

500Strand length: negligible if exceed bp Base composition: GC / AT content

Chemical environment: monovalent cation

formamide or urea

Factorsonenergy required for strandseparation

Melting Temperature

Tm as a measure for duplex stability Hybridization at Ti lower than Tm

to promote heteroduplex formation

Calculation of Tm Hybrids Tm (°C)

- DNA DNA 8 1 .5 + 1 6 .6 (log10

TT T+]a ) 041+ . (%GCb - ) 500/Lc

- DNA RNA or 798 185. + . (log10

[Na+]a ) + 0.58 (%GCb)

- RNA RNA 118+ . (%GCb)2 - 820/Lc - oligo DNA or < 2 0 : 2 ( ln )-oligo RNAd - 2035 22 1 46For nucleotides: + . (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: virusgenome

Southern/Northern Hybridization

Dot Blot Hybridization

Slot Blot Hybridization

Colony Hybridization

DNAMicroarray

- 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