W4L7 - Nucleic Acid Hybridisation Flashcards
Techniques based on NA Hybridisation
PCR Southern Blot Northern Blot Microarray Bead-based arrays
NA Hybridisation
The binding of two pieces of ss NA to one another
Binding is mediated by H bonds between complementary base pairs
What are the factors that the degree of hybridisation is mediated by?
NA base composition NA length Degree of homology between NA strands Hybridisation temperature Salt concentration surrounding medium
Factors that Affect Hybridisation - NA Base Compostion
Binding of A and T mediated by two H bonds
Binding of C and G mediated by 3 H bonds
Therefore C-G bonds are more stable than A-T and require more energy to break them
Factors that Affect Hybridisation - NA Length
For specificity, nucleic acids should be at least 20 bp in length
Longer NAs more stable once hybridised
Factors that Affect Hybridisation - Degree of Homology
Greatest degree of binding occurs when the two NA sequences are identical (i.e. homologous)
Binding decreases as the % sequence identity decreases
Factors that Affect Hybridisation - Hybridisation Temperature
ds NA denatures as temperature increases
Melting temp of NA is the temperature at which 50% of the NA is denatured
Decreasing the reaction temperature promotes DNA hybridisation, but can also decrease specificity
Conversely, increasing the reaction temperature promotes DNA denaturation, but increases specificity
Factors that Affect Hybridisation - Salt Concentration
Negatively charged phosphate groups in each of the NA strands repel each other
This repulsive force is overcome by H bonds
Monovalent cations (i.e. Na+) shield the phosphate groups, decreasing their repulsive force
Therefore, hybridisation is promoted by increasing salt concentration, but also decreases binding specificity
Conversely, decreasing salt concentration decreases hybridisation but increases specificity
Southern Blotting (after initial steps)
After transfer of DNA to the membrane, the membrane is incubated in a pre-hybridisation solution
The membrane is then incubated with a probe, which hybridises with the target of interest
After hybridisation, the membrane is washed to remove non-specifically bound probe
After the washes, probe label needs to be detected
Depending on label used, next perform autoradiography to view labels
What is a Probe?
A piece of DNA whose sequence is complementary to the target sequence
Labelled so it can be detected
Examples of Labels Used in Southern Blotting
Radioactive
- dNTPs with attached radioactive labels
- very sensitive, but have associated safety and environmental concerns
Non-radioactive
- e.g. Biotin-labelled dNTPs which can be detected with streptavidin-HRP and a HRP substrate
- e.g. Digoxigenin (DIG)-labelled dNTPs which are detected with an alkaline phosphate-labelled anti-DIG antibody and an alkaline phosphate substrate
Northern Blotting
Technique used to analyse RNA on a membrane
Basically same as Southern Blot except that RNA is separated on the gel
Microarray Analysis
Allows performance of a large number of hybridisation experiments at once
Probes spotted onto glass
DNA to be analysed is fragmented, fluorescently labelled, added to the chip and allowed to hybridise
Unbound test DNA washed off
Hybridised DNA detected via fluorescence
Bead Arrays
DNA to be analysed is fluorescently labelled (with a colour that isn’t represented in the population of beads)
DNA binding to the bead can be detected by flow cytometry
Bead Array - Luminex System
Approx. 100 different beads each containing a unique combination of red and infrared fluorophores
Each bead labelled with a unique probe or antibody
DNA is labelled with phycoerythrin (PE), which fluoresces at a different wavelength to the beads
Labelled DNA is mixed with the beads and allowed to hybridise
Beads analysed using flow cytometry
If DNA is bound, both the bead and PE fluorescence is detected
If DNA not bound, only the bead fluorescence is detected
