Biotech - Nucleic Acid Detection

Question 1

Two components of biosensors?

Answer 1

Molecular Recognition Element
Transducer

Question 2

Examples of molecular recognition elements?

Answer 2

Enzymes, antibodies, polymers, organelles, aptamers…

Question 3

Transducer

Answer 3

This reports MRE-analyte interaction

Question 4

Aptamers

Answer 4

Artificial chemical antbodies generated from randomized nucleic acid library.

Question 5

Why are aptamers good in biosensing?

Answer 5

They can form tertiary structures and be modifed to include reactive groups.

Question 6

Examples of detectable signals in biosensors?

Answer 6

Electrochemical, mechanical, piezoelectric or fluoresence

Question 7

Chromophores

Answer 7

Molecules in a given material absorbing particular wavelengths of visible light, conferring colour on the material

Question 8

Examples of chromophores?

Answer 8

Ethidium Bromide
Nucleobase analogs(2-aminopurine(A) and isoxanthopterin(G)

Question 9

How is fluoresence given to nucleic acids?

Answer 9

Chromophores

Question 10

How are fluorophores bound?

Answer 10

Covalenetly attached to DNA/RNA either end of the NA chain or internal bases

Question 11

Molecular Beacons

Answer 11

This is an ss bi-labelled fluoresence probe in a stem loop conformaiton

Question 12

What are the units of MB?

Answer 12

A fluorophore and a quencher

Question 13

Quencher

Answer 13

These are substances absorbing energy from a fluorophore and re0emitting it as heat.

Question 14

How do MB work?

Answer 14

Stem loop structure with two confrimations(open and cloed), in closed state, quencher and fluorophore close, open state are seperated

Question 15

What are the types of MB?

Answer 15

Dual-Fluorophore
Wavelength-Shifting MB

Question 16

Dual Fluorophore

Answer 16

This are labelled MB assessed at two different wavelengths using FRET

Question 17

Fluoressence Resonance Energy Transfer

Answer 17

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

Question 18

Why is DFMB good?

Answer 18

Reduce chance of false positve reading from other factors activating duplex(activating when both MB are close)

Question 19

Wavelength Shifting MB

Answer 19

Labelled with two fluorophore and one quencher, the FP being a FRET pair, where excitation of one induces emissions of the second.

Question 20

Why does WSMB increase sensitivity?

Answer 20

Stokes shift

Question 21

Stokes shift

Answer 21

This is the difference between band maxima positions of absorption and emission sepctra of the same electronic transistion

Question 22

Function of quencher…

Answer 22

Reduce background fluoressence, that being fluoressence prior to target addition

Question 23

How can MB be used in PCR?

Answer 23

Annealing step when amplicon is present in low amounts, detecting increase as PCR goes on

Question 24

Ethidium Bromide

Answer 24

Allows visualization of dsDNA in gels

Question 25

Chemistry of ehitidium bromide?

Answer 25

Emits orange light(0.5mm) due to core phenanthridine when excited

Question 26

Mechanism of ETHBRO emmission?

Answer 26

Intercalating DNA results from hydrophobic envrionmente between base pairs, the ethidium cation shedding water molecules associated(natural quenchers)

Question 27

Green Fluoresent Protein

Answer 27

Naturually occuring chromophores having same dipeptide tyrosine-glycine double bond linked rings.

Question 28

What is the structure of a chromophore?

Answer 28

Double bonds or aromatic rings capable of delocalization of the electrnos.

Question 29

Why is delocalization in chromophores important?

Answer 29

Creates energy states absorbing specific wavelengt, which can transfer to molecules, exciting electron to higher energy staes

Question 30

What is an example of a chromophore?

Answer 30

Retinal

Question 31

Retinal

Answer 31

This converts light entering the eye into electrical signals your optic nerve sends to the brain to create images.

Question 32

What is the structure of Retinal?

Answer 32

Derive from VitA, having a polyene chain with a cyclic end group forming a schiff base with a lysie residue of rhodopsin protein.

Question 33

Schiff Base

Answer 33

Compounds characterised by double bond linking carbon and nitrogen atom.

Question 34

Why can retinal absorb visible spectrum light?

Answer 34

Alternating single and double bonds of the polyene chain.

Question 35

What structural changes occur in Retinal?

Answer 35

Isomerization of double bond between C11-C12 from cis to trans inducing protein CC, triggering cascade events.

Question 36

How do photons interact with Retinal?

Answer 36

Induce electron excitation to higher levels, causing CC due to polarized dipole moment, leading to cascade down polyenee then C11-C12 isomerization.

Question 37

Structure of ETH-B?

Answer 37

Planar, hereocyclic molecule with two N atoms, which intercalate between DNA.

Question 38

What causes Fluoresence Resonance Electron Transfer?

Answer 38

When two fluorophores come into close proximity and emission spectra of one overlaps with absorption spectra of another.

Question 39

What is the structure of GFP?

Answer 39

238 AA barrel structure enclosing a central chromophore, where light excitation causes chemical changes and green light emission.

Question 40

How is Eth-B visualised?

Answer 40

UV transilluminator.

Question 41

UV Transilluminator

Answer 41

This uses UV radiation to visualise proteins, DNA, RNA and their precursors in GE.

Question 42

Stokes Shift

Answer 42

This refers to the difference between wavelength of absorbed light and wavelength of emitted light representing energy lost.

Question 43

How does UV Illuminator work?

Answer 43

Emits UV at 302nm excitaing fluorophore into higher states then reduction to ground state emitting.

Question 44

What do transducers do in biosensors?

Answer 44

Detect and measure biochemical reactions.

Question 45

What are the components of the biosensor?

Answer 45

A sensing element interacting with target molecule and transducer converting signal into a measurement.

Question 46

How does transducer work?

Answer 46

Binding recognition element, like an antibody, inducing CC or hybridizing with NA.

Question 47

Biological Sensing ELement

Answer 47

This portion recognises and binds the target biomolecule.

Question 48

What is an example of a biosensor?

Answer 48

Glucose oxidase reacting with blood glucose generating electrical signal by converting glucose to hydrogen peroxide.

Question 49

Where are quenchers used?

Answer 49

NA detection.

Question 50

Structure of Molecular Beacons?

Answer 50

Hairpin-shaped probe with a fluorophore and quencher at opposite ends.

Question 51

What is the purpose of a quencher?

Answer 51

Fluoresence is quenched in the absense of a target sequence.

Question 52

What does hybridization with a target sequence cause in MB?

Answer 52

CC seperating quencher and fluorophore, resulting in a signal.

Question 53

What can fluorophores be used for?

Answer 53

Gene expression analysis in ISH and qPCR