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 RadioisotopeHalf-lifeDecay-typeEnergy of emission 3 H12.4 years  MeV 32 P14.3 days  MeV 33 P25.5 days  MeV 35 S87.4 days  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 temperatureLow salt concentrationHigh denaturant concentration High strigency Low strigency Low temperature Sequence G/C content Sequence lenght Tm Low denaturant concentration High salt concentration Perfect match complementary sequences Perfect match non-complementary sequences

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Filter hybridization techniques 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/cm 2 ) –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