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MLSCI 262 > 360 - Molecular Techniques > Flashcards

360 - Molecular Techniques Flashcards

1
Q

The target n________ _____d for detection will determine the starting material for extraction.

A

nucleic acid

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2
Q

The selection of starting material is dependent on this

A

the target nucleic acid

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3
Q

potential target nucleic acids

A

chromosomal DNA, mitochondrial DNA, circulating fetal DNA, and microbial DNA & RNA

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4
Q

The three basic steps in DNA extraction and purification:

A

cell disruption, separation of DNA from cellular material, and concentration of the DNA

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5
Q

how are DNA cell lysates produced?

A

DNA cell lysates are produced by disrupting cell walls and membranes

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6
Q

Lysis solutions usually contain one or more of the following reagents:

A

chaotropic salts – guanidinium isothiocyanate, urea, and sodium dodecyl sulphate (SDS); detergents – SDS; and an alkaline denaturant – sodium hydroxide

NOTE: these agents denature proteins resulting in the disruption of cell membranes and the release of the cellular contents, including DNA

porteinase K can be added

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7
Q

This is also added to lysis buffers to chelate metal ions which are required as cofactors for DNases

A

EDTA

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8
Q

This can be added to extraction to remove residual RNA

A

RNase

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9
Q

Processing before lysis: tissue biopsies

A

They can be disrupted by homogenization using stainless steel beads in a bead mixer or by grinding liquid nitrogen frozen tissue with a mortar or pestle

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10
Q

T or F. Nucleic acids can be extracted from formalin fixed paraffin embedded tissue

A

T!

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11
Q

Gram-positive bacteria require enzymatic digestion with THIS before cell lysis

A

lysozyme

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12
Q

Fungal cell walls can be disrupted by enzymes or by using c______ t_________ b_____ in a bead mixer

A

carbon tungsten beads

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13
Q

how can cell lysates be further purified to separate DNA from other cellular components?

A

can be cleared by liquid extraction/purification methods like phenol/chloroform or by solid phase isolation like magnetic silica beads

  • liquid extraction/purification methods = cellular components are precipitated by centrifugation or removed by filtration
  • magnetic bead extraction/purification method = binding of the DNA to the bead surface by adsorptive or ionic interactions
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14
Q

Qiagen Symphony

A

an automated nucleic acid purification platform

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15
Q

how does the Qiagen Symphony work?

A
  • samples incubated in a lysis buffer containing a chaotropic salt, and usually, Proteinase K
  • cells lyse and nucleic acids + other cellular components released
  • magnetic silica beads added to lysate, and nucleic acids adsorb to the silica
  • magnetic rod transfers the bound nucleic acid material to a series of reaction vessels where the DNA is washed to remove contaminants
  • DNA eluted in a low salt buffer.
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16
Q

DNA and nucleotides absorb at which wavelength

A

260 nm

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17
Q

how do we calculate nucleic acids in a pure solution?

A

50 μg/mL of double-stranded DNA has an A260 of 1
33 μg/mL of single-stranded DNA has an A260 of 1
40 μg/mL of single-stranded RNA has an A260 of 1

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18
Q

T or F. Both nucleic acid and proteins absorb UV light at 260 and 280 nm.

A

T!

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19
Q

how can protein contamination of nucleic acid solutions be assessed?

A

by analyzing the ratio of absorbance at 260:280 nm

pure dsDNA has an A260/A280 ratio of 1.7 to 1.9

pure single-stranded RNA has an A260/A280 ratio of approximately 2.0

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20
Q

T or F. The A260/A280 ratio is affected by alkaline pH

A

F! acidic pH; slightly alkaline buffer is the preferred solvent

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21
Q

These contaminants can be detected by analyzing the absorbance at 230 nm

A

carbohydrates, phenol and guanidine

The A260/A230 ratio of pure nucleic acid preparation will be greater than the A260/A280 ratio

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22
Q

what is a Nanodrop?

A

assess nucleic acid concentration and purity but
used instead of a spec (Nanodrop is faster & more convenient!)

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23
Q

how does a Nanodrop work?

A
  • small volumes of the sample (1-2 μL) are pipetted onto the end of a fibre optic cable
  • a second cable is placed on the opposite side of the drop of sample to form a small gap
  • a pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear charged couple detect array is used to analyze the light after passing through the sample
  • a computer algorithm calculates the concentration of nucleic acid
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24
Q

endpoint or conventional PCR

A

any PCR protocol which uses agarose gel electrophoresis to visualize the PCR amplicons

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25
Q

This uses fluorescent labels to monitor the production of PCR amplicons as they are synthesized

A

quantitative PCR aka real-time PCR

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26
Q

PCR is the combination of three processes that occur at different temperatures:

A

denaturation, annealing/hybridization, and extension

  • one round of each = cycle
  • standard PCR = 25 to 40 rounds
  • number of cycle rounds required is dependent on the amount of template used
  • thermal cycler
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27
Q

denaturation

A
  • 92 - 95°C the hydrogen bonds of double-stranded DNA break and single-stranded DNA is formed
  • single-stranded DNA serves as the template for a heat resistant DNA polymerase
  • 10 to 30 seconds denaturation is sufficient for most targets; GC rich targets can require longer for complete denaturation
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28
Q

This prevents non-specific DNA replication at lower temperatures

A

hot start Taq DNA polymerase
- enzyme is inactivated by antibodies or chemical modifications until the denaturation temperature is reached
- Hot Start PCR reaction will start with an extended initial denaturation period before cycling

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29
Q

describe annealing

A
  • temperature of the reaction is decreased to around 50 to 60°C for approximately thirty seconds
  • temperature is controlled by the melting point (Tm) of the primers and the ionic concentration of the reaction
  • starting point for an annealing temperature is the primer Tm - 5°C
  • primers bind to their complementary sequences on the single-stranded DNA template
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30
Q

During this step, DNA polymerase to bind the free 3’ end of the newly formed double-stranded DNA and initiates DNA replication

A

annealing

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31
Q

The Tm of a primer can be calculated using the following formula:

A

Tm = 4(G + C) + 2(A + T)

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32
Q

DNA polymerase forms double-stranded DNA by adding complementary free deoxynucleotides to the 3’ end of the annealed primer

A

extension
= DNA has been doubled

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33
Q

The extension temperature of a PCR is dependent on the…

A

type of DNA polymerase used
Taq polymerase active at 72C

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34
Q

General rule for extension time

A

1 minute per 1 kilobase and 30 seconds for targets less than 1 kilobase

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35
Q

template DNA

A

A gene or portion of a gene that serves as the template on which many new copies (amplicons) of the DNA are created

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36
Q

T or F. If you have1 μg of sample genetic DNA it is equivalent to 1 μg of template DNA.

A

F! It is not

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37
Q

the enzyme that replicates DNA

A

DAN polymerase

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38
Q

DNA polymerase

A
  • replicates DNA
  • each cycle of PCR doubles the copies of the target DNA
  • many types
  • Taq polymerase is used for endpt PCR
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39
Q

dNTPs

A
  • mixture of the four dNTPs is used to provide the building blocks for the new DNA strands created by PCR
  • there is an optimal concentration for dNTPs in the reaction mixture; Otherwise, Taq polymerase can be inhibited.
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40
Q

optimal working pH of Taq

A

8.3

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41
Q

what do buffers do in PCR

A
  • maintain pH
  • provide co-factors for enzyme activity (eg. Mg 2+)
  • include salts that aid in the hybridization of primer to template DNA

NOTE: if concentration of salt is too high, polymerase will be inhibited

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42
Q

what are primers?

A

single-stranded oligonucleotides typically between 18 - 30 nucleotides in length with between 40-60% GC content

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43
Q

the sequence of the primer is _______________ to an upstream or downstream region of the target DNA

A

complementary

44
Q

Taq polymerase recognizes the free ___’ end of the bound primer to initiate ______ ___________.

A

3’ end; DNA replication

45
Q

T or F. Two primers are necessary, and they must be complementary to opposite strands of a DNA target sequence and have similar melting temperatures (Tm)

A

T!

46
Q

these ions are a required cofactor for Taq polymerase activity

A

Mg 2+
- If the concentration is too high, replication of DNA will be inhibited
- usual concentration for most PCR is 1.5 - 2 mM

47
Q

purpose of master mixes

A
  • to reduce the error introduced by repeat pipetting of small volumes
  • performed in a clean room; all reagents excluding template are mixed
  • mixed into a single vessel
  • then aliquoted into reaction vessels
  • outside clean room = template is added to rxn vessels
  • master mixes prepare without enzyme
  • stored at -20C
48
Q

reverse transcription PCR/ RT-PCR

A

template is RNA instead of DNA
- RNA converted to cDNA by enzyme reverse transcriptase = first strand synthesis
- cDNA used as template for PCR rxn

49
Q

first strand synthesis process

A

heat -> hybridize -> reverse transcription -> cDNA

50
Q

RT-PCR template

A
  • either total RNA or RNA sub-fractions like mRNA can be used as a template
  • RNA heated to denature secondary structures (eg hairpins)
51
Q

RT-PCR primers

A

can be template specific, random oligomers, or poly dT (for eukaryotic specific mRNA targets)

52
Q

reverse transcriptase for RT-PCR

A
  • most labs = commercial genetically modified reverse transcriptase: RNA-dependent DNA polymerases
  • normally isolated from a variety of retroviral sources such as AMV or MMLV
  • MMLV better suited for RT-PCR bc lower endogenous RNase activity
53
Q

AMV

A

Avian myeloblastosis virus

54
Q

MMLV

A

Moloney murine leukemia virus

55
Q

dNTPs for RT-PCR

A

mixture of four dNTPs used to provide building blocks for the new DNA strands created by PCR

56
Q

RT buffer

A
  • each type of reverse transcriptase has an optimal pH range at which they will react (just like Taq at 8.3)
  • often contain reducing agents to inhibit the RNA template from forming secondary structures
57
Q

One-step vs Two-step RT-PCR

A

one-step = cDNA and PCR synthesized in same rxn vessel; uses target (gene) specific priers; more time consuming; less likely to be contaminated as rxn vessel is never opened

two-step = cDNA is synthesized in one tube and transferred to a second rxn vessel for PCR; advantageous w their multiple (gene) targets and allows for storage of cDNA for later use

58
Q

DNA and RNA molecules have a constant charge to mass ratios; thus, the migration rate of nucleic acids is dependent on the…

A

length of nucleic acid

59
Q

T or F. The size of the pores in the agarose decrease as the concentration of agarose increases

A

T! by altering the concentration of agarose the ability to resolve DNA molecules of different lengths can be enhanced

60
Q

what can happen if the buffering capacity of the electrophoresis buffer is insufficient?

A

the cathode can become alkaline and the anode acidic

61
Q

the two most common DNA buffers

A

Tris-acetate-EDTA (TAE)

Tris-borate-EDTA (TBE); more expensive but higher buffering capacity

62
Q

These are added to DNA samples before loading the gel (pipetting the DNA into the well)

A

gel loading buffers
- mixture of dyes and either FFicoll, glycerol, or sucrose
- dye component usually bromophenol blue and xylene cyanol (adds colour to sample and aids the loading process)

63
Q

T or F. Gel loading dyes migrate towards cathode

A

F! They migrate towards anode at predictable rates which helps us to monitor electrophoresis to ensure DNA sample of interest has migraed sufficiently for optimum results

64
Q

Ficoll purpose

A

increases density of DNA sample to ensure sample sinks in the gel well and is not lost in buffer

65
Q

Nucleic acids can be visualized in an agarose gel using special dyes. This is the most common dye used

A

ethidium bromide
- an intercalating dye; it binds in between the complementary base pairs of double-stranded DNA

66
Q

The fluorochrome of ethidium bromide is typically excited with __ light and is observed as _____ light.

A

UV; red

67
Q

commercial alternatives to ethidium bromide (teratogen, mutagen and toxin)

A

Sybr Green and Gel Red
- also intercalating so also mutagens

68
Q

Molecular weight markers/standards, also known as DNA ladders, are composed of …

A

nucleic acid fragments of known sizes and concentrations

69
Q

what are molecular weight markers used for?

A

to calculate sample molecular weights

to monitor the progress of an electrophoretic run

to estimate the concentration of the sample

NOTE: a marker should be included in every gel

70
Q

a technique used for DNA sequencing and fragment analysis

A

capillary gel electrophoresis

71
Q

before CGE, the sample nucleic acid must be labelled with …

A

fluorescent tag by a PCR reaction

the PCR must be cleaned to remove unincorporated label and reagents

72
Q

explain CGE

A
  • long, thin silica tube with polyimide coating
  • internal diameter = 20 to 180 um; with length = 20 to 80 cm
  • heat dissipated quickly through thin walls = voltage 100X than gel
  • 1-2 μL containing between 1 pg/mL to 1 μg/mL of the labelled template injected into the capillary by electrokinetic injection
  • DNA migrate through a ‘flowable polymer’ to the anode based on their mass to charge ratio
  • as molecules migrate towards the anode, they pass an exposed area of the capillary = fluorescent label on nucleic acid is excited by a laser, and the emitted light is detected
73
Q

injection timing in CGE

A

affects signal intensity and resolution

accurate timing essential for reproducible results

74
Q

Nucleotides are strung together by _____________bonds which join…

A

phosphodiester bonds join 3’ hydroxyl group of one dNTP to a 5’ phosphate of an adjoining dNTP

75
Q

what are ddNTPs?

A

Dideoxyribonucleotides
- lack 3’ hydroxyl groups and are unable to form phosphodiester bond
- if a ddNTP is incorporated during replication, DNA replication terminates

76
Q

In dideoxy sequencing, the target DNA is amplified by…

A

PCR
- mastermix includes both dNTPs and fluorescently labelled ddNTPs
- PCR replication of the target DNA proceeds as normal; however, when a ddNTP is randomly incorporated, replication is terminated
- multiple cycles of amplification = DNA strands of various lengths will be generated, each terminating with a labelled ddNTP

77
Q

what happens once the amplification program has ended?

A
  • the sequencing reactions are cleaned to remove any unincorporated nucleotides and other reagents
  • cleaned sequencing reactions are capillary electrophoresis
  • fluorophore attached to the ddNTP will emit a wavelength of light which is detected after pasing fluorescence excitation light source
  • computer converts the detector signal into relative fluorescent units and assigns a base code (AGCT) to the signal
  • data visualized as an electropherogram
78
Q

quantitative PCR is also known as..

A

real-time PCR

79
Q

quantitative PCR

A
  • real-time
  • does not need detection of amplification products by electrophoresis
  • qPCR products are fluorescently labelled, allowing ‘real time’ detection of amplification products
  • amplicons can be detected by intercalation of fluorescent dyes such as Sybr green OR hybridization of fluorescently labelled probes using an integrated thermal cycler and detector system
80
Q

advantages to qPCR

A
  • better sensitivity
  • faster testing
  • reduced risk of contamination
81
Q

disadvantages of qPCR

A

cost involved in purchasing specialized equipment & probes as well as the difficulties encountered in optimizing the technique

82
Q

T or F. Sybr Green is specific when it binds to dsDNA

A

F! non-specific and it emits a fluorescent signal when excited

83
Q

how can SyBr green binding in qPCR be detected?

A
  • during the extension steps of the amplification program
  • the fluorescent signal intensity increases with the accumulation of amplicon
84
Q

main advantage of Sybr Green (qPCR)

A

less expensive than target-specific probes

85
Q

main disadvantage of Sybr Green (qPCR)

A

lack of specificity
non-specific PCR products can contribute to the signal

86
Q

The most common qPCR probe techniques

A

Hydrolysis and Dual Hybridization probes

NOTE: both probe types are based on FRET

87
Q

hydrolysis probes

A
  • 5’ nuclease
  • an oligonucleotide sequence complementary to the target sequence
  • modified with a fluorophore attached to the 5’ end and a quencher attached to the 3’ end
  • intact probe does NOT fluoresce
  • during extension phase = Taq cleaves the probe separating fluorophore and quencher moiety
  • separation of the fluorophore and the quencher results in detectable fluorescence signal that is proportional to the amount of accumulated PCR product
  • commercial name Taqman
88
Q

dual hybridization probes

A
  • PCR with fluorescence resonance energy transfer (FRET) probes uses two labelled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion
  • two probes bind = fluorophores in close proximity, five nucleotides = energy transfer from a donor fluorophore to an acceptor fluorophore
  • fluorescence is detected during the hybroidization phase of PCR and is proportional to the amount of PCR product
89
Q

the FRET system uses…

A

two primers
two probes

90
Q

Analysis of qPCR requires…

A

plotting fluorescent signal intensity against cycle number

NOTE: signal detected during the initial 3 -15 cycles of PCR is background noise (baseline of fluorescence; subtracted from fluorescence value obtained from amplification products)

91
Q

where is threshold value set in qPCR?

A

set within the linear region of the amplification curve

  • any signal above the threshold value = true amplification product signal
  • threshold cycle (Ct) or crossing point (Cp) is the cycle at which the amplification plot crosses the threshold
92
Q

In qPCR, the concentration of DNA in a patient sample can be determined by generating a…

A

standard curve

93
Q

How is a standard curve for qPCR generated?

A
  • dilution series of known template concentrations are amplified
  • plot template concentrations against their respective crossing points
  • standard curve can then be used to determine the initial starting amount of the target template in the patient sample
94
Q

The CAP requires _______ controls for each qPCR run:

A

three
positive, negative, and no-template controls

ALSO: assays which are based on the presence or absence of PCR products must include an internal positive control

95
Q

qPCR: positive control

A
  • contains target nucleic acid of sufficient quantity and purity for amplification
  • validates that the PCR conditions were sufficient to amplify and detect the target of interest
96
Q

qPCR; negative control

A
  • a nucleic acid sample in which the target sequence is not present
  • this control validates the specificity of the PCR reaction
97
Q

qPCR: no-template control

A
  • contains all the reagents necessary for PCR except for the target nucleic acid; water is substituted to make up the volume difference
  • detection of PCR products indicates nucleic acid contamination
98
Q

for PCR to be valid…

A

positive control pos and both negative and NTC = neg

99
Q

for PCR to be valid…

A

positive control pos and both negative and NTC = neg

100
Q

what is an internal positive control?

A

used to test for the presence of PCR inhibitors
- the internal control is simultaneously extracted and amplified with the template nucleic acid
- internal positive controls (IC) can be endogenous template normally found in the specimen (e.g. ß-actin, actin, and glyceraldehyde-3-phosphate dehydrogenase)
- exogenous controls are spiked into samples either during nucleic acid extraction or before PCR amplification (e.g. MS2).

101
Q

PCR rxn with negative internal pos control and negative target

A

A PCR reaction with both a negative internal positive control and a negative target is suggestive of inhibition. The result of this individual reaction is invalid

102
Q

RT-PCR control

A

additional control required for reverse transcription PCR
- set up at transcription step of rxn
- reverse transcriptase enzyme omitted from rxn
- absence of enzyme, RNA cannot be transcribe to DNA = should be NO DNA detected at end of PCR phase of assay
- presence of PCR product = presence of contaminating DNA in RNA template

103
Q

Transcription mediated amplification (TMA)

A

utilized for the detection of Chlamydia trachomatis and Neiserria gonorrheae on the Panther instrument
- assay detects rRNA
- prokaryotic organisms lysed in a buffer (also protects rRNA)
- rRNA is bound by a capture oligo
- oligos are complementary to the target rRNA
- capture oligos also have a poly-adenosine sequence which hybridizes to a magnetic particle that has complementary sequence
- after capture of rRNA targets, a magnetic field is applied and the magnetic particles (with and without hybridized rRNA) are immobilized and washed

104
Q

T7 and TMA process

A
  • oligo (TMA)
  • hybridizes to target rRNA
  • 5’ end of the T7 oligo encodes the T7 promoter sequence and the 3’ end of the oligo binds the target RNA and serves as a primer for a reverse transcriptase
  • after creation of cDNA from the RNA target, the RNase H activity of the reverse transcriptase digests the RNA of the RNA:DNA hybrid molecule
  • second oligo, non T7, binds the cDNA and the DNA dependent DNA polymerase activity of the reverse transcriptase synthesis the second strand of DNA
  • T7 RNA polymerse binds to the now incorporated T7 promoter and synthesises multiple copies of RNA, identical to the target
  • newly synthesised RNA is then amplified by the reverse transcriptase and the T7 and non-T7 oligos
    = transcribed by T7 RNA polymerase
  • RNA products are detected by single stranded DNA probes labeled with acridium esters (chemiluminescence)
105
Q

three functions of reverse transcriptase in TMA

A
  • RNA-dependent DNA polymerases
  • RNaseH activity
  • DNA-dependent DNA polymerase activity

MLSCI 262 (21 decks)

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