Hybridization Formats Flashcards
What are the 3 hybridization formats?
solution - homogenous
solid - mixed phase
in-situ
Components of mixed phase system and function?
solid transport - target DNA immobilized
solution - probe
solid support types?
Nitrocellulose
latex beads
magnetic beads
Explain the process of dot blot method
denaturation immobilization pre-hybridization probes are added washing xray expose
Why the filter is pre-hybridized?
to -bg noise. omit non specific binding sites
How and what stringency is applied?
high
What may be the reasons for bg noise?
bad washing
inefficient blocking
Why southern blotting is used?
To detect specific dna sequences
Explain the process of southern blotting.
restriction digestion agarose gel electro. blot on a gel on a sponge - buffer move by capillary membrane is separated & baked prehybridization probs adding xray
What to do to hybridize RNA? What is done differently in n. blotting?
formaldehyde in gel - resolve 2ry str.s.
diazobenzyloxymethyl ppr
What are the alternatives for southern blotting?
microtiter wells for immobilization
hybridization in dried agarose gel
What are the applications of solid hybridization?
genome analysis
gene discovery & mapping
diagnostics
forensics
What are the advantage and limitation of solution hybridization?
How come sol. hybri. is the fastest?
- fastest rate
- difficult to separate
-probe finding target chance is high
Explain the process of solution hybridization?
hybridization
HAP added & mixed well - HAP binds to ds DNA
cetrifugation
wash the pellet
quantification - colorimetry / scintillation counter
In situ hybridization
For what it is carried out?
- to identify seq.s in the cells/tissues themselves
In-situ hybridization
how specimens are kept?
fixed on micro. slides - formalin fixed paraffin embedded tissues
In-situ hybridization
What are the solid support and the solution?
- cells
- probe
In-situ hybridization
What are the uses?
- Histology & cytology labs
- detection of viral seq.s - their intracellular location & no. of viral seq.s per cell
- diagnostics - detect chromo. translocations, deletions, cancer, FISH
-Research - localizing genes and their expression, -mapping genes
Chromosome In-situ hybridization
What are the uses?
- cytogenetic analysis
- diagnose chromo. abnormalities
Explain the process of chromo. In-situ hybridization? And as to why each is added.
- micro. slides of cells in meta/prometaphase prepared(why)
- treated with RNAse & proteinase K(why)
- denatured by formamide
- hybridized with probes
- washing
- Detection using FISH using a fluorescent microscope
How to use tissues in In-situ hybridization?
- paraffin removed
- mounted on a micro. slide
- hybridized with probes - cDNA or RNA(higher sensivity but unstable)
- Do FISH
How to prepare a blood culture for FISH?
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