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Nucleic Acid Hybridization

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Nucleic Acid Hybridization Nucleic acids Complementary bases Hybridization Complementary strands from any sources Reversible reaction DNA/DNA or DNA/RNA or RNA/RNA – PowerPoint PPT presentation

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Title: Nucleic Acid Hybridization


1
Nucleic Acid Hybridization
Nucleic acids Complementary bases Hybridization
Complementary strands from any sources Reversible
reaction DNA/DNA or DNA/RNA or RNA/RNA
2
Denaturation
Denature helix separation Hydrogen bonds broken
/ Strands unwind Double strands to Single strands
3
Denaturation
4
Denaturation
Heat 100 C for a short period (completely
denatured at 90 C) 97 C salt Alkaline pH gt
11.3 (0.3 N NaOH)
5
Denaturation
Organic solvent Urea and formamide directly
reacting with bases inhibit normal base
pairing reversible reaction
6
Denaturation
Organic solvent formaldehyde irreversible
denaturation form covalent bond with NH2 group
7
Renaturation
Renaturation / Hybridization / Reassociation Base-
pairing reaction of complementary strands Slow
cooling at 65 C Fast cooling (100 --gt 0 C) stay
separated
8
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
9
Renaturation
Two steps of base-pairing reaction
II Zippering / Annealing step Fast Extend
base-pairing reaction over the whole strands
10
Hybridization
AT vs CG regions
11
Hybridization
Factors affecting hybridization Rate of
reaction Criterion or helix stability
12
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
13
Hybridization Rate
Factors affecting rate
II Temperature Melting temperature Salt, GC
content, Organics Maximum rate Tm - 25
14
Melting Temperature
Melting curve at 260 nm
Temperature at which DNA is half folded
15
Melting Temperature
Tm (AT-rich) lt Tm (GC-rich)
16
Hybridization Rate
Factors affecting rate
III Fragment Length optimal length at 450
nt Too short easy mismatch Too long very
slow rate
17
Hybridization Rate
Factors affecting rate
IV Organic solvent concentration Denaturing
agent higher Conc slightly lower Rate V
Solvent Viscosity higher Viscosity lower Rate
18
Hybridization Rate
Factors affecting rate
VI GC composition higher GC content slightly
higher Rate VII pH 5-9 no effect gt 11 (13)
denature
19
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
20
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
21
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
22
Hybridization
Hybrid formation Considered rate and
criterion Hybrid specificity Considered
hybridization stringency
23
Stringency
Conditions for hybridization Effect of degree of
mismatch High stringency best match Low
stringency some mismatch
24
Stringency
25
Stringency
26
Renaturation analysis
Evaluation of degree of genetic similarity
between organisms Evaluation of genome
complexity
27
Renaturation analysis
DNA with high amounts of satellite DNA Renature
much faster When compared to DNA with Mainly
single sequences Regardless of genome size
28
Renaturation analysis
Multiple-copy sequence of Genome eg. repetitive
sequence Easy nucleation step Quick hybridization
29
Renaturation analysis
Complex genome High amounts of single
sequences Long reaction period
30
Renaturation analysis
Eukaryote 4 DNA groups Foldback DNA Highly
repetitive DNA Moderately repetitive DNA Unique
/ Single copy DNA
31
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)
32
Nucleic acid probe
Sequence with known molecular identity Homologous
probe same source Heterologous probe different
source
33
Nucleic acid probe
DNA genomic DNA (by cloning or
PCR) complementary DNA RNA transcription of DNA
inserted in plasmid Synthetic oligonucleotide sp
ecific to target sequence sometimes as a set of
degenerate probes
34
Nucleic acid probe
35
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 32P (or
others) to terminal nucleotides
36
Probe labeling
Nick Translation
37
Probe labeling
Random Primed Labeling
38
Probe labeling
Kinase end labeling
39
Probe labeling
Fill in labeling by Klenow
40
Probe labeling
Riboprobe / RNA probe
41
Types of Label
Isotopic label Commonly used 32P, 33P, 35S or
3H Non-isotopic label Direct label Fluorescene
dye Indirect label Digoxygenin
Biotin-Strepavidin
42
Choices of Label
Sensitivity Resolution Probe stability Safety Ease
of Use
43
Radioactive Label
44
Radioactive Label
Radio-labeled nucleotide Autoradiographic
detection Radiation intensity --gt signal 32P
Highly sensitive / Low resolution
45
Non-Radioactive Label
Safe / Easy / High resolution / Low
sensitivity Direct Label Fluorescene dye /
Fluorophore Indirect Label Biotin-Strepavidin
Digoxigenin Required conjugated marker
46
Non-Radioactive Label
Detection Fluorescence Colorimetric
assay Alkaline phosphatase NBT
BCIP Chemiluminescence assay HRP H2O2
luminol
47
Fluorophores
48
Indirect Label
49
Indirect Label
50
Nucleic Acid Hybridization
Identification of closely related
molecules Probe homogeneous population of
identified molecules Target heterogeneous
population of nucleic acid
51
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
52
Nucleic Acid Hybridization
Denaturation of double strands by heating by
alkaline treatment Annealing of complementary
strands Formation of Homo or Heteroduplex
53
Nucleic Acid Hybridization
54
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
55
Melting Temperature
Tm as a measure for duplex stability Hybridization
at Ti lower than Tm to promote heteroduplex
formation
56
Calculation of Tm
Hybrids Tm (C) DNA-DNA 81.5 16.6
(log10Naa) 0.41 (GCb) - 500/Lc DNA-RNA
or 79.8 18.5 (log10Naa) 0.58
(GCb) RNA-RNA 11.8 (GCb)2 - 820/Lc
oligo-DNA or For lt20 nucleotides 2 (ln)
oligo-RNAd For 20-35 nucleotides 22 1.46
(ln )
57
Blotting
Transfer of Nucleic acid onto solid
support Membrane filter Nylon /
Nitrocellulose By capillary force, vacuum or
electroblot
58
Blotting
59
Hybridization
Southern electrophoresed DNA Northern
electrophoresed RNA Dot blot unfractionated
target Slot blot big volume / unfractionated
target Colony bacterial genome Plaque virus
genome
60
Southern/Northern Hybridization
61
Dot Blot Hybridization
62
Slot Blot Hybridization
63
Colony Hybridization
64
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
65
Microarray VS Northern
66
Microarray / DNA chip
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