L6 Hybridization Flashcards
Using specific probes that are labelled specific sequences of DNA can be identified
Blotting/Hybridization Techniques
Transfer of an RNA sample separated and identified using DNA or RNA probes
Northern Blot
Transfer of a DNA sample separated and identified using DNA or RNA probes
Southern Blot
Transfer of a protein sample separated and identified typically using an antibody
Western Blot
ssDNA that will base pair with a complementary sequence of either RNA or DNA
Annealing/hybridization
Probes (detection with probes)
Stringency: heat, chemical, salt affect binding sensitivity and specificity
Annealing/hybridization
- Radioactive (P32) label
- Chemiluminescent compound
- Fluorescent compound
- Enzymatic label
ID of target detected/identified by labeling probes with
Separated Nucleic Acids are blotted onto _____ ______ by suction of buffer through gel and paper
nitrocellulose paper
Separate DNA fragments by ___ and ___ ________.
RE, gel electrophoresis
ASO (stands for?)
Allele-specific Oligonucleotide
How many probes does ASO have for each allele?
2
Cystic fibrosis - loss of ____________ leads to one form of the disease
phenylalanine 508
Probes
- AsOn = WT (wild type)
- ASOx = mutant cystic fibrosis gene (loss of phenylalanine 508)
ASO for Cystic Fibrosis Detection
RNA:DNA hybrids are captured onto a microtiter well coated with capture antibodies specific for RNA:DNA hybrids
Capture hybrids
- DNA is denatured
- Hybridized to an RNA probe
- Captured by bound anti-DNA/RNA antibodies
Chemiluminescence detection of hybrid molecules (DNA/RNA)
PCR steps
- Denaturation
- Annealing
- Extension (elongation)
Denaturation temperature
94
Extension (elongation) temperature
72