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CHAPTER SIX Nucleic acid hybridization: principles and applications. 생물정보학협동과정 강민호. Nucleic Acid Hybridization. - PowerPoint PPT Presentation
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CHAPTER SIXNucleic acid hybridization: principles and applications
생물정보학협동과정강민호
Nucleic Acid Hybridization
• Nucleic acid hybridization is a fundamental tool in molecular genetics which takes advantage of the ability of individual single-stranded nucleic acid molecules to form double stranded molecules (that is, to hybridize to each other)
- A labeled nucleic acid - a probe - to identify related DNA or RNA molecules
- Complex mixture of unlabeled nucleic acid molecules- the target
-Base complementarity with a high degree of similarity between the probe and the target.
Standard nucleic acid hybridization assays
Types of probes
Probes
• DNA labelling– 5’– 3’– Uniform labeling
• Nick translation• Random primer• PCR-mediated labeling
• RNA labelling– In vitro transcription of a cloned DNA insert
• Different probes– Radioactive labeling or isotopic labeling– Nonradioactive labeling or nonisotopic labeling
Kinase end-labeling of oligonucleotides
Fill-in end labeling
Nick translation
Random primed labeling
Riboprobes
Characteristics of radioisotopes commonly used for labeling DNA and RNA probes
Radioisotope Half-life Decay-type Energy ofemission
3H 12.4 years - 0.019 MeV
32P 14.3 days - 1.710 MeV 33P 25.5 days - 0.248 MeV 35S 87.4 days - 0.167 MeV
Nonisotopic labeling and detection
• The use of nonradioactive labels has several advantages:– safety– higher stability of a probe– efficiency of the labeling reaction– detection in situ– less time taken to detect signal
• Major types– Direct nonisotopic labeling (ex. nt labeled with a fluorophore)– Indirect nonisotopic labeling (ex. biotin.-streptavidin system)
Structure of fluorophores
Fluorescence microscopy
Common Fluorophores
Structure of digoxigenin-modified and biotin-modified nucleotides
Indirect nonisotopic labeling
Nucleic acid hybridization- formation of heteroduplexes
Denaturation of DNA results in an increase of optical density
Factors affecting Tm of nucleic acid hybrids
• Destabilizing agents (ex. formamide, urea) • Ionic strenght• Base composition (G/C%, repetitive DNA) • Mismatched base pairs• Duplex lenght
Different equations for calculating Tm for: • DNA-DNA hybrids• DNA-RNA hybrids• RNA-RNA hybrids• Oligonucleotide probes
Stringency
High temperature Low salt concentration High denaturantconcentration
High strigency
Low strigency
Low temperature
Sequence G/C content
Sequence lenght
Tm
Low denaturantconcentration
High salt concentration
Perfect matchcomplementarysequences
Perfect matchnon-complementarysequences
The identification of specific sequences in a complex mixture.
Filter hybridizationtechniques
Filter hybridization methods
Bacteriophage blotting Benton-Davis
Bacterial colony blotting Grunstein-Hogness
Slot/Dot blotting
Northern analysis Southern analysis
Filters or Membranes
• Nitrocellulose• Nylon• Positive charged nylon (hybond)• PVDF (hydrophobic polyvinylidene difloride)
• Different properties:– Binding capacity (mg nucleic acids/cm2)– Tensile strenght– Mode of nucleic acid attachment– Lower size limit for efficient nucleic acid retention
Dot blot or slot blot
Principles of Southern blot
Northern Blot
Colony blot hybridization
In situ hybridization
• Chromosome in situ hybridization– Metaphase or protometaphase chromosomes
are probed with labeled DNA . The DNA can be labeled with a fluorochrome (FISH).
• Tissue in situ hybridization– Sliced or whole mounted preparations can be
probed with RNA probes to detect mRNA expression
Tissue In situ hybridization
Gridded clone hybridization
Construction of DNA and oligo microarrays
Gene expression profiling by hybridization
Summary I
• Hybridization is due to complementarity of DNA strands.
• DNA can be labeled various ways
• Isotopic and non isotopic
• Hybridization can detect identical or similar sequences.
Summary II
• A variety of techniques utilize hybridization of DNA or RNA probes– ASO– Southern Blot, RFLP, VNTRs, Mutation
detection, deletion detection– Northern Blot, tissue specific expression– In situ hybridization
• Chromosome location and integrity• Tissue specific expression
Summary III
• Colony hybridization can be used to identify specific clones. Once you have one clone you can find others that hybridize to it.
• Screening of gridded clones . One can identify genomic clones homologous to a cDNA or identify cDNA expressed in a cell line.
• Microarrays can be used in many ways to analyze gene expression in various cell types, in response to various stimuli.