DNA and hybridisation Flashcards
what do nucleotides make up?
DNA and RNA
What does DNA compromise of?
4 nucleotides joined together.
- nitrogenous base
- ribose sugar
- phosphate group
what are the 4 nucleotides that DNA consist of?
cytosine
thymine
guanine
adenine
which nucleotides are purines
adenine
guanine
which nucleotides are pyrimidines
thymine
cytosine
how many hydrogen bonds between cytosine and guanine
3
how many hydrogen bonds between thymine and adenine
2
describe DNA structure
antiparallel strands
negatively charged phosphates on outside
bases on inside
at what absorbance can denaturation be optically measured
260nm
what is the melting temperature (Tm)
the point at which 50% of all strands seperate
what does TM depend on? (6)
hydrogen bonds GC content length of DNA molecule salt conc PH mismatches
what does more GC mean for Tm
more GC = more H bonds = higher Tm
what does longer molecule length mean for Tm
higher tm, due to more h bonds in longer molecule = more strength.
stops past 300bp
what does high salt conc mean for Tm
higher tm
high salt conc = stable structure = overcomes destabilising effect of mismatched base pairs
what does less hydrogen bonds do to the Tm?
lowers Tm
define mismatch
a base pair combination that is unable to form hydrogen bonds
do mismatches lower Tm
yes
-reduces no. of h-bonds
shorter stretches of double stranded sequences
what is reversal of denaturation?
formation of structure favours energy minimisation driven by change in free energy
similarities in renaturation and hybridisation?
-involve DNA molecules that have been introduced to each other
both duplex formation
why do complementary bases have higher tm
thermodynamically favoured
no mismatching
high stringency on tm?
higher tm
stable complementary base pairing
application of complimentarity and hybridisation?
northern blotting southern blotting microarrays dideoxy and next gen sequencing PCR cloning
what does nucleic acid hybridisation allow?
identifying presence of nucleic acids containing specific sequence of bases
allows absolute or relative quantitation of these sequences in a mixture
what is a probe
single stranded DNA or RNA
between 20-1000 bases
labelled with fluorescent molecule
nucleic acid blotting techniques
northern blotting
analysis of mana or DNA
flaws of nucleic acid blotting techniques
limited technology = detect one gene at a time
small no of samples
gel based techniques are messy and long
explain northern/southern blotting
- extract RNA or DNA
- gel electrophoresis
- transfer to nylon membrane
- add labeled probe, hybridises mRNA
- detect hybridisation
- nylon membrane captures nucleic acid
- mass flow through gel capillary action carries nucleic acid
what are microarrays?
ordered assembly of thousands of nucleic acid probes
how do microarrays work?
- probes fixed to solid surface
- sample is hybridised to probes
- RNA extracted, labelled and hybridised.
used to compare gene expression
microarrays and SNPs
Detects 2.5 million SNPs simultaneously
- used in genome wide association studies
