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