2- DNA and Hybridisation Flashcards
the chemical and molecular makeup and arrangement of DNA
polynucleotide polymer of repeating nucleotides - ACGT - which consist of three key components:
deoxyribose pentose sugar, phosphate group attached to C5 and nitrogenous base attached to C1
sugar phosphate backbone is held together by phosphodiester bonds formed between phosphate groups and deoxyribose linking the 5’ OH and 3’ phosphate of nucleotides
nitrogenous bases are single/double ring structures with carbon and nitrogen - different polar groups allow the specificity of Watson-Crick pairing; CG and AT. sequence of nitrogenous bases carries genetic info.
how the structure and composition of DNA influences its chemical properties and behaviour in solution
hydrophilic sugar phosphate backbone due to phosphodiester bonds, hydrophobic interactions within the internal arrangement of bases due to base stacking - allows DNA to be soluble in water whilst protecting the hydrophobic bases
H bonds between complementary nitrogenous bases due to opposite charge distribution creating dipoles. adds stability to duplex
van der Waals depends on the proximity of molecules to one another - small individually but contribute greatly to stability
overall negative charge of DNA due to the phosphate group - attracted to positively charged molecules
properties contributing to duplex formation
complementary base pairing
- held together by H bonds. CG form three, AT form two, making CG the stronger pair
antiparallel strands
- run in opposite orientations and allow complementary bases to line up
base stacking
- interactions between bases (hydrophobic and van der Waals) increase stability
what is the denaturation of DNA?
denaturation is the separating of a double-stranded DNA molecule into single stranded
what are the two methods for inducing DNA denaturation?
induced by disrupting H bonds:
heating the molecules to 95 degrees
molecules being put into a solution of strong acid, urea or alkali
how is DNA denaturation measured?
measured by hyperchromicity
- the ability of single stranded molecules to absorb more UV light than double stranded molecules. measured optically at absorbance 260nm
as temperature increases, more of the duplex separates, optical density decreases
what is Tm? how does it relate to denaturation? what factors affect it - e.g. lead to an increase in Tm?
Tm is the temperature at which 50% of the molecules have separated/ denatured. a higher Tm means a more stable structure.
affected by CG content, molecule length, salt conc, pH and mismatched bases
- cytosine=guanine content
CG form three H bonds as opposed to two by AT. more CG within a duplex, more H bonds, higher Tm
- molecule length
longer molecule, more H bonds, higher Tm. doesn’t make much different beyond 300bp however.
- salt conc.
higher salt conc increases Tm - overcomes the destabilising effect of mismatched base pairs as salt ions interact with the molecule. some duplexes with mismatches are only stable at high salt conditions, which means more hybridisation but less specificity.
- pH
alkaline/high pH (OH- ions) can disrupt H bonds, decrease Tm and destabilise pH
- mismatched bases
unable to form H bonds - more mismatches, less H bonds and distorts duplex making it less stable, lower Tm
what is renaturation? under what conditions does it occur?
renaturation is the reverse of denaturation and similar to hybridisation in that two DNA molecules are introduced to one another to form a duplex
position of equilibrium between de- and renaturation needs to be in favour of energy minimisation driven by a change in free energy - e.g. by cooling or neutralisation
what is stringency? how do high and low stringency conditions affect complementary and specificity within duplexes?
stringency is the ability to limit and manipulate conditions to select perfectly matched duplexes - manipulates specificity of a duplex
high stringency conditions form perfectly matched duplexes with high complementarity = determined by a temperature near Tm or low salt conc
low stringency conditions form mismatched duplexes with low specificity - lower temp than Tm or high salt conditions, but the kinetics are faster for hybridisation
what is a probe? how is it used in hybridisation? what can it be used for?
probe - a single stranded DNA or RNA molecule made chemically and labelled with a fluorescent/luminescent molecule
in hybridisation, a probe is used to be detect unique sequences in a gene - designed to be complementary to that specific region. under high stringency conditions, the probe will form a perfect match with the target region and hybridise with specificity.
it can be used to capture a DNA segment, amplify it or quantify the number of molecules in sample containing a sequence complementary to the primer
examples of techniques that use hybridisation. disadvantage to these techniques?
chromosome painting of chromosome spreads
PCR and qPCR
Sanger sequencing
in-situ hybridisation of tissue sections
- detect individual nucleic acids a few genes at a time, not a scalable activity
describe microarrays, next generation sequencing and exon capture as high throughput techniques
high throughput techniques occur on a larger scale - can investigate more complex systems and use higher sample sizes
microarrays = use labelled DNA/RNA probes complementary to a specific target sequence of interest. the probe is immobilised/ covalently fixed onto a surface and hybridised with the sample of interest. the level of fluorescent measured indicates the level of hybridisation.
next generation sequencing = parallel sequencing of millions of molecules captured on a surface by hybridisation
exon capture = probes designed to be complementary to target sequences/exons - captured by hybridisation, isolated and sequenced
what is complementarity?
the basis of specificity in DNA
