Biotech - Nucleic Acid Detection Flashcards
Two components of biosensors?
Molecular Recognition Element
Transducer
Examples of molecular recognition elements?
Enzymes, antibodies, polymers, organelles, aptamers…
Transducer
This reports MRE-analyte interaction
Aptamers
Artificial chemical antbodies generated from randomized nucleic acid library.
Why are aptamers good in biosensing?
They can form tertiary structures and be modifed to include reactive groups.
Examples of detectable signals in biosensors?
Electrochemical, mechanical, piezoelectric or fluoresence
Chromophores
Molecules in a given material absorbing particular wavelengths of visible light, conferring colour on the material
Examples of chromophores?
Ethidium Bromide
Nucleobase analogs(2-aminopurine(A) and isoxanthopterin(G)
How is fluoresence given to nucleic acids?
Chromophores
How are fluorophores bound?
Covalenetly attached to DNA/RNA either end of the NA chain or internal bases
Molecular Beacons
This is an ss bi-labelled fluoresence probe in a stem loop conformaiton
What are the units of MB?
A fluorophore and a quencher
Quencher
These are substances absorbing energy from a fluorophore and re0emitting it as heat.
How do MB work?
Stem loop structure with two confrimations(open and cloed), in closed state, quencher and fluorophore close, open state are seperated
What are the types of MB?
Dual-Fluorophore
Wavelength-Shifting MB
Dual Fluorophore
This are labelled MB assessed at two different wavelengths using FRET
Fluoressence Resonance Energy Transfer
This is a distant dependent physical process where energy is transferred non-radiatively from an excited molecular fluorophore to another fluorophore by dipole-dipole coupling
Why is DFMB good?
Reduce chance of false positve reading from other factors activating duplex(activating when both MB are close)
Wavelength Shifting MB
Labelled with two fluorophore and one quencher, the FP being a FRET pair, where excitation of one induces emissions of the second.
Why does WSMB increase sensitivity?
Stokes shift
Stokes shift
This is the difference between band maxima positions of absorption and emission sepctra of the same electronic transistion
Function of quencher…
Reduce background fluoressence, that being fluoressence prior to target addition
How can MB be used in PCR?
Annealing step when amplicon is present in low amounts, detecting increase as PCR goes on
Ethidium Bromide
Allows visualization of dsDNA in gels
Chemistry of ehitidium bromide?
Emits orange light(0.5mm) due to core phenanthridine when excited
Mechanism of ETHBRO emmission?
Intercalating DNA results from hydrophobic envrionmente between base pairs, the ethidium cation shedding water molecules associated(natural quenchers)
Green Fluoresent Protein
Naturually occuring chromophores having same dipeptide tyrosine-glycine double bond linked rings.
What is the structure of a chromophore?
Double bonds or aromatic rings capable of delocalization of the electrnos.
Why is delocalization in chromophores important?
Creates energy states absorbing specific wavelengt, which can transfer to molecules, exciting electron to higher energy staes
What is an example of a chromophore?
Retinal
Retinal
This converts light entering the eye into electrical signals your optic nerve sends to the brain to create images.
What is the structure of Retinal?
Derive from VitA, having a polyene chain with a cyclic end group forming a schiff base with a lysie residue of rhodopsin protein.
Schiff Base
Compounds characterised by double bond linking carbon and nitrogen atom.
Why can retinal absorb visible spectrum light?
Alternating single and double bonds of the polyene chain.
What structural changes occur in Retinal?
Isomerization of double bond between C11-C12 from cis to trans inducing protein CC, triggering cascade events.
How do photons interact with Retinal?
Induce electron excitation to higher levels, causing CC due to polarized dipole moment, leading to cascade down polyenee then C11-C12 isomerization.
Structure of ETH-B?
Planar, hereocyclic molecule with two N atoms, which intercalate between DNA.
What causes Fluoresence Resonance Electron Transfer?
When two fluorophores come into close proximity and emission spectra of one overlaps with absorption spectra of another.
What is the structure of GFP?
238 AA barrel structure enclosing a central chromophore, where light excitation causes chemical changes and green light emission.
How is Eth-B visualised?
UV transilluminator.
UV Transilluminator
This uses UV radiation to visualise proteins, DNA, RNA and their precursors in GE.
Stokes Shift
This refers to the difference between wavelength of absorbed light and wavelength of emitted light representing energy lost.
How does UV Illuminator work?
Emits UV at 302nm excitaing fluorophore into higher states then reduction to ground state emitting.
What do transducers do in biosensors?
Detect and measure biochemical reactions.
What are the components of the biosensor?
A sensing element interacting with target molecule and transducer converting signal into a measurement.
How does transducer work?
Binding recognition element, like an antibody, inducing CC or hybridizing with NA.
Biological Sensing ELement
This portion recognises and binds the target biomolecule.
What is an example of a biosensor?
Glucose oxidase reacting with blood glucose generating electrical signal by converting glucose to hydrogen peroxide.
Where are quenchers used?
NA detection.
Structure of Molecular Beacons?
Hairpin-shaped probe with a fluorophore and quencher at opposite ends.
What is the purpose of a quencher?
Fluoresence is quenched in the absense of a target sequence.
What does hybridization with a target sequence cause in MB?
CC seperating quencher and fluorophore, resulting in a signal.
What can fluorophores be used for?
Gene expression analysis in ISH and qPCR
