DNA complementarity hybridisation & application Flashcards
What is the basic structure of DNA?
Phosphate, nitrogenous base and pentose sugar (Deoxy ribose)
What is the basic structure of RNA?
Phosphate, nitrogenous base and pentose sugar (ribose)
What is the structure of the nitrogenous base?
Either a single or double ring of nitrogen with polaroid charged groups that result in complimentary base pairing
What is the Structure of the Pentose sugar?
- 5 carbon that form a cyclical structure with oxygen bridge
- Nitrogenous base bind to carbon 1
- Phosphate group binds to carbon 5
- Hydroxyl group at carbon 3
What is the polymer and monomer of DNA?
Polymer: Nucleic acid
Monomer: Nucleotides
What are the four nucleotides of DNA?
Cytosine, Thymine, Guanine, Adenine
What are the four nucleotides of RNA?
Cytosine, Uracil, Guanine, Adenine
Which nucleotides are Purines?
Adenine and Guanine.
-single nitrogen ring means they form 2 hydrogen bonds
Which nucleotides are Pyrimidines?
Cytosine, Thymine, Uracil.
Double nitrogen ring means they for 3 hydrogen bonds
What determines the specificity of pairing
Charged polar groups
What does each complimentary pair contain?
Each pair contains a purine and a pyrimidine
How many hydrogen bonds does each base pair have?
Cytosine and Guanine form 3 hydrogen bonds
Thymine and Adenine form 2 hydrogen bonds
Which base pairing is stronger?
Guanine and Cytosine are stronger as they form a larger number of hydrogen bonds
What bonds are present in a DNA chain?
- Sugar phosphates linked by phosphodiester bonds
- Base stacking: Hydrophobic interactions from the arrangement of the bases means there is internalized strength that excludes water
- Van der Waal forces are individually small but contribute to overall stability
What determines the stability of DNA?
- The free energy of the molecule.
- Energy minimization makes double stand DNA dynamic, but can still be influenced by its molecular environment
What are the 3 forms of DNA?
A, B,Z
What is the most common form of DNA?
B
What provide stability to DNA?
Hydrogen bonds, and base stacking
What allows for bases to be stacked?
Antiparallel strands
What charge is DNA?
Negative due to the phosphate groups, which is useful for gel electrophoresis
What can cause for DNA to be denatured?
Heat+ Chemicals (Urea, or strong alkali)
What is Tm?
Melting temperature of which 50% of the strands are denatured
What is Hyperchromicity?
As the temperature increases the absorbance increases as there is more single stranded DNA which absorbs more UV light then dDNA
How does heat and chemicals denature DNA?
Disrupt the hydrogen bonds
What does denaturation depend on?
the stability of structure determined by sequence of bases
What does the Tm value depend on?
- GC content
- Length of DNA molecule
- Salt concentration
- pH (alkali is a denaturant)
- mismatches (unmatched base pairs)
How does GC content affect Tm?
More GC bonds, more hydrogen bonds, higher Tm
Formula for GC content
%GC=( G+C)/ (G+C+A+T)x100
How does Molecule length affect Tm?
More hydrogen bonds, more stability greater Tm, until beyond 300bp were Tm value plateaus
How does Salt concentration affect Tm value?
Increase [Na] means increase in stability of the structure and a greater Tm
How does salt increase stability?
Salt reduces the specificity of base pairing and go negates the effect of mismatch base pairing thus increasing Tm
How does pH affect Tm?
High pH destabilises the duplex so lowers the Tm
How does alkali affect stability?
Oh ions disrupt H bond and thus reduces stability
What is a mismatch?
A base pair combination that is unable to form hydrogen bonds
How does mismatch affect Tm?
Reduces number of Hydrogen bonds, distorts the structure and adjacent base pairing, thus reducing Tm
What is renaturation?
The reverse of denaturation, formation of the structure favours energy minimization favoured by free energy
What facilitated renaturation?
Slow cooling and neutralisation
What is Hybridisation?
Formation of a duplex structure of two DNA molecules that have been introduced to one another
What is the basis of specificity?
- Complementarity and Tm.
- Perfect matches have a higher Tm and are more thermodynamically stable (This property can be exploited to form complimentary molecules with no mis-matches.
what is stringency?
The manipulation of condition that limit hybridisation between imperfectly matched sequences
What conditions are present under high stringency?
Temperature near Tm and low salt concentration
What nucleic acid based techniques rely on specificity and complementarity?
- Northern blotting
- Southern blotting
- Microarrays
- Dideoxy and Next generation sequencing
- PCR
- Cloning
What is the main aim of hybridisation techniques?
- Identification of the presence of a Nucleic acid containing a specific sequence
- Allows the absolute or relative quantitation of these sequences in a mixture
How do we achieve the main aim of the hybridisation techniques?
using a probe/ primer and allowing for hybridisation to occur between the desired sequence (can be used in diagnostics)
What is a probe?
- A ssDNA or RNA
- Labelled with fluorescent or luminescent molecules
What is Northern blotting?
- An adaptation of southern Blotting
- Analysis of mRNA or DNA from a cell, tissue or organ
- Separation of a gene by gel electrophoresis and using a probe to detect for any hybridisation
What is the problem with Northern and Southern Blotting?
- Limited technique as it only detects one gene at a time and small number of samples
- Time consuming and messy
- Largely superseded by PCR or other techniques
What are microarrays?
- An ordered assembly of thousands of nucleic acid probes
- Probes are fixed to the surface then sample of interest is hybridised to it
- Any hybridisation of the probes identifies the molecules present
- Simultaneously measuring 50,000 different transcripts from a cell, tissue or organ
What are the uses of microarrays?
- Compare gene expressions eg during drug trials (determines the changes in specific gene)
- Help identify genes that relate to a specific disease
- Detect SNPs (used in GWAS)
