DNA complementarity hybridisation & its application Flashcards
What is DNA?
Polymer or polynucleotide comprising of 4 nucleotides joined together
What does the nitrogenous base comprise of?
A single or double ring, which contains nitrogen and carbon
What is attached to carbon 5 of the ribose sugar?
A phosphate group
Where is the hydroxy group on the ribose sugar?
Carbon 3
Where is the nitrogenous base on the ribose sugar
carbon 1
What are the 4 nucleotides?
Cytosine, Guanine, Thymine, Adenine
What are the 2 molecular structures that the 4 nucleotides fall into?
Pyrimidines
Purines
What are the structural differences between pyrimidines and purines?
Pyrimidine - a single nitrogen ring
Purine- a double nitrogen ring
What are the nucleotides that are pyrimidines?
Cytosine and thymine
What are the nucleotides that are purines?
Guanine and adenine
What are the watson and crick base pairs?
Adenine ad Thymine
Cytosine and Guanine
How many hydrogen bonds form between the base pairs?
Guanine and cytosine form 3 hydrogen bonds
Thymine and Adenine form 2 hydrogen base pairs
Which base pair is stronger?
Guanine and cytosine because they have 3 hydrogen bonds
In RNA which base pair is stronger?
Guanine and Cytosine is still the stronger base pair.
Adenine and Uracil only have 2 base pairs
What molecular structure is uracil?
A pyrimidine like Thymine
What links the sugar phosphate groups?
Phosphodiester bonds
What bonds are present within DNA?
Phosphodiester
For base stacking - Hydrophobic interactions
Van der Waals forces (which are weak but help with stability)
What is the form of DNA that is most common?
B form
What determines the stability of DNA?
Free energy of the molecule and energy minimisation
What can influence the stability of DNA
The molecular environment
How is double stranded DNA formed?
From 2 anti parallel strands that means they have opposite orientation
What overall charge does DNA have?
Negative due to the phosphate groups. This fact is used for gel electrophoresis
DNA denatured
Double strand molecules to a single strand molecule
What can cause DNA to denature?
The disruption of Hydrogen bonds within the double helix
What causes the hydrogen bonds within DNA to break?
When DNA is heated
Or when DNA is in a strong alkali, formamide or urea
What is hyperchromicity?
Increased absorption of light at 260 nm on denaturation
What is Tm/ melting temperature?
Point at which 50% of all strands separate.
What is special about single stranded DNA?
Hyperchromicity - Single strand DNA absorbs more UV light than double stranded DNA
What does Tm depend on?
GC content Length of DNA molecule Slat conc (molecular environment) pH (alkali denatures DNA) Mismatched Base pairs
What does a higher GC content mean?
More Hydrogen bonds = higher Tm
What does more hydrogen bonds mean for stability?
There is greater stability?
Is there a difference in stability after 300 bp?
No, after 300 bp there is no increase in Tm / stability
What cation influences stability?
All of them but sodium ions is the most common
What does a higher [Na+] mean for Tm?
A higher Tm, as salt conc increases stabilisation.
Higher Salt conc also over comes how unstable mismatched base pair are
What else does a high salt also do?
Reduces the specificity of base pairing
What does an alkali solution do to DNA?
Alkali dissociates into its ions.
The OH- ion disrupts the hydrogen bonds in DNA
Higher pH means?
A lower Tm
What is mismatch?
A base pair combination that is unable to form a hydrogen bond
Low no of H bonds?
Low Tm
What else does DNA mismatch do to DNA?
Distorts the structure and destabilise adjacent base pairing
How do you reverse denaturation?
Slow cooling
Neutralisation
What is renaturation / hybridisation?
Formation of duplex structure of two DNA molecules that have been introduced to one another
Are there different factors that influence renaturation and hybridisation?
No they are both examples of duplex formation
What contributes to a high Tm?
Perfect base pairs
What is stringency?
Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
Why would we want low stringency?
Kinetics of hybridisation are much faster under low stringency conditions
What determines high stringency?
Temperature near the Tm or a low salt conc
What are the 6 nucleic acid based techniques?
- Northen blotting
- Southern blotting
- Microarrays
- PCR
- Cloning
- Dideoxy and next gen sequencing
Specific aspects of Hybridisation techniques?
Identifies the presence of NA containing a specific sequence of bases
What are uses of probes?
Capture specific species of DNA
Amplify segments of DNA
Or to quantify the no of molecules present
What is a probe?
ssDNA or RNA
About 20 -1000 bases in length
Labelled with a fluorescent or luminescent molecule
The limitation of northern blotting
One gene at a time and a small sample size
messy and time consuming
PCR is better
Northern and southern blotting
- Uses DNA or RNA that has been separated by gel electrophoresis.
- Transferred to a mass capillary flow of a buffer from a reservoir with nylon membrane
- Captured by and covalently bonded to the membrane and then hybridised with a labelled probe
Microarrays
An ordered assembly of thousands nucleic acid probes
-They are fixed to a solid surface, then the sample of interest is hybridised to the probes
Where are the probes fixed to?
A solid surface like a glass matrix or silicon
What are microarrays used for?
Gene expression profiling for drug treated cells and untreated cells
What are GWAS?
Genome Wide Association studies
Microarrays with SNPs are used for GWAS
What is needed for microarrays?
RNA
Labelled with a fluorescent molecule
hybridised to the array and amount and location of the label measured
