DNA hybridisation Flashcards
what is a pentose sugar in DNA
5 carbon (1-5) that form a cyclical structure with an oxygen bridge and a hydroxyl OH group on carbon 3
what is the structure of a nitrogenous base
a ring structure composed of carbon and nitrogen
Where does the nitrogenous base join the pentose sugar in a nucleotide?
carbon 1
where does phosphate group lie on pentose sugar
carbon 5
what are the purines (double ringed)
guanine adenine
what are the pyrimidines (single ringed)
thymine cytosine
Why is Cytosine-guanine pairing stronger than adenine- thymine/uracil pairing?
There is an extra Hydrogen bond between C-G than A-T/U
what is base stacking and what does it do
base stacking has hydrophobic interactions - arrangement of bases set above each other internalised to the structure and excludes water
What is the significance of Van der Waals forces in DNA?
individually small but contributes to the stability
What determines DNA stability?
The structure stability is determined by the free energy of the molecule and energy minimisation just as in protein structure.Stability is derived from the H bonding and internal arrangement of bases gaining additional stability by base stacking
VDW forces also impacts stability
Why does DNA have a negative charge?
the negatively charged phosphates are external giving DNA an overall negative charge
What is meant by denaturing DNA?
Conversion of a double stranded molecule → single stranded molecules by disruption of Hydrogen bonds within the double helix
When does DNA denaturing occur?
DNA in solution heated to energise bonds
- Or induced by strong alkali / urea, formamide forms randomly structured coil
How is denaturing measured?
Denaturation can be measured optically by absorbance at 260nm
What is hyperchromicity?
As temp increases, duplex melts and optical density (absorption) increases. This is because single stranded DNA absorbs UV light at a greater extent than double stranded DNA = hyperchromicity - absorption of UV light increases on denaturation
what does denaturation of a DNA duplex depend on
duplex stability determined by its sequence of bases.
What is the Tm?
Point at which 50% of all strands separate is called the melting temperature or Tm
What is the significance of Tm?
Tm is specific to a duplex with a given sequence - can use this to control the formation of a short duplex used as a primer or a probe
What factors determine the Tm of a DNA molecule?
Stability and thus Tm of a molecule is determined by 5 factors:
GC content
Length of a molecule
Salt concentration (molecular environment)
pH (alkali is a denaturant)
Mismatches (unmatched base pairs)
How does the GC content affect Tm?
Higher GC content = more H bonds = higher Tm
3 H+ bonds in G:C vs 2 in A:T
∴ more G:C pairing = more H+ bonds
How would you calculate the % of GC base pairs in a DNA strand?
%GC = (G+C / G+C+A+T) x 100
How does molecule length affect the Tm?
Longer the contiguous duplex, the higher Tm and stability
More H bonds, so more stable.
However a length beyond 300bp contributes little to no more to the Tm and stability.
How does the salt concentration affect Tm?
Salt stabilises DNA duplexes, so higher the NA+ (sodium ion) concentration, the higher the Tm
How does increasing [salt] overcome base pairing mismatches?
Increasing the salt concentration stabilises the structure and increases the Tm and thus overcomes the destabilising effect of mismatched base pairing, reducing specificity
What is the effect of a high [salt] of DNA?
High salt reduces the specificity of base pairing at a given temperature.
x
x
How does (high) pH affect the Tm?
Chemical denaturants disrupt hydrogen bonds e.g. Alkali, formamide, urea
NaOH ⇆ Na+ + OH- = high pH
OH- disrupts H bond pairing
Fewer hydrogen bonds = Lower Tm
high pH destabilises DNA
What is a mismatch?
a base pair combination that is unable to form hydrogen bonds
What effect does mismatches have on the Tm of DNA?
Reduces Number of Hydrogen bonds, Fewer = lower Tm
What effect does mismatching have on the structure of a DNA duplex?
Mismatches also distorts the structure and destabilises adjacent base pairing
What is renaturation?
The reversal of denaturation
Explain renaturation in terms of free energy
Formation of structure favours energy minimisation driven by change in free energy DG
What is renaturation facilitated by?
Slow Cooling
Neutralisation
Explain how renaturation and hybridisation differ?
Renaturation :
forming a duplex from single strands that were once already a double helix
Hybridisation :
formation of a duplex structure of 2 DNA molecules that have been introduced to one another
e.g., a short synthetic DNA (or primer) and genomic DNA
Outline what a perfect match would involve
- Have a higher Tm
- Thermodynamically favoured over Mismatches
- Property can be used to form a complementary molecule
with no mismatches
How can we prevent mismatches forming between two molecules?
by performing a hybridisation at the Tm of the duplex by hybridising at a temperature near to the perfectly matched duplex.
What is stringency and what does it allow us to do?
Stringency is the concept of manipulating the conditions to select duplexes with a perfect match only
allows us to manipulate (increase) specificity
Explain the outcome of hybridisation under high stringency
Only complementary sequences are stable (no mismatches) determined by a
- Temperature near Tm
- Low salt concentration
What is the effect of hybridisation under low stringency?
multiple duplexes form containing different mismatch pairings
kinetics of hybridisation are much faster under low stringency conditions, high salt conc
Which methods use Complementarity and hybridisation techniques?
Northern blotting
Southern blotting
Microarrays
Dideoxy and Next Gen Sequencing
PCR
Cloning
All rely on complementary base pairing specificity and avoidance of mismatches using Tm + manipulating conditions of hybridisation
What is the significance of Nucleic Acid Hybridisation Techniques?
to indicate the presence or absence of specific sequences of bases in a mixture of nucleic acids.
So essentially the nucleic acid hybridisation techniques
Identify the presence on nucleic acids containing a specific sequence of bases to capture specific sequences of DNA
Allows the absolute or relative quantitation (quantifying the amount) of these sequences in a mixture
Why are nucleic acids so important for hybridisation?
Hybridisation uses the ability of NA to form specific duplexes
What is a probe?
Labelled short DNA molecules (oligonucleotides) that are used to detect unique sequences and are part of a gene A ssDNA (or RNA) molecule Typically 20 – 1000 bases in length
How are probes identifiable?
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
what is a primer
Used in an enzymatic reaction to prime the reaction
Short single stranded polynucleotide, between 16-30 nucleotides
Explain how probes are used to identify specific base sequences
Probes used to detect nucleic acids are designed to be complementary to a specific target gene sequence region which is unique to that gene, which under high stringency conditions form a duplex
How are probes able to identify specific sequences?
Uses the complementarity and hybridisation of labelled nucleic acids
What is Northern Blotting?
Northern Blotting is a technique adapted from Southern Blotting for analysing the genes that are expressed by a cell tissue or organ
northern blotting limitation
it can only detect one gene at a time and small numbers of samples
The gel based techniques are time consuming and messy and as a consequence is superseded by quantitative PCR or other techniques
Outline the technique of Northern Blotting
Southern / Northern blotting uses DNA / RNA which is
separated by gel electrophoresis
- Transferred by mass capillary flow to a nylon membrane
- Covalently bond to membrane and then hybridised with
a labelled probe - The probe can be visualised by fluorescence
What is the use of Northern Blotting?
Analysis of mRNA or DNA
What is a microarray and what kind of method is it?
An ordered assembly of thousands nucleic acid probes
can also assess millions of SNPs
A comparative methodology - cannot find an absolute measurement of gene expression.
Explain how microarrays work
Probes are fixed to a solid surface such as silicone or glass matrix, then sample of interest is hybridised to the probes
Simultaneously measuring 50,000 different transcripts in a Cell, Tissue or Organ
What are microarrays used for?
microarray might be used for gene expression profiling for example a comparison of drug treated cells and untreated cells (compares gene expression)
Outline the process of microarrays
- RNA is extracted
- Labelled
- Hybridised to array and amount + location of label is
measured - This tells us how much of each and everyone of the
transcripts in the human genome are being expressed
compare microarrays and blotting techniques
Microarrays compare many different conditions simultaneously. Have advantage over blotting techniques in that it can simultaneously measure the expression of many different transcripts and compare multiple experimental conditions whereas northern blotting can only measure single gene targets.
How many SNPs can be detected from 1 persons DNA?
DNA from 1 person on 1 microarray
Detects 2.5 million SNPs simultaneously
What is an SNP?
Single-nucleotide polymorphism
most common type of genetic variation among people
Each SNP represents a difference in a single nucleotide
