DNA Complementarity Hybridisation and it's Application Flashcards
What are DNA and RNA made up of?
Nucleotides
What are nucleotides comprised of ?
Nitrogenous Base
Phosphate
Pentose Sugar
What is a nitrogenous base?
A ring structure composed of carbon and nitrogen. Comprising of either a single or double ring
What is a Pentose sugar?
A Pentose sugar is a 5 carbon cyclical structure with an oxygen bridge
At which Carbon is the Nitrogenous base attached to?
C1
At which Carbon is the phosphate group attached to?
C5
Which carbon is the hydroxyl group attached on?
C3
Between which carbons is there a oxygen bridge?
C1 and C4
Through what bond does the phosphate and the next nitrogenous base attach by?
Phosphodiester bond
How many nucleotides is DNA made up of and what are they?
DNA is made up of 4 nucleotides
- cytosine
- thymine
- adenine
- guanine
Which of the nucleotides are Pyrimidines ?
Cytosine and thymine
Which of the nucleotides are Purine?
Adenine and Guanine
What provides the specificity of base pairing?
The difference in the structures due to the charged and polar groups
In RNA what substitutes Thymine?
Uracil
DNA double helix is formed due to what?
- Hyrdogen bonding of Watson’s and Cricks base pairing
- Bonding between amine and carboxyl gorups between A-T or C-G
- Each pair there is a single purine and pyrimidine
What is the difference between A-T and C-G pairing
The number of hydrogen bonds formed
Why is C-G pairing stronger?
C-G forms 3 hydrogen bonds whereas A-T forms 2
More hydrogen bonds
What does base pairing contribute to in terms of the DNA structure?
It explains the stability of the double helix structure
What are the 3 structures of the Nucleotide Chain of DNA?
- Sugar Phosphates
- Base Stacking
- Van der Waals froces
What are the Sugar phosphates linked by?
Phosphodiester bonds
Through what interactions does base stacking occur? What is exculded thorugh this?
Hydrophobic interactions
Arrangement of bases set above eachother internalised to the strucure and exlcudes water
How big are the Van der Waal forces?
Individually small but still contributes to the stability
What is the backbone structure formed from?
Phosphodiester Linkage
How is the stability of the structure determined?
It is determined by the free energy of the molecule and energy minimisation
How is Stability of the helix derived?
Stability is derived from:
- hydrogen bonding of the bases
- internal arrangement and addition stability of base stacking
- alongside vdW
Where does the stability come from in a single stranded DNA molecule?
From the sugar phosphate backbone
From what strands is the double helix formed from?
Two antiparallel strands
What does having antiparallel strand mean?
They have an opposite orinetation
- bases are on the inside forming stacked bases
- the negatively charged phopshates external givign dNA an overall negative charge
Why is it important tohave charged phosphate groups on the otuside?
Importantn for the interation with proteins
What is Denautirng
Denatriing is the breaking of hydrogen bonds inot its consitiuent starnds
conversion of double strnaded molecule into a single stranded molecule
During what condidiotns are the hydrogne bonds disrupted?
- DNA is heated
- Induced by strong alkali or urea
On denaturaion what type of strand does it form?
Randomly structured coil
How can denaturation be measured?
It can be measured optically by abd=sorbace at 260nm
What is Hyperchromacity?
Increased absorption of light at 260nm on denatutration
Single stranded DNA absorbs UV lgiht to a greater extent than double standed DNA
What is the Melting Temperature or Tm?
Point at which 50% of all strands have sperarated
The Tm is specific to what?
The Tm is specific to an individual double helix
Use this charactersitics to control formation of the duplex
What does the denautartion of a specififc DNA depend on?
Depedns upon the stability of the specific structure
Name 6 conditions that Tm is dependent on.
- Hydrogen Bonds
- G-C base pairing content
- Length of DNA molecule
- Slat concerntrarion
- pH
- Mismatches
What is the relationship between G-C content and Tm?
The higher G-C content = more hydrogen bonds = higher Tm
Why does G-C content cause a higher Tm?
G-C is stronger than A-T
There are 3 H bonds rather than 2
therefore more H bonds to break which requires a higher temperature
How do you calculate the %GC
(G+C) / (G+C+A+T) X 100
What is the relationship between Tm and Molecule length?
The longer the contiguous duplex the higher the Tm
How does having a longer duplex cause a higher Tm?
There are more hydrogen bonds within the molecule therefore a greater stability
At which point in the molecule does hydrogen bonds stop contributing to the stability?
Beyond 300 base pairs
What is the relationship between Tm and Salt Concentration?
Increasing the salt concentration stabilises the structure increases the Tm and therefore overcomes the destabilising effect of mismatched base pairing
What does salt do to DNA?
Salt stabilises DNA duplexes
Compare the scenarios of having High salt concentrations/ low concentration and mismatched base pairing
High Salt Concentration:
- a duplex containing mismatches can form and be stable at a given temperature in the presence of this condition
Low Salt Concentration:
- the same duplex would be unstable and dissociate at the same temperature in low salt
Mismatch base pairings also affect what?
They affect adjacent base pairings as a consequence of distortion of the helix
This encourages the unzipping of the molecule
What is the relationship between Tm and pH?
The higher the pH, the lower the Tm
Why does a high pH cause a lower Tm?
OH- ions are released which disrupts H bond pairinfs
Fewer H bonds = Lower Tm = lower stability of the structure
What do chemical denaturants do?
Disrupts hydrogen bonds
What does a high pH do to a DNA duplex?
It destabilises the DNA duplex
What is a mismatch?
A mismatch is a base pair combination that is unable to form hydrogen bonds
What are the three effects that mismatches have?
- reduces the number of hydrogen bonds formed within a duplex
- distorts the duplex and destabilises adjacent base pairs
- leads to zipping and unzipping
What is the relationship between Tm and Mismatches?
More mismatches = less hydrogen bonds = lower Tm
Shorter contiguous stretches of double stranded sequence = lower Tm
What is the effect of reducing the change in free energy on duplex formation?
Formation of a duplex less energetically favourable
What is the process name for the reverse of denaturation?
Renaturation
What facilitates Renautration?
Facilitated by reversal of the factors that influence denaturation
What does renaturation depend on?
It depends upon the energy of the system and corresponding molecules
Occur as a result of a change in free energy
What are the two conditions of renaturation?
- Slow cooling ( cool down the heating)
- Neutralisation (neutralise the Alkali)
What is the difference between renaturation and hybridization?
- Renaturation : formation of a duplex molecule originally from a duplex molecule before
- Hybridization: the formation of a duplex molecule by introducing a 2nd molecule ( involves 2 DNA molecules being introduced to each other)
What is Stringency?
Stringency is manipulating conditions by limiting hybridization between imperfectly matched sequences allowing us to manipulate specificity.
Explain the Stringency concept.
Stringency is manipulating the conditions to select duplexes with a perfect match only
What are the results of a Low Stringency?
Conditions: High Salt , Low Temp
- multiple duplex with different mismatch pairing
What are the results of a High Stringency?
Conditions: Temperature near Tm , Low Salt Concentration
Promote formation of non-mismatch pairing
Name some Nucleic Acid Based Technology that uses hybridisation.
- Northern Blotting
- Southern Blotting
- Microarrays
- Dideoxy and Next Gen Sequencing
- PCR
- Cloning
Explain how Nucleic Acid Hybridisation technique works.
- identifies the presence of nucleic acids containing specific sequences of bases
- allows the relative quantitation of these sequences in a mixture
- uses labelled molecules which are complementary to molecules under investigation
What is the term used for a labelled nucleic acid?
Probe
How are Probes used ?
Probes that are used are complementary to a specific region of a target gene sequence
How many bases are probes?
Probes are between 20 - 1000 bases in length dependent on the technique used
What is a Probe?
A single stranded DNA molecule or RNA
Labelled with fluorescent molecules
What is the technique where thousands of probes are used simultaneously?
Microarrays
Of what technique is Northern Blotting an adaptation from?
Southern Blotting
What is Northern Blotting?
Analysis of mRNA or DNA within an population
What is a limitation of Northern Blotting?
Only detects one gene at a time and small number of samples
Time consuming and messy
What techniques superseded Northern Blotting?
Quantitative PCR or Microarrays
How does Northern Blotting work?
- Separates DNA or RNA via gel electrophoresis
- transferred to a nylon membrane via mass capillary flow
- covalently bond to the membrane and then hybridised with a labelled probe
- Probe can be visualised
What are microarrays?
An ordered assembly of thousands nucleic acid probes
How do microarrays work?
Probes are fixed to a solid surface
Then sample if interest is hybridised ti the probes
What can microarrays be used for?
Microarrays can be used for gene expressions profiling
Explain the process of gene expression profiling.
- RNA is extracted
- Labelled
- Hybridised to the array and the amount and location of the label is measured
- Results: Quantity of how much and who in the human genome are being expressed
