DNA Hybridisation: DNA Complementarity, Hybridisation And Its Application Flashcards
Where does the nitrogenous base of DNA join the sugar?
C1
Where does the phosphate group of DNA join the sugar?
C5
Where does the hydroxyl group of DNA join the sugar?
C3
Which carbons in the pentose ring is the oxygen bridge between?
C1-C4
Which are the pyrimidine bases?
Cytosine and thymine
Which are the purine bases?
Guanine and adenine
What is base stacking caused by?
Hydrophobic interactions that change the arrangement of bases set above each other on the inside of the DNA structure
How many hydrogen bonds does the C-G base pairing form?
3
How many hydrogen bonds does the A-T base pairing form?
2
Under what conditions does DNA denature?
When the DNA in solution is heated or exposed to a strong alkali and urea
What does the single DNA strand look like?
A randomly coiled string
How can you measure the denaturation point of DNA?
Using optical density at 260nm
What happens to the absorbance of DNA at higher temperatures?
Increases
Why does the absorbance of DNA increase at higher temperatures?
Because single stranded DNA absorbs more
What is hyperchromicity?
Increased absorption of light at 260nm on denaturation
What is the melting temperature (Tm) of DNA?
The point at which half of the DNA strands have separated
What factors determine the melting temperature of DNA?
GC content, length of DNA molecule, salt concentration, pH and mismatches
How does GC content determine the melting point of DNA?
Higher GC content (more guanine-cytosine bps) -> more H bonds -> higher melting point
How does length of the DNA molecule determine the melting point of DNA?
Longer = higher melting point
Why does length of the DNA molecule determine the melting point of DNA?
More H bonds means it’s more stable
How does salt concentration determine the melting point of DNA?
Salt stabilises the DNA helix so higher sodium conc increases the melting point
What does high salt conc reduce in DNA at a given temperature?
The specificity of base pairing
How does pH affect the melting point of DNA?
Alkalinity disrupts the H bonds and lowers the melting point
How do mismatches affect the melting point of DNA?
More mismatches mean a lower melting point
Why do mismatches affect the melting point of DNA?
Less H bonds, so less stable
What’s the opposite of denaturation?
Renaturation
What is renaturation facilitated by?
Slow cooling and neutralisation
What is the basis for specificity of DNA renaturation?
Complementarity
What is stringency?
Limiting hybridisation between imperfectly matched sequences
What does stringency allow us to manipulate?
Specificity
What is high stringency determined by?
A temperature near the melting point or a low salt conc
What happens to DNA at high stringency?
Only complementary sequences are stable
What techniques do complementarity and hybridisation underlie?
Northern and southern blotting, microarrays, dideoxy (Sanger) and next gen sequencing, PCR and cloning
What is northern and southern blotting?
An analysis of mRNA or DNA
What limits the use of northern and southern blotting?
It can only detect one gene at a time in small numbers of samples, which makes it slow. Also gel based so time consuming and messy
How do microarrays work (basic)?
Thousands of nucleic acid probes fixed to a solid surface, then a sample of interest is hybridised to the probes
What do nucleic acid hybridisation techniques allow?
The absolute or relative quantitation of sequences in a mixture
Basically, how do nucleic acid techniques work?
Label a probe and hybridise it in a mixed population of DNA. This captures specific DNA sequences
How does hybridisation form specific duplexes?
Using the complementarity of nucleic acids
What are the characteristics of a probe DNA molecule?
Single stranded, 20-1000 bases in length, labelled with a fluorescent or luminescent molecule
What is the process of northern or southern blotting?
Extract DNA or RNA
Gel electrophoresis
Transfer to nylon membrane
Add labelled probe and let it hybridise to the sample
Detect hybridisation using electrophoresis
