Hybridization

Denaturation

• separation of 2 strands

• double stranded to single stranded

Hybridization

• joining of 2 complementary strands

• single stranded to double stranded

Hybridization purpose

• detection of a specific DNA or RNA sequence within your sample

• VERY important technique in molecular

• probe and target have complementary sequences

  • in optimized conditions the probe and target hybridize, anneal to each other

    • temperature, hybridization buffer

Target

• DNA (gDNA, PCR product)

• RNA

Probe

• DNA

• RNA

• oligonucleotides

  • small pieces of DNA 18-50 bp long

• must be complementary to target DNA

  • antiparallel

  • not identical

• specific for target

• must be labeled for visualization

Probe rules

• need to recognize target ONLY

• longer the probe, the more specific

• shorter sequences can be useful for finding small mutations

• hybridization conditions (stringency) affect the bidning

  • high = less forgiving

  • temp, pH, salt concentration

Probe must be labeled

• labeling makes probe visible

  • high specific activity (small amt, highly visible)

• radioactivity: 32P

• Biotin

• Fluorescent

• Enzymes

Hybridization types

• southern blot

• northern blot

• “in situ” hybridization

• fluorescent “in situ” hybridization. FISH

• Dot blot/ reverse dot blot/ slot blot

• microarrays

• solution hybridization

Southern blot

• developed by edwin southern

• allows analysis of any specific gene or region without having to separate it from a complex background

Southern Blot info

• target and probe is DNA

• information obtained:

  • presence of a particular sequence in genome

  • size of DNA fragment

Southern blot steps

• DNA purification, fractionation, electrophoresis

• denaturation

• transfer onto membrane

• hybridization with labeled DNA probe

• detection

Gel prep before transfer to membrane

Depurination

• fractionate DNA

  • acid solution

Denaturation

• separation of DNA strands. Obtain single strand of DNA

  • alkaline, high salt solution

Neutralization

• Neutralize gel

  • tris, high salt solution

Transfer to membrane

• membrane: DNA binding media that gives solid support

  • nitrocellulose membrane

  • nylon membrane. Neutral or + charged

  • special membrane

Southern blot: DNA transfer

• electrophoresis

• vacuum transfer

• electrophoretic

Hybridization details

• once DNA is transferred to membrane, hybridization of probe determines which DNA fragment is seen

  • membrane

  • hybridization buffer

    • contains blocking agents to reduce bg (BSA, RNA, DNA, dry milk)

  • labeled probe

  • temps: 37-65°C (2-16hrs)

Hybridization steps - washes

• low stringency. High salt, room temp

• high stringency. Low salt, high temp

hybridization steps - detection

• X-ray film

• color development

Southern blot applications

• genetics, oncology (translocations, gene rearrangements)

• typing/classification of organisms

• cloning/verification of cloned DNA

• forensic, parentage testing (RFLP, VNTR)

Northern blot

• target: RNA

• probe: DNA

• information obtained:

  • presence/abundance of mRNA (lvls of gene expression), tRNA, rRNA

  • size of RNA

    • alternative splicing

Filter based hybridization technologies

Northern blot steps

• RNA purification (obtain mRNA)

• no restriction digests

• gel electrophoresis

• RNA transfer onto membrane

• hybridization of membrane with labeled DNA probe

• detection

Dot blot

• simplified southern/northern blot

• no gel electrophoresis or transfer

• mixture of molecules is applied directly on a membrane, followed by probe hybridization

• no complex blotting procedures → time saving

• no information about size of detected molecule

• only confirms the presence or absence of molecules detected by probe

DNA microarray technology

• used to investigate multiple genomic sites simultaneously

  • small, solid support

  • thousands of diff unlabeled DNA probes

  • specimen DNA is labeled and hybridized to probes

  • “reverse dot blot” methods

DNA microarrays uses

• genome-wide expression analysis

  • which mRNA is produced?

• CGH: comparative genome hybridization

  • analyzes genomic DNA

  • detect chromosomal duplication/deletion

In situ hybridization

• target: DNA or RNA

• probe: DNA

• cells, tissues onto slides

• information obtained:

  • presence of DNA or RNA sequence

  • correlation with histopathology

FISH: fluorescent in situ hybridization

• probe: DNA

• target: DNA

• metaphase chromosomes or interphase nuclei

• probes. fluorescent labeled

  • various types of probes used

• detect chromosomal abnormalities

  • translocations, deletions, aneuploidies

  • detect and/or monitor a disease

Different FISH probes