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Biology - Biotechnology > Biotech - Nucleic Acid Detection > Flashcards

Biotech - Nucleic Acid Detection Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Two components of biosensors?

A

Molecular Recognition Element
Transducer

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2
Q

Examples of molecular recognition elements?

A

Enzymes, antibodies, polymers, organelles, aptamers…

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3
Q

Transducer

A

This reports MRE-analyte interaction

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4
Q

Aptamers

A

Artificial chemical antbodies generated from randomized nucleic acid library.

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5
Q

Why are aptamers good in biosensing?

A

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

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6
Q

Examples of detectable signals in biosensors?

A

Electrochemical, mechanical, piezoelectric or fluoresence

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7
Q

Chromophores

A

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

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8
Q

Examples of chromophores?

A

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

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9
Q

How is fluoresence given to nucleic acids?

A

Chromophores

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10
Q

How are fluorophores bound?

A

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

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11
Q

Molecular Beacons

A

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

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12
Q

What are the units of MB?

A

A fluorophore and a quencher

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13
Q

Quencher

A

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

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14
Q

How do MB work?

A

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

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15
Q

What are the types of MB?

A

Dual-Fluorophore
Wavelength-Shifting MB

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16
Q

Dual Fluorophore

A

This are labelled MB assessed at two different wavelengths using FRET

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17
Q

Fluoressence Resonance Energy Transfer

A

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

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18
Q

Why is DFMB good?

A

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

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19
Q

Wavelength Shifting MB

A

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

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20
Q

Why does WSMB increase sensitivity?

A

Stokes shift

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21
Q

Stokes shift

A

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

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22
Q

Function of quencher…

A

Reduce background fluoressence, that being fluoressence prior to target addition

23
Q

How can MB be used in PCR?

A

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

24
Q

Ethidium Bromide

A

Allows visualization of dsDNA in gels

25
Q

Chemistry of ehitidium bromide?

A

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

26
Q

Mechanism of ETHBRO emmission?

A

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

27
Q

Green Fluoresent Protein

A

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

28
Q

What is the structure of a chromophore?

A

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

29
Q

Why is delocalization in chromophores important?

A

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

30
Q

What is an example of a chromophore?

A

Retinal

31
Q

Retinal

A

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

32
Q

What is the structure of Retinal?

A

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

33
Q

Schiff Base

A

Compounds characterised by double bond linking carbon and nitrogen atom.

34
Q

Why can retinal absorb visible spectrum light?

A

Alternating single and double bonds of the polyene chain.

35
Q

What structural changes occur in Retinal?

A

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

36
Q

How do photons interact with Retinal?

A

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

37
Q

Structure of ETH-B?

A

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

38
Q

What causes Fluoresence Resonance Electron Transfer?

A

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

39
Q

What is the structure of GFP?

A

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

40
Q

How is Eth-B visualised?

A

UV transilluminator.

41
Q

UV Transilluminator

A

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

42
Q

Stokes Shift

A

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

43
Q

How does UV Illuminator work?

A

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

44
Q

What do transducers do in biosensors?

A

Detect and measure biochemical reactions.

45
Q

What are the components of the biosensor?

A

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

46
Q

How does transducer work?

A

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

47
Q

Biological Sensing ELement

A

This portion recognises and binds the target biomolecule.

48
Q

What is an example of a biosensor?

A

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

49
Q

Where are quenchers used?

A

NA detection.

50
Q

Structure of Molecular Beacons?

A

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

51
Q

What is the purpose of a quencher?

A

Fluoresence is quenched in the absense of a target sequence.

52
Q

What does hybridization with a target sequence cause in MB?

A

CC seperating quencher and fluorophore, resulting in a signal.

53
Q

What can fluorophores be used for?

A

Gene expression analysis in ISH and qPCR

Biology - Biotechnology (7 decks)

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