Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental tool in molecular genetics...
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![Page 1: Nucleic Acid Hybridization Nucleic acid hybridization is a fundamental tool in molecular genetics which takes advantage of the ability of individual single-stranded.](https://reader035.fdocuments.net/reader035/viewer/2022062320/56649d0a5503460f949dc64c/html5/thumbnails/1.jpg)
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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)
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- 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
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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
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Kinase end-labeling of oligonucleotides
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Fill-in end labeling
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Nick translation
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Random primed labeling
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Riboprobes
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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
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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)
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Structure of fluorophores
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Structure of digoxigenin-modified nucleotides
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Indirect nonisotopic labeling
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Nucleic acid hybridization- formation of heteroduplexes
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Ultraviolet absortion spectrum of DNA
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Denaturation of DNA results in an increase of optical density
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Melting curve of a specific DNA sequence
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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
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• Temperature• Ionic strenght• Destabilizing agents• Mismatched base pairs• Duplex lenght• Viscosity• Probe complexity• Base composition• pH
Factors affecting the hybridization for nucleic acids in solution (annealing)
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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
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06_10.jpg
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Filter hybridizationtechniques
Filter hybridization methods
Bacteriophage blotting Benton-Davis
Bacterial colony blotting Grunstein-Hogness
Slot/Dot blotting
Northern analysis Southern analysis
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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
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Principles of Southern blot
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Southern Blotting Apparatus
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Depurination/Denaturation of DNA
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Typical hybridization solution
• High salt solution (SSC or SSPE)
• Blocking agent (Denhardts, salmon sperm
DNA, yeast tRNA)
• SDS
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Southern Applications
• Detection of DNA rearrangements and deletions found in several diseases
• Identification of structural genes (related in the same species (paralogs) or in different species (orthologs))
• Construction of restriction maps
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Southern applications- example
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Colony blot hybridization-1
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Colony blot hybridization-2
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Colony blot hybridization- example