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MOLECULAR BIOLOGY - PREFINAL > NUCLEIC ACID AMPLIFICATION > Flashcards

NUCLEIC ACID AMPLIFICATION Flashcards

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1
Q
  • Most common nucleic acid amplification method
A

POLYMERASE CHAIN REACTION

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2
Q

What the 3 categories in Nucleic acid amplification?

A

TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION

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2
Q

Advantages of PCR:

A

User-friendly, more automated, and more amenable to use

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3
Q

o Copy of the target DNA
o Products of PCR
o Multiple copies produced

A

AMPLICONS

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4
Q

Discovered in 1983 by Kary Mullis
o Escherichia coli plasmid pBR322 (1st to be amplified)
o Method was called “polymerase-catalyzed chain reaction”

A

PCR

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4
Q
  • 1st to be amplified
A

Escherichia coli plasmid pBR322

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5
Q
  • Application of heat to separate dsDNA (template) into 2 single strands
  • Denatured by heat
    ▪ Temperature is enough to break hydrogen bonds
A

DENATURING (90-96C)

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6
Q

▪ Temperature is enough to break hydrogen bonds
▪ Application of heat to separate dsDNA (template) into 2 single
strands

A

DENATURING

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7
Q

DNA TEMPLATE is derived from?

A

▪ Patient’s genomic DNA
▪ Patient’s mtDNA
▪ DNA from viruses, bacteria, parasites

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8
Q

90-60 CELSIUS

A

DENATURING

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9
Q

50-70 CELSIUS

A

ANNEALING

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10
Q

Binding of primers to ssDNA template by hybridization
▪ Most crucial step
▪ 50oC to 70oC, 20sec – 90 sec

A

ANNEALING

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10
Q

Primers are also known as?

A

OLIGODEOXYNUCLEOTIDES

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11
Q

This step determine the specificity of PCR.

A

ANNEALING

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12
Q

-More specific & hybridize at slower rate

A

LONG PRIMERS

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13
Q
  • Less specific & hybridize at faster rate
A

SHORT PRIMERS

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14
Q

2 TYPES OF PRIMERS:

A

FORWARD , REVERSE

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15
Q
  • Artifact composing of 2 primers binding onto each other
  • Double the size of the utilized primer
  • Occur where there are 3 or more complementary happens at 3’ end of primers
A

PRIMER DIMERS

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15
Q

(68oC - 75oC)

A

ELONGATION/EXTENSION

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15
Q
  • Aberrant primer binding
  • Primers bind to unintended sequences
A

MISPRIMING

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15
Q

WHAT ARE THE FACTORS THAT MAY AFFECT THE EXACT Tm

A
  1. Reactive conditions
  2. Salt concentrations
  3. Template conditions
  4. Secondary structure (internal folding & hybridization between DNA
    strands)
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16
Q

DNA polymerase helps here but should be thermostable
o Obtained from bacteria (thermophilic bacteria)
▪ DNA synthesis occurs
▪ Addition of nucleotides (primer extension)

A

EXTENSION/ELONGATION

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17
Q

10sec – 60sec

A

EXTENSION/ELONGATION

18
Q

20-60 secs

A

DENATURING

19
Q

20-90 secs

A

APPEALING

20
Q
  • Enzyme for the addition of nucleotides to the hybridized primer
  • High temperature in denaturation step will inactivate the enzyme
A

DNA POLYMERASE

21
Q

TYPES OF DNA POLYMERASE
➢ Mainly utilized in PCR
➢ Obtained from Thermus aquaticus
➢ Thermostable
➢ By Randall Saiki
➢ Commonly used

A

Taq POLYMERASE

22
Q

Taq POLYMERASE is obtained from?
- THERMOSTABLE

A

THERMUS AQUATICUS

22
Q

TYPES OF DNA POLYMERASE
➢ Utilized in reverse-transcriptase PCR (RNA)
➢ Obtained from Thermus thermophilus

A

Tth POLYMERASE

22
Q

Tth POLYMERASE is obtained from?

A

Thermus thermophilus

23
Q

TYPES OF DNA POLYMERASE
➢ Tli polymerase
➢ Allows Taq or Tth to generate large products
(30,000 bases)
➢ Obtained from Thermococcus litoralis

A

VENT POLYMERASE

23
Q

VENT POLYMERASE is obtained from?

A

Thermococcus Litoralis

24
Q
  • Incorporates ddNTPS
  • Chain termination sequencing
A

THERMOSEQUENASE AND 17 SEQUENASE

24
Q

MODIFIED TAQ VERSIONS:
- 2x the half-life of Taq in high temperature
- Broader MgCl2 concentrations
- For allele specific PCR and Amplification of GC content

A

STOFFEL FRAGMENT

25
Q

Why is vigorous agitation of enzyme is not recommended?

A
  • may alter the structures of enzyme and will denature irreversibly and air bubbles may damage
    it
25
Q

PCR REAGENTS MASTER MIX:

A

buffer + DNA template + DNA polymerase + dNTPs (deoxyribonucleotide triphosphates) + KCl + MgCl + Primers

26
Q
  • It Directs DNA synthesis to the desired region
A

PRIMERS

26
Q
  • Divalent cation, required by the enzyme
A

1.5 mM MgCl2

27
Q

Building blocks that extend the primers

A

dATP, dCTP, dGTP, dTTP (dNTPs)

27
Q

Buffer to maintain optimal pH (8-9.5) for the enzyme reaction

A

Tris, pH 8.4

28
Q
  • Monovalent cation (salt), for optimal hybridization of primers to template
A

KCl

29
Q
  • A mixture of 4 equimolar deoxynucleotide triphosphates
  • 0.1-0.5 mM concentration of each
  • Concentration and purity may affect PCR results
  • High concentrations (10-100 mM) are recommended for storage to
    present breaking down to dNDP & dMNP = inhibit PCR
  • dNDP(diphosphate) & dMNP (monophosphate) can also be a result
    of poor manufacturing and contamination of heavy metals
A

DEOXYRIBONUCLEOTIDE BASES

30
Q
  • provide optimal conditions for enzyme activity (pH 8-9.5)
  • increased salt concentration may denature long DNAs slower than short DNAs
A

PCR BUFFERS

31
Q

▪ Lower denaturing temperature

A

Formamide (1% - 10%)

32
Q

PCR EQUIPMENT
o 1st PCR system conducted
o Replaced by automated methods and thermostable
enzymes

A

WATER BATHS & HEAT BLOCKS

33
Q

PCR EQUIPMENT
- For rapid and automatically ramp to the required temperatures

A

THERMAL CYCLERS/THERMOCYCLERS

34
Q

PCR products can be analyzed by:

A

o Gel/capillary electrophoresis
o Nucleic acid analysis
o Other molecular biology tests

35
Q

PCR CONTROLS
- AKA contamination control / reagent blank
- Lacks DNA template

A

NEGATIVE CONTROLS

36
Q

PCR CONTRTOLS
- Ensure
o Enzyme is active
o Buffer is optimal
o Primers are priming right sequences
o Thermal cycles are cycling properly

A

POSITIVE CONTROLS

37
Q

PCR CONTROLS
- With a DNA template that has no target sequence
- There should be no annealing

A

NEGATIVE TEMPLATE CONTROLS

37
Q

PCR CONTROLS
- Contain a primer binding site
- Determine false negative (amplification failure)

A

AMPLIFICATION CONTROLS

38
Q
  • Common in open tube methods
  • Most common sources:
    o Products of previous amplifications (may aerosolized if
    uncapped)
A

PCR CONTAMINATION

39
Q

WHAT IS THE MOST COMMON SOURCE OF PCR CONTAMINATION?

A

Products of previous amplifications (may aerosolized if
uncapped

40
Q

CONTROL METHODS
o 10% bleach (7 mM sodium hypochlorite)
* For decontamination of workspaces
o Enzymatic method
* UNG for previous amplifications (enzyme: uracil-Nglycosylase)
o Wipe tests
* To determine concentration

A

CHEMICAL CONTAMINATION CONTROL

41
Q

CONTROL METHODS
o Separate pre- & post-PCR areas
o Positive airflow, airlocks
o Equipment, PPE, & reagents should be dedicated to either
pre- or post-PCR
o UV for decontamination
o Psoralens
o Attach T, U, C, in DNA under UV (prevent
denaturation & amplification)

A

PHYSICAL CONTAMINATION CONTROL

42
Q

CONTROL METHODS
* For decontamination of workspaces

A

10% bleach (7 mM sodium hypochlorite)

43
Q

CONTROL METHODS
* To determine concentration

A

WIPE TEST

MOLECULAR BIOLOGY - PREFINAL (3 decks)

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