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MLSCI360 > MOLECULAR TECHNIQUES > Flashcards

MOLECULAR TECHNIQUES Flashcards

1
Q

Explain the principle of Qiagen Symphony

A
  • automated nucleic acid purification
  • lysis buffer containing chaotropic salt (Proteinase K) lyse cells and cellular components are released
  • magnetic silica beads are added; nucleic acids adsorb to silica
  • a magnetic rod transfers beads to a series of reaction vessels; DNA is washed to remove contaminants
  • DNA eluted in 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 and identify the thermocyler program steps used

A
  • agarose gel electrophoresis used to PCR amplicons
  • denaturation, annealing/ hybridization, extension
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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

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16
Q

What can occur if [Mg2+] is too high in endpoint PCR ?

A

DNA replication can be inhibited

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17
Q

Why is a master mix used in endpoint PCR ?

A

Reduces error introduced by repeated pipetting of small volumes

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18
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
  • no enzymes are added
  • DNA template is added outside the clean room
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19
Q

Describe the 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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20
Q

Describe the annealing step in PCR thermocyler program

A

At 50 to 60°C:
- 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)
- takes approx. 30 sec
- primers bind to complementary sequences on ssDNA template

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21
Q

How is Tm of PCR primers calculated ?

A

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

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22
Q

Describe the extension step in PCR thermocyler program

A

At 72°C (dependent on which polymerase used ie. Taq):
- DNA polymerase adds free deoxynucleotides to 3’-end of annealed primer = forms dsDNA

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

  • template DNA is doubled
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23
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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24
Q

What is first strand synthesis ?

A

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

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25
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

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26
Q

What is reverse transcriptase ?

A

It is a RNA-dependant DNA polymerase used in RT-PCR
- 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

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27
Q

Is AMV or MMLV better suited for RT-PCR ? Why ?

A

Moloney Murine Leukemia Virus is better suited bc it has lower endogenous RNAse activity

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28
Q

What do RT buffers contain ?

A

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

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29
Q

Differentiate one-step vs two-step RT-PCR

A

One-step:
- cDNA and PCR 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

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30
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

31
Q

If agarose = 0.5%,

Range of separation of linear DNA = ?

A

1-30 kb

32
Q

If agarose = 1.0%,

Range of separation of linear DNA = ?

A

500 bp - 10 kb

33
Q

If agarose = 1.5%,

Range of separation of linear DNA = ?

A

200 bp - 3 kb

34
Q

If agarose = 2.0%,

Range of separation of linear DNA = ?

A

100 bp - 2.5 kb

35
Q

Identify the 2 most common DNA electrophoresis buffers

A
  1. Tris-acetate-EDTA (TAE)
  2. Tris-borate-EDTA (TBE)
36
Q

Which is more expensive: TAE or TBE ? Why ?

A

TBE is more expensive bc it has a higher buffer capacity

37
Q

Why is loading dye added in DNA gel electrophoresis ?

A
  • add color to DNA samples for loading process
  • dyes migrate towards anode at predictable rates = monitors sufficient migration
  • ficoll, glycerol, or sucrose component increases density of DNA sample and ensures sample sinks down/ is not lost in the buffer
38
Q

Describe the dye used for visualization in DNA gel electrophoresis

A
  • Ethidium bromide is an intercalating dye
  • It binds between complementary bp of dsDNA
  • fluorochrome is excited by UV light and observed as red light
39
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
40
Q

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

A

In DNA gel electrophoresis, DNA samples migrate from cathode (black) to anode (red).

41
Q

Describe capillary gel electrophoresis

A
  • used for DNA sequencing and fragment analysis
  • nucleic acid sample must be labelled with a fluorescent tag by PCR reaction
  • uses long silica tube reinforced with polyimide coating
  • thin walls efficiently dissipates heat = allows HIGH VOLTAGE = FASTER
  • small volumes of sample are introduced via electrokinetic injection
  • DNA migrates through flowable polymer based on mass to charge ratio TOWARDS ANODE
  • fluorescent label is excited by a laser = emitted light is detected
42
Q

Describe Sanger Sequencing

A
  • aka DNA termination sequencing due to random incorporation of fluorescently-labeled ddNTPs
  • ddNTPs 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
43
Q

Describe quantitative PCR

A
  • aka real-time PCR
  • does not require electrophoresis for detecting amplification products
  • qPCR products are fluorescently-labeled, allowing for “real-time” detection
  • intercalation of SYBR green dyes in dsDNA = signal intensity increases with accumulation of products
44
Q

Pros and Cons of qPCR, target-specific probes vs SYBER green

A

Pros:
- better sensitivity
- faster testing
- reduced risk of contamination
- SYBER green is less expensive than target-specific probes

Cons:
- target-specific probes are more expensive
- SYBER green lacks specificity; binds non-specifically to dsDNA = false positive signal

45
Q

Identify 2 most common qPCR probe techniques

A
  1. Hydrolysis probes (5’ nuclease)
  2. Dual hybridization probes
46
Q

Describe qPCR hydrolysis probes

A
  • “Taqman”; 5’-nuclease
  • probe is an oligonucleotide sequence 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 the probe = FLUORESCENT SIGNAL
  • detectable fluorescent signal is proportional to amount of PCR product
47
Q

Describe qPCR dual hybridization probes

A
  • PCR with fluorescence resonance energy transfer (FRET)
  • uses 2 labeled oligonucleotide probes that bind PCR product in close proximity (5 nucleotides) = ENERGY TRANSFER from donor fluorophore to acceptor fluorophore
  • FLUORESCENCE IS DETECTED AT HYBRIDIZATION proportional to PCR product
  • FRET uses 2 primers and 2 probes
48
Q

Analysis of qPCR requires plotting __ against __.

A

Analysis of qPCR requires plotting fluorescent signal intensity against cycle number.

49
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

50
Q

What is the threshold cycle/ crossing point ?

A

The cycle at which the amplification plot crosses the threshold

51
Q

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

A

A true amplification product signal

52
Q

The concentration of DNA in a patient sample can be determined by generating a __.

A

The concentration of DNA in a patient sample can be determined by generating a standard curve.

53
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
54
Q

What does a positive control do ?

A

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

55
Q

What does a negative control do ?

A

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

56
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

57
Q

What does an internal positive control do ?

A
  • tests presence of PCR inhibitors
  • is simultaneously extracted and amplified with DNA template = POSITIVE
58
Q

Identify 2 types of positive internal controls

A
  1. Endogenous; found in specimen ie. actin
  2. Exogenous; added into samples during extraction or before PCR amplification ie. MS2
59
Q

What additional control is required for reverse transcription PCR ?

A
  • REVERSE TRANASCRIPTASE is NOT ADDED = NEGATIVE
  • RNA cannot be transcribed to cDNA
  • presence of PCR product indicates contamination
60
Q

What is transcription mediated amplification used to detect ?

A
  • Chlamydia trachomatis rRNA
  • Neisseria gonorrhoeae rRNA
61
Q

How is the rRNA in transcription mediated amplification obtained ?

A
  • buffer lyses C. trachomatis and N. gonorrhoeae and protects rRNA
  • capture oligo binds complementary rRNA
  • poly-adenosine sequence of capture oligo hybridizes to magnetic particle
  • magnetic field is applied and captured targets are washed
62
Q

Describe the 3 functions of reverse transcriptase in transcription mediated amplification (TMA)

A
  1. RNA-dependent DNA polymerase = converts RNA to cDNA
  2. RNAse H activity = digests the RNA of RNA:cDNA hybrid
  3. DNA dependent DNA polymerase = synthesizes second strand of DNA
63
Q

When do capture oligo T7 and non T7 bind during transcription mediated amplification (TMA) ?

A
  • T7 binds to target rRNA; provides 3’ for reverse transcriptase TO FORM cDNA
  • non T7 binds cDNA; provides 3’ for DNA dependent reverse transcriptase TO FORM dsDNA
64
Q

How are RNA products in transcription mediated amplification (TMA) detected ? What is this called ?

A
  • RNA products are detected by ssDNA probes labeled with acridinium esters = CHEMILUMINESCENCE
65
Q

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

A

~1.8

66
Q

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

A

~2.0

67
Q

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

A

Approximately 2.0 - 2.2

68
Q

Why may A(260/280) be low ?

A
  • residual phenol or other reagents from extraction
  • protein contamination
69
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

70
Q

Why may A(260/230) be low ?

A
  • carbohydrate carryover
  • residual phenol or guanidine HCl from extraction
  • glycogen was used in the precipitation
71
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
72
Q

What is a chaotropic agent ? Give an example.

A
  • molecules that distrupt intramolecular bonds that fold proteins together (hydrogen bonding between water molecules) = denaturant
    Eg. GUANIDINE
73
Q

What substance is most commonly used in enzymatic digestion ?

A

Proteinase K; cleaves adjacent carboxylic groups

MLSCI360 (22 decks)

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