DNA Hybridisation Flashcards
What are the different components of DNA?
Nucleotides make up DNA and RNA- A, G, C and T
Pentose sugar:
5 carbons that form a cyclical structure with oxygen bridge
Carbons are numbered 1-5
Nitrogenous base joined to carbon-1
Phosphate group joined to carbon 5
Hydroxyl group carbon 3
What are the different types of bonds in a nucleotide chain of DNA?
Sugar phosphates- linked by phosphodiester bonds
Base stacking- hydrophobic interactions lead to an arrangement of bases set above each other internalised to the structure and excludes water
Van der Waals forces- individually small but contributes to the stability
3’ to 5’ linkage between the pentose ring
What does it mean when DNA is denatured?
Conversion of a double stranded molecule → of single stranded molecules
Disruption of Hydrogen bonds within the double helix
Occurs when DNA in solution is heated
Can also be induced by strong alkali or urea
How can denaturation be measured?
Denaturation can be measured optically by absorbance at 260nm
What is hyperchromicity?
Hyperchromicity- Increased absorption of light at 260nm on denaturation
What is meant by Tm?
The point at which 50% f all strands separate is called the melting temperature or Tm
What are the different factors affecting Tm?
Tm depends largely on hydrogen bonds: GC content Length of DNA molecule Salt concentration i.e. the molecular environment pH (alkali is a denaturant) Mismatches (unmatched base pairs)
Why does GC content affect Tm and how can we calculate it?
Higher GC content= more hydrogen bonds= higher Tm
%GC =[ (G+C)/(G+C+A+T) ] x 100
where G is the number of guanine nucleotides, C is the number of cytosine nucleotides etc
Three h-bonds in G:C vs two in A:T pair
How does molecule length affect Tm?
The longer the contiguous duplex, the higher Tm
More hydrogen bonds within the molecule means greater stability
However little further contribution beyond 300 bp
How does salt concentration affect Tm?
Salt stabilises DNA duplexes
High [Na+] = High Tm
Increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing
High salt reduces the specificity of base pairing at a given temperature
How does pH affect Tm?
Chemical denaturants disrupt hydrogen bonds
Alkali, formamide, urea
NaOH —->
How do mismatched affect Tm?
A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
Reduces Number of Hydrogen bonds, Fewer = lower Tm
Shorter contiguous stretches of double stranded sequence = lower Tm
Mismatches also distorts the structure and destabilises adjacent base pairing
What is renaturation and how can it occur?
Denaturation is reversible- we call this Renaturation
Reversal of denaturation:
Formation of structure favours energy minimisation driven by change in free energy ΔG
Facilitated by:
Slow Cooling
Neutralisation
What is stringency in terms of DNA hybridisation?
Perfect matches have a higher Tm
Are thermodynamically favoured over Mismatches
We can use this property to form a complementary molecule with no mis-matches
Stringency:
Manipulating conditions- limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
What are complementarity and hybridisation useful in?
Complementarity and hybridisation are at the heart of many nucleic acid-based techniques: Northern blotting Southern blotting Microarrays Dideoxy and Next-Gen Sequencing PCR Cloning
How can nucleic acid hybridisation be used?
Nucleic acid hybridisation techniques:
Identifies the presence of NA containing a specific sequence of bases
Allows the absolute or relative quantification of these sequences in a mixture
Hybridisation uses the ability of NA to form specific duplexes
Uses the complementarity and hybridisation of labelled nucleic acids
These molecules are referred to as “probes”
What is a probe?
An ssDNA (or RNA) molecule
Typically 20-1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously
What are the examples of nucleic acid blotting techniques? (What is northern and southern blotting)
Northern blotting an adaptation of Southern blotting:
Analysis of mRNA or DNA
Limited technique only detects one gene at a time and small number of samples
The gel based techniques are time consuming and messy
Largely superseded by quantitative PCR or other techniques
Recall the steps of Northern and Southern blotting?
Extract RNA or DNA
Gel electrophoresis
Transfer to nylon membrane
Add labelled probe
Probe hybridises to mRNA transcript in sample
Detect hybridisation
Mass flow trough gel by capillary action passively carries the nucleic acid
Potential applied across the gel electrophoretically transfers the -ively charged nucleic acid Nylon or nitrocellulose membrane capture nucleic acid and hybridises it with a label probe.
More typically today the gel is sandwiched by two electrodes and a voltage is applied across the two electrodes. The negatively charged DNA migrates towards the positive electrode and is transferred to the membrane electrophoretically. Again the DNA or RNA is captured by and covalently bonded to the membrane and then hybridised with the label probe.
What are microarrays?
An ordered assembly of thousands nucleic acid probes
Probes are fixed to a solid surface, then sample of interest is hybridised to the probes
Simultaneously measuring 50,000 different transcripts in a cell, tissue or organ
Probes are fixed to a solid surface such as glass or silicone
What are microarrays used for?
Microarrays can also assess millions of SNPs
DNA from 1 person on 1 microarray
Detects 2.5 million SNPs simultaneously
Result- homozygous or heterozygous for each SNP e.g. rs1333049: CC, GG or CG
Used in Genome Wide Association studies
