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CSMLS > CHEM Molecular Techniques > Flashcards

CHEM Molecular Techniques Flashcards

1
Q

Explain the principle of Qiagen Symphony

A
  • AUTOMATED nucleic acid EXTRACTION
  • chaotropic salt (Proteinase K) lyse cells = cellular components are released
  • MAGNETIC SILICA BEADS are used to ADSORB NUCLEIC ACIDS
  • a magnetic rod transfers beads to a series of reaction vessels; DNA is washed to remove contaminants
  • DNA eluted by a low salt buffer
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2
Q

Concentration of dsDNA when A(260) = 1.000

A

50 µg/mL

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3
Q

Concentration of ssDNA when A(260) = 1.000

A

33 µg/mL

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4
Q

Concentration of ssRNA when A(260) = 1.000

A

40 µg/mL

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5
Q

If a solution of dsDNA has an A(260) of 0.81, what is the concentration of dsDNA ?

A

(50 µg/mL / 1.000) = ( x / 0.810), so x = 40.5 µg/mL

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6
Q

List the reagents used in endpoint PCR

A
  • DNA polymerase
  • dNTPs
  • PCR buffer
  • Primers
  • MgCl2

NOTE: Master Mixes can include all reagents to reduce repetitive measurements

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7
Q

What is the function of Taq polymerase ?

A
  • used in endpoint PCR
  • replicates template DNA
  • recognizes 3’ end of primer
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8
Q

What can occur if [dNTPs] is not optimal ?

A

Taq polymerase can be inhibited

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9
Q

What is the function of PCR buffers ?

A
  • maintains optimal pH = 8.3 for Taq polymerase
  • provides cofactors (Mg2+) for enzymatic activity
  • provides salt for annealing/ hybridization
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10
Q

What can occur if [salt] is too high in endpoint PCR ?

A

Taq polymerase can be inhibited

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11
Q

Which formula is used to calculate the reagent amounts required for endpoint PCR ?

A

C1V1 = C2V2

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12
Q

What is endpoint-PCR ? ID the 3 thermocycles

A

Agarose gel electrophoresis used to amplify nucleic acids:
1. Denaturation (95°C)
2. Annealing/ hybridization (60°C)
3. Elongation (65°C)

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13
Q

Describe the unidirectional workflow requirements of a molecular lab

A
  • amplicons produced in the post-room cannot re-enter the pre-room
  • this also applies to PPE and equipment
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14
Q

How many primers are necessary for endpoint PCR ?

A
  • two primers that are complementary to opposite strands of DNA target sequence
  • must have similar melting temperature (Tm)
  • provides 3’ end for Taq polymerase
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15
Q

What is the function of Mg2+ in endpoint PCR ?

A

A required cofactor for Taq polymerase

NOTE: but if [Mg2+] is too high, DNA replication can be inhibited

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16
Q

Why is a master mix used in endpoint PCR ?

A

Reduces error introduced by repeated pipetting of small volumes

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17
Q

How is the master mix prepared in the “clean room” ? What is added outside of the clean room ?

A
  • all reagents EXCEPT the template is added into a single vessel in the clean room = master mix
  • DNA template is added outside the clean room
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18
Q

Denaturation step in the PCR thermocyler program

A

At 92 - 95°C:
- dsDNA breaks to form ssDNA
- 10 to 30 sec, but GC-rich targets can take longer
- hot start Taq DNA polymerase prevents non-specific DNA replication at lower temp. until desired denaturation temp. is reached

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19
Q

Annealing step in PCR thermocyler program

A

At 50 to 60°C:
- primers bind to complementary sequences on ssDNA template
- takes ~30 sec
- specific temp is determined by melting point (Tm) of primer and ionic concentration of rxn = (Tm - 5°C)
Tm = 4(G+C) + 2(A+T)

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20
Q

How is Tm of PCR primers calculated ?

A

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

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21
Q

Extension step in PCR thermocyler program

A

At 72°C:
- template DNA is doubled
- DNA polymerase adds free dNTDs to 3’-end of annealed primer = forms dsDNA

a). one min/ 1kb
b). 30 sec for targets < 1kb

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22
Q

What is RT-PCR ?

A
  • reverse transcription PCR uses an RNA template instead of DNA
  • RNA is converted to cDNA (complementary DNA) via reverse transcriptase = first strand synthesis
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23
Q

What is first strand synthesis ?

A

When RNA is converted to complementary DNA (cDNA) via reverse transcriptase in RT-PCR

24
Q

Can mRNA be used as the template in RT-PCR ?

A

yes, RNA sub-fractions like mRNA can be used instead of total RNA

25
Q

What is reverse transcriptase ?

A
  • an RNA-dependant DNA polymerase used in RT-PCR

NOTE: normally isolated from Avian Myeloblastosis Virus (AMV) or Maloney Murine Leukemia Virus (MMLV)*

*MMLV is better for RT-PCR bc it has lower endogenous RNAse activity

26
Q

What do RT buffers contain ?

A

Reverse transcription buffers:
- REDUCING AGENTS to inhibit RNA template from forming secondary structures with itself
- cofactors for enzyme activity (Mg2+)
- salts that aid in hybridization of primer to template cDNA

27
Q

Differentiate one-step vs two-step RT-PCR

A

One-step:
- cDNA and PCR products are synthesized in the same reaction vessel
- uses target (gene)-specific primers
- faster bc less pipetting steps
- lower possibility of contamination bc reaction vessel is never opened

Two-step:
- cDNA is synthesized in one tube and transferred to a second reaction vessel for PCR
- advantageous when there are multiple (gene) targets
- allows storage of cDNA for later use

28
Q

The size of the pores in agarose __ as the concentration of agarose __

A

The size of the pores in agarose DECREASES as the concentration of agarose INCREASES

29
Q

Which is more expensive: TAE or TBE ? Why ?

A

Tris-borate-EDTA (TBE) is more expensive bc it has a higher buffer capacity

TAE =Tris-acetate-EDTA

30
Q

Why is loading dye added in DNA gel electrophoresis ?

A
  • adds color to DNA samples to facilitate loading process
  • dyes migrate towards anode at predictable rates = monitors sufficient migration
  • increases density of DNA/ensures sample sinks down into well
31
Q

Describe the dye used for visualization in DNA gel electrophoresis

A
  • ETHIDIUM BROMIDE is an intercalating dye
  • binds between complementary bp of dsDNA
  • fluorochrome is excited by UV light and observed as red light
32
Q

Why are DNA ladders used ?

A
  • used as a reference to calculate sample molecular weights
  • monitors progress of electrophoresis run
  • estimates concentration of sample
33
Q

In DNA gel electrophoresis, DNA samples migrate from __ to __.

A

In DNA gel electrophoresis, DNA samples migrate from CATHODE (black) to ANODE (red).

34
Q

Describe DNA capillary gel electrophoresis

A
  • nucleic acid sample is FLUORESCENTALLY-labelled
  • uses long silica tube reinforced with polyimide coating
  • thin walls efficiently dissipates heat = allows HIGH VOLTAGE = FASTER
  • small sample volumes of sample inserted via electrokinetic injection
  • DNA migrates in flowable polymer based on mass:charge from cathode TO ANODE
  • fluorescent label is excited by a laser = emitted light is detected
35
Q

Describe Sanger Sequencing

A
  • “DNA termination sequencing”
  • random incorporation of fluorescently-labeled ddNTPs
  • ddnNTPs lack 3’ hydroxyl (OH) group = DNA replication terminates
  • DNA strands of various lengths are formed (each with a terminal ddNTP)
  • capillary electrophoresis will detect the emitted light from fluorophores attached to terminal ddNTPs
  • each signal is assigned a base code (GCAT) = visualized as an electropherogram
36
Q

Describe quantitative PCR

A
  • “Real-time PCR”
  • NO electrophoresis to detect amplification products
  • qPCR products are fluorescently-labeled (SYBR green) = “real-time” detection
  • intercalation of SYBR green dyes in dsDNA = signal intensity increases with accumulation of products
37
Q

Pros and Cons of using SYBR green in qPCR ?

A

Pros:
- faster testing
- SYBER green is less expensive than target-specific probes

Con: SYBER green lacks specificity; binds non-specifically to dsDNA = false positive signal

38
Q

Identify 2 most common qPCR probe techniques

A
  1. Hydrolysis probes (5’ nuclease)
  2. Dual hybridization probes
39
Q

Describe qPCR hydrolysis probes

A
  • “Taqman” is a 5’-nuclease
  • probes are complimentary to target
  • modified with a 5’-fluorophore and 3’-quencher (complex does not fluoresce)
  • during EXTENSION phase of qPCR = Taq DNA polymerase 5’-3’ exonuclease activity CLEAVES PROBE = FLUORESCENT SIGNAL
  • detectable fluorescent signal is proportional to amount of PCR product
40
Q

Describe qPCR dual hybridization probes. When is a signal detected ?

A
  • PCR with fluorescence resonance energy transfer (FRET)
  • uses 2 labeled probes that bind PCR product when in close proximity = ENERGY TRANSFER from donor fluorophore to acceptor fluorophore
  • during HYBRIDIZATION phase of qPCR = FLUORESCENCE detected = is proportional to PCR product
  • FRET uses 2 primers and 2 probes
41
Q

Analysis of qPCR requires plotting __ against __.

A

Analysis of qPCR requires plotting FLUORESCENT SIGNAL INTENSITY against CYCLE NUMBER.

42
Q

T or F: the signal detected during the initial 3-15 cycles of PCR is background noise

A

TRUE; the signal detected during the initial 3-15 cycles of PCR is background noise

43
Q

What do values above the threshold represent in a qPCR analysis plot ?

A

A true amplification product signal

44
Q

Identify the 4 controls required in a PCR run

A
  1. Positive control = POSITIVE
  2. Negative control = NEGATIVE
  3. No-template control = NEGATIVE
  4. Internal positive control = POSITIVE
45
Q

What does a positive control in PCR do ?

A

Validates that PCR conditions were sufficient to amplify and detect the target = POSITIVE amplification of target

46
Q

What does a negative control do ?

A

Nucleic acid sample does NOT contain target = NEGATIVE for target but amplification of DNA works

47
Q

What does a no-template control do ?

A

The NTC contains all reagents EXCEPT for a DNA template = should be NEGATIVE; detection of PCR products indicates contamination

48
Q

What does an internal positive control do ?

A
  • tests presence of PCR inhibitors
  • is simultaneously extracted and amplified with DNA template = POSITIVE
49
Q

What additional control is required for reverse transcription PCR ?

A
  • RTase is NOT ADDED = NEGATIVE
  • RNA cannot be transcribed to cDNA
  • presence of PCR product indicates contamination
50
Q

What A(260/280) is considered “pure” for DNA ?

A

~1.8

51
Q

What A(260/280) ratio is considered “pure” for RNA ?

A

~2.0

52
Q

What A(260/230) ratio is considered “pure” for both DNA and RNA ?

A

Approximately 2.0 - 2.2

53
Q

Why may A(260/280) be low ?

A
  • residual phenol or other reagents from extraction
  • protein contamination
54
Q

T or F: High A(260/280) ratios are indicative of purity problem

A

FALSE; high A(260/280) ratios are NOT INDICATIVE OF PURITY PROBLEMS
- can indicate issue with spectrophotometer

55
Q

Why may A(260/280) be high ?

A
  • not associated with contamination; MORESO PROCEDURAL ERRORS
  • using an inappropriate blanking solution ie. not similar ionic strength as sample solution
56
Q

What is a chaotropic agent ? Give an example.

A
  • molecules that distrupt hydrogen bonding between water molecules (intramolecular bonds that fold proteins together) = DENATURANT
    Eg. GUANIDINE
57
Q

What substance is most commonly used in enzymatic digestion (DNA extraction)?

A

Proteinase K; cleaves adjacent carboxylic groups

CSMLS (46 decks)

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