Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization Complementary strands...
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Transcript of Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization Complementary strands...
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
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 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
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
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
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
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
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
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
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 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
Hybridization
Southern: electrophoresed DNA Northern: electrophoresed RNA
Dot blot: unfractionated target Slot blot: big volume / unfractionated target
Colony: bacterial genome Plaque: virusgenome
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