Hybridization Flashcards
Renaturation of DNA
- reannealing of ssDNA to dsDNA
- also called hybridization
Hyperchromic effect
increase in absorbance of DNA when it is denatured
The bases become unstacked and can thus absorb more light.
Hypochromic effect
decrease in absorbance of DNA when it hybridizes
Tm (melting temperature)
temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured
= 4 x (number of G/C) + 2 x (number of A/T)
Hybridisation Technique
- heat and separate DNA
- attach ssDNA to filter
- add ssDNA second sample to filter bound DNA
- hybridisation occurs if the two strand have any related sequences
Result: more closely related sequences = more hybrid molecules and high proportion of labelled DNA bound to filter
Factors affecting Tm
Increases:
- increasing G-C content
- increasing Na+ content
- increasing hybrid length
Probe Sequence
use to search for identical or similar sequences in the sample
Stringency of Hybridisation
High stringency: temperature of hybridisation closer to Tm for perfect match
- find exact sequence
Low stringency: temperature of hybridisation much lower than Tm for related sequences
Homologous vs Heterologous Hybridisation
Homo: probe and target contain identical sequences (high stringency)
Hetero: probe and target contain similar but non identical sequences (low stringency)
Nucleic Acid Labelling
- labelling DNA can be generated by end labelling of random priming
- can be isotopic or nonisotopic labelling (direct or indirect)
END labelling: polynucleotide kinase (forward or exchange reactions) with Klenow (fill in reaction)
*check this mechanism**
Random Priming
- 6 base long fragments
- random sequence
- denature and anneal in presence of mixture of random nucleotides
- Klenow subunits of DNA polymerase elongates sequence
Blotting
transfer of nucleic acid/protein to a solid membrane support
buffer movement upwards takes sample with it to botom surface of membrane (stained)
Southern Blotting
- DNA sample, ssDNA/RNA probe
- DNA separated by size on agarose gel + transferred on nitrocellulose, nylon membrane
- radio labelled DNA fragment (probe) incubated with membrane
- hybridised strands detected via film exposure
- used to detect specific DNA sequence within DNA samples
- good sensitivity and efficiency
- used for things like fingerprinting or genome mapping
Northern Blotting
- RNA analysis, ssDNA/RNA probe
- RNA separated by size on agarose gel and transferred onto membrane
- radiolabelled fragment (probe) incubated with membrane hybridized bands visualised
- used to identify what is transcribed and the level of transcription
- RNA needs to be stretched and relaxed
Colony Hybridization
- colony (phage) screening
- if a bacterial colony or plaque is potentially carrying a fragment of interest, it can be screened via hybridization
DNA Microarrays
- thousands of genes hybridised simultaneously
- Probe: DNA complementary to gene of interest is generated and positioned in microscopic quantities on solid surfaces at specific positions
- Target: DNA generated by sample RNA is fluorescently tagged and allowed to hybridize to DNA chip. 2 sample target populations analysed simultaneously
Monitor fluorescence with laser excitation. Different target DNA samples have different fluorophores
In Situ Hybridisation
- RNA/DNA fixed and probed in situ, ie. in position it occupies in living tissue
- cut into sections, hybridized with probe, examine with microscope
- used to examine levels of expression/transcription
FISH
- chromosome painting
- fluoresence in situ hybridisation
- chromosome specific probes
laboratory technique for detecting and locating a specific DNA sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it. - gene identification on a chromosome
- can be used to visualise abnormalities
