DNA and Hybridisation Flashcards
What type of polymers are DNA and RNA?
Polynucleotide Polymers
What does the DNA consist of?
A Pentose sugar
Nitrogenous base
Phosphate group
Describe the 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 on carbon 3
How many Nucleotides make up the DNA?
Which groups are they divided into?
Purines - Adenine, Guanine
Pyramidines - Cytosine, Thymine
What do the hydrogen bonds in DNA form the basis of?
Watson Crick base pairing rule
- Opposite charge distribution results in formation of dipoles
Which nucleotides bind to each other?
How many Hydrogen bonds form between them?
Adenine - thymine = 2 Hydrogen bonds
Cytosine - Guanine = 3 Hydrogen bonds
Describe the nucleotide chain of DNA
- Sugar Phosphates: linked by phosphodiester bonds
- Base stacking : hydrophobic interactions ->arrangement of bases set above each other internalised to the structure & excludes water
- Van der Waals forces: Individually small but contributes to the stability
Describe the double stranded DNA.
- Forms 2 anti-parallel stands
- Negatively charged phosphates on the outside
- forms a double helix shape
How can the DNA be denatured? (Basis)
DNA double helix + Chemicals/Heat = Denatured
How can the DNA be denatured? (STEPS)
- 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?
- It can be measured optically by absorbtion at 260nm through Hyperchromicity.
- Increased absorption of light at 260nm On denaturation Point at which 50% of all the strands are separate is called the melting temperature or Tm
- The Tm of a duplex is dependent upon the sequence and composition of its bases
What does TM largely depend on in terms of hydrogen bonds?
- GC content
- Length of DNA molecule
- Salt Concentration
- pH (alkali is a denaturant)
- Mismatches (unmatched base pairs)
What is the correlation between TM & GC content? Give the equation
- The higher the GC content = The more hydrogen bonds = The higher the TM
- %GC = TBC
How does the Molecule length affect the TM?
- The longer the contiguous duplex, The higher the TM
- More hydrogen bonds within the molecule will have greater stability
- However little further contribution beyond 300 bp
How does the salt concentration affect the TM?
- Salt stabilises DNA duplexes
- High [Na+] = High TM
- Increasing the salt concentration stabilises the DNA which increases TM and thus overcomes the destabilising effect of the mismatched base pairing
How does the salt concentration affect the TM? (PART 2)
High salt reduces the specificity of base pairing at a given temperature
How does the pH affect the TM?
- Chemical denaturants disrupt hydrogen bonds
- Alkali, Formamide, Urea
- NaOH -> Na+ + OH-
- OH- disrupts the H bond pairing
- Fewer hydrogen bonds = Lower TM
What is a Mismatched base pairing?
A mismatch is defined as a base pair combination that is unable to form hydrogen bonds
How does Mismatches affect the TM?
- Reduces the number of hydrogen bonds, Fewer H bonds = lower Tm
- Shorter contiguous stretches of double stranded sequence = lower Tm
- Mismatches also distorts the structure and destabilises adjacent base pairing
How can denaturation be reversed?
- Duplex formation is an equilibrium
- Denaturation is reversible, This is called renaturation
- Formation of structure favours energy minimisation driven by change in free energy DG
What is Renaturation facilitated by?
- Slow cooling
- Neutralisation
How is Complementarity and Tm the basis of specificity?
- Perfect matches have a higher Tm
- Are thermodynamicslly favoured over mismatches
- We can use this property to form a complementary molecule with no mis-matches
Describe Stringency
- Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
(VD)
Describe the conditions of Stringency
- Under high stringency: Only complementary sequences are stable determined by:
- Temperature near Tm
- Low salt concentration
What are the Nucleic acid hybridisation techniques?
- Identifies the presence of Nucleic Acids containing a specific sequence of bases
- This allows the absolute or relative quantitation of these sequences in a mixture
What is the Hybridisation technique?
- Hybridisation uses the ability of Nucleic acids to form specific duplexes
- Uses the complementarity and hybridisation of labelled nucleic acids
- These molecules are referred to as “probes”
What is a Probe?
- A 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
Describe the hybridisation technique in more detail
- In-situ hybridisation of tissue sections
- Chromosome painting of chromosome spreads
- Analysis of mRNA or DNA by PCR/qPCR
- Sanger/dideoxy sequencing
- These techniques are not very scalable and only detects few genes at a time and small numbers of samples
Describe the Genome wide technique
- Frequently we want to analyse thousands of segments of DNA or RNA in each sample.
- So hybridisation has been employed in high throughput techniques like microarrays and next generation sequencing
- Both techniques are used for measuring gene expression or genomic composition
Describe the High Throughput techniques
Microarrays:
- An ordered assembly of thousands of nucleic acid probes
- Probes are fixed to a solid surface, then sample of interest is hybridised
Next Generation sequencing:
- is the parallel sequencing of millions of molecules captured in a surface by hybridisation
