DNA Hybridisation Flashcards
What makes up DNA and RNA?
- Phosphate
- NItrogenous Base
- Pentose Sugar
What is the structure of a Nitrogenous Base?
A ring structure (single or double ring) composed of caron and nitrogen (occasionally oxygen).
What is the strucutre of a Pentose Sugar?
- 5 carbons
- Acyclical structure with oxygen bridge
Which carbon does the nitrogenous base, phosphate group and hydroxyl group join to respectively?
Nitrogenous base joins to carbon 1.
Phosphate group joins to carbon 5.
Hydroxyl group joins to carbon 3.
How is the phosphate group attached to carbon 5?
Via an ester bond
How many hydroxyl group are in RNA?
2
What are the four nucleotides in DNA?
Pyrimidines:
- Cytosine
- Thymine
Purines:
- Guanine
- Adenine
What is the difference in the structure of pyrimidines and purines?
The difference resides in the charged or polar groups providing the specificity of base pairing.
How is the RNA duplex structure formed?
Uracil substitues Thymine and base pairs with Adenine in RNA to form duplex structure.
What are the complementary bases?
Cytosine - Guanine
Thymine - Adenine
How many hydrogen bonds are between C-G and T-A?
3 and 2 respectively
What does the nucleotide chain of DNA form?
It forms a double helix which can take on different combinations.
What is the most common form of DNA?
B-DNA
What forms the backbone of DNA?
From a phosphodiester linkage which connects the 3’ and 5’ prime carbons of the deoxyribose sugar of DNA
What determines the stability of a structure?
The free energy of the molecule and energy minimisation.
What stabilises the structure of DNA?
- Hydrogen bonding of the bases
- Internal arrangement
What other than hydrogen bonding contributes to the stability of DNA?
- Sugar phosphates
- Base stacking
- Van der Waals forces
What is base stacking?
The hydrophobic interactions between the arrangement of bases set above each other internalised to the structure and excludes water from the internal environment of the structure
What gives DNA its overall negative charge?
The bases on the inside forming stacked bases and the negatively charged phosphates are on the outside giving DNA an overall negative charge.
What is the overall negative charge of DNA useful for?
It is used for electrophoresis - in a highly negative environment will migrate to the positive electrode.
What happens when DNA is denatured?
When DNA is denatured, there is conversion of a double stranded molecule to single stranded molecules.
What happens when there is a disruption of the hydrogen bonds?
The hydrogen bonds can be denatured or broken down into its constituent strands.
What structure is formed when DNA is denatured?
- A randomly structured coli
What conditions does DNA need to be under to denature?
- DNA solution is heated
- Induced by strong alkali or urea
How is denaturation of DNA measured?
- It can be measured optically by absorbance at 260nm.
- Take the solution, heat it up and measure the optical density
What is hyperchromicity?
Single stranded DNA absorbs UV light to a greater extent than double stranded DNA
What does the denaturation of a specific DNA depend on?
- The stability of the specific structure
What is the melting temperature Tm?
- The temperature at which 50% of the molecules (all strands separate) have melted
What does the Tm depend on?
Hydrogen bonds
What are the 5 factors that the Tm of a DNA molecule is determined by?
- GC content
- Length of DNA molecule
- Salt concentration
- pH
- No. of mismatches
The higher GC content….
…the more hydrogen bonds, the higher Tm
The longer the contiguous duplex….
…. the higher the Tm
Why is the Tm higher when there is a longer contiguous duplex?
There is more hydrogen bonds within the molecule greater stability.
When does the length have a dimishing return?
There is a diminishing return on this and a length beyond about 300 base pairs contributes little or no more to the stability.
What does salt do to the DNA duplexes?
It stabilises DNA duplexes.
The higher [Na+]…
… the higher Tm.
What overcomes the destabilising effect of mismatched base pairing?
- Increasing the salt concentration stabilises the structure increases the Tm
When is a duplex unstable and/or stable?
A duplex is stable at a given temperature in the presence of high salt concentration whilst the same duplex would be unstable and dissociate at the same temperature in low salt.
What do chemical denaturants do to DNA?
They disrupt hydrogen bonds and are easy to add to a solution and can denature the DNA present in that solution.
Give examples of chemical denaturants
Alkali, formamide, urea
What is adding alkali pH the same as?
It is the same as adding OH groups.
When OH is added to DNA strands?
They are small enough to penetrate into the molecule and break the hydrogen bonds - resulting in dissociate of the two strands.
What is mismatch?
A base pair combination that is unable to form hydrogen binds.
What do mismatch base pairs do to the DNA duplex?
This reduces number of H bonds, fewer means lower Tm. The shorter contiguous stretches of double stranded sequence, meaning lower Tm.
What is renaturation?
- The reverse of denaturation.
- The formation of structure favours energy minimisation driven by change in free energy.
What facilitates renaturation?
Slow cooling
Neutralisation
Define hybridisation
The formation of duplex structure of two DNA molecuels that have been introduced to one another.
What is the similarity between hybridisation and renaturation?
Both involve two DNA molecules taht have been introduced to each other. The molecule has not previously been part of the duplex of that molecule.
Why are perfect matches formed?
- Have a higher Tm
- Are thermodynamically favoured over mismatches
- Can use this property to form a complementary molecule with no mismatches
What is stringency?
The concept of manipulating the conditions to select duplexes with a perfect match only.
How are conditions manipulated?
Temperature is near Tm or there is a low salt concentration whih mean only complementary sequences are formed stabely.
Define northern blotting
used for the identification of RNA species within a dense population of RNA
define southern blotting
used for the identification of DNA species within a dense population of DNA
Microarrays
measure the absence or presence of particular species within a population of molecules.
What are nucleic acid hybridisation techniques used for?
- Identify the presence of nucleic acid containing specific sequence of bases
- Allows the absolute or relative quantitation of these sequences in a mixture
What is a probe?
- A ssDNA (or RNA) molecule
- Typically 20-1000 bases in length
- Labelled with fluorescent or luminescent molecule
- In some techniques, thousands or millions of probes are used simultaneously.
Disadvantages of northern blotting
- Analysis of mRNA or DNA
- Limited technique only detects one gene at a time and small numbers of samples
- The gel-based techniques are time-consuming and messy.
- Largely superseded by quantitative PCR
How are microarrays carried out?
- An ordered assembly of thousands of 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.
How many SNPs can microarrays detect simultaneously?
2.5 million
When are microarrays used?
In Genome wide association studies
