nucleic acid hybridisation Flashcards
what is nucleic acid hybridisation?
when single-stranded DNA or RNA molecules anneal to complementary DNA or RNA.
what factors contribute to the melting temperature of DNA?
- The chemical environment - chemical denaturants will decrease the melting temperature
- The composition of the strand - the more As and Ts the lower the temperature is likely to be, the more Gs and Cs the higher the temperature is likely to be
- The length of the DNA strand - the longer the strand, the higher the temperature is likely to be, the shorter the strand, the lower the temperature is likely to be
which techniques use DNA hybridisation?
- Chromosome in situ hybridisation
- Colony blotting
- Dot blotting
- Gene editing (CRISPR/Cas9 system)
- Microarrays
- Northern blotting
- PCR
- Southern blotting
Which enzyme is used in PCR?
taq polymerase
whats the difference between northern and southern blotting?
Northern blotting has RNA on the filter membrane, Southern blotting has DNA.
what are the probes used in northern or southern blotting?
can be fluorescent or radio-labelled
what affects hybridisation stringency?
- Decreasing the temperature decreases stringency.
- Increasing NaCl concentration decreases stringency
- Increasing the temperature increases stringency
what is stringency?
more specific interactions and decreased likelihood of non-specific binding
a better match between the probe and the target RNA or DNA molecule)
what can microarrays be used for?
- mutation screening
- identifying single nucleotide polymorphisms
- to examine the levels of gene expression
what does CRISPR require to work?
Cas9
double-stranded DNA target
guide RNA
PAM sequence.
what’s the steps of northern blotting?
- Restriction digest
- Run sample on a gel
- Transfer DNA to a filter membrane
- Add the hybridisation probe
- Develop the result.
