Principle of Northern Blotting
RNA is isolated from the sample. RNA is put in the electrophoresis. RNA is transfer to membrane with labeled your probes. You mix the probes with you rmembrane. If perfect match, the probe will fix to the membrane hybridized. Visualization of labeled RNA on X-ray film.
What about Northern Blotting is different from Southern Blotting?
Southern Blotting uses restriction enzymes. Northern Blotting doesn't use restriction enzymes because enzymes don't cut RNA.
Principle of Western Blots
- immobilized targets are proteins
- (utilizes the theory similar to Southern blots)
- According to the molecular weights of various proteins, target proteins are SEPARATED on SDS-polyacrylamide gels (SDS-PAGE)
- SDS is a denaturing reagent that makes all proteins uniformly negatively charged such that the rates of migration are solely depends on their sizes
- Proteins in the polyacrylamide gels are next blotted to a nitrocellulose membrane and then fixed
- Protein-containing membrane is future probed with protein-specific antibody, in order to detect the protein of interest
- Detection of antibodies can be carried out by ELISA
Enzyme Linked Immunoassay
is a sandwich reaction where you'll use a primary antibody to bind to a protein of interest then you use the secondary antibody will catch on the primary antibody where the substrate would bind to the secondary antibody - there is chemiluminescent.
The difference with Western Blots and Northern/Southern Blots
Western Blots - use a polyacryamline gel
Northern & Southern - use agarose gel
You can do a Dot Blots or Slot Blots. (quicker than Southern Blotting)
- GOOD THING: you can analyze simultaneously on a dot or slot blot.
- Can test for only ONE GENE or gene product or address one kind of genetic aberration
- MULTIPLE SPECIMENS can be analyzed for ONE GENE.
- technologies are applied to analyze expression, mutation, amplification and deletion
Array-Based Hybridization - SLOT BLOTS
- Extracted DNA is denatured and the ssDNA is bound to a positively charged nylon membrane
- After the DNA is bound to the membrane, a probe complementary to the gene of interest is applied and allowed to hybridize to the DNA
- the hybridized complex is detected by Colorimetry or chemiluminescence methods
- The amount of DNA in the sample is estimated by comparison of the density of the band or bands observed to that of the standards
Allele-Specific Oligomer (ASO) Hybridization (I)
- a dot blot method utilizes the theory similar to Southern blots. Specimen DNA was first amplified by PCR, immodilized on a pair of membranes, and then each membrane is respectively.
- Utilizes the differential outcomes in hybridization between target DNA and the probe with short sequences of about 20 bases. Failed hybridization is due to one or two mismatches, and it can be distinguished from those with excellent hybridization due to a "perfect match".
- Probes used in this assay are synthetic single-stranded with normal or mutant target DNA sequences.
- At specific annealing temperatures and conditions (stringencies), given probes wil NOT bind to one or two mismatached based. Therefore, probes display differential binding to either mutant or to wild-type target DNA sequences.
- Applications include: detection of BRCA1 and BRCA2 genees in inherited breast cancer.