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
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
Structure of digoxigenin-modified nucleotides
Indirect nonisotopic labeling
Nucleic acid hybridization- formation of heteroduplexes
Ultraviolet absortion spectrum of DNA
Denaturation of DNA results in an increase of optical density
Melting curve of a specific DNA sequence
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
• 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)
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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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
Principles of Southern blot
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Southern Blotting Apparatus
Depurination/Denaturation of DNA
Typical hybridization solution
• High salt solution (SSC or SSPE)
• Blocking agent (Denhardts, salmon sperm
DNA, yeast tRNA)
• SDS
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
Southern applications- example
Colony blot hybridization-1
Colony blot hybridization-2
Colony blot hybridization- example