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MOLECULAR BIOLOGY > 4. NUCLEIC ACID AMPLIFICATION - LAB > Flashcards

4. NUCLEIC ACID AMPLIFICATION - LAB Flashcards

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1
Q

make many copies of target DNA

A

NUCLEIC ACID AMPLIFICATION

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2
Q

Three Categories of Nucleic acid amplification

A
    1. Target amplification systems
    1. Probe amplification system
    1. Signal amplification system
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3
Q

category of NAA that targets nucleic acid based (PCR)

A

Target amplification systems

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4
Q

category of NAA that is probe specific for target sequence

A

Probe amplification system

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5
Q

is a short region of double-stranded DNA

A

TARGET

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6
Q

Involves making many copies of a specific DNA sequence

A

TARGET AMPLIFICATION

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7
Q

is the first and prototypical method for amplifying target nucleic acid

A

PCR

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8
Q

PCR Discovered by

A

Kary Mullis in 1983 (Nobel Prize Awardee)

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9
Q

Involves making copies of a target sequence to such a level (in the millions of copies) that they can be detected in vitro (be visualized on an agar plate).

A

Target Amplification

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10
Q

PCR

The first successful amplification was a short fragment of the

A

Escherichia coli plasmid (pBR322

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11
Q

The first practical application is the amplification of amino acid
sequence of beta- globin and analysis for diagnosis of patients with

A

sickle cell anemia

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12
Q

PCR products

A

Amplicons

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13
Q

binds beside the target sequence

A

Primer

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14
Q

binds directly to the target sequence

A

Probe

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15
Q

AMPIFICATION PROGRAM Components:

A

DNA template
Short oligonucleotide primers
Nucleotides
Polymerase
Buffers

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16
Q

Short oligonucleotide primers types

A

FORWARD AND REVERSE

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17
Q

useful in addition of dNTPs to growing strands

A

Polymerase

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18
Q

maintain and stabilize condition→ pH (stabilize enzyme) and Mg, NaCl for temp

A

Buffers

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19
Q

ELEMENTS OF PCR CYCLE

A
    1. Denaturation
    1. Annealing
    1. Extension/Elongation
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20
Q

Denaturation Temperature (°C)
Time (sec)

A

90-96 temp
20-60 time

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21
Q

Annealing Temperature (°C)
Time (sec)

A

50-70 temp
20-90 time

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22
Q

Extension/Elongation Temperature (°C)
Time (sec)

A

68-75 temp
10-60 time

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23
Q

always bind to the minus strand or anti-sense strand (TEMPLATESTRAND)

A

Forward Primer

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24
Q

Forward Primer formation of amplicons

A

Forward from 3’ to 5

25
Q

binds to plus strand or sense strand (NON-TEMPLATE)

A

Reverse Primer

26
Q

Reverse Primer formation of amplicons

A

3’ to 5’ pero baliktad kasi yung strand kaya reverse ok????

27
Q

Tm formula for annealing

A

Tm = 4x #GC + 2x#AT

28
Q

attachment of primer away from the target sequence

A

Mispriming

29
Q

Binding of primers onto each other through short (2- to 3-base)

homologies at their 3’ ends and the copying of each primer sequence

A

Primer Dimer

30
Q

DNA TEMPLATE Sources:

A
  • Patient’s genomic or mitochondrial DNA
  • Viruses
  • Bacteria
  • Fungi
  • Parasites
31
Q

DNA POLYMERASES used in PCR

A

Taq polymerase
Tth polymerase
Vent polymerase

32
Q
  • most known polymerase for PCR
  • Thermostable enzyme (stable even at high temp)
A

Taq polymerase

33
Q

Taq polymerase Isolated from the thermophilic bacterium

A

Thermus aquaticus

34
Q

Tth polymerase From

A

Thermus thermophilus

35
Q

Has reverse-transcriptase activity, so it can be used in reverse transcriptase PCR

A

Tth polymerase

36
Q

Allows Taq or Tth polymerase to generate large products over 30,000 bases in length.
- exonuclease, removing non-complementary and unnecessary bases, exons especially for very long DNA bases

A

Vent polymerase

37
Q

Proofreading enzyme

A

Vent polymerase

38
Q
  • Lacking the 289 N-terminal amino acids of Taq polymerase

recommended for allele- - specific PCR and for amplification of regions with high GC content

A

Stoffel fragment

39
Q

Modified Polymerase Enzymes

A

Stoffel fragment

40
Q

Accessory components

A
  • Bovine serum albumin (10 to 100 µg/mL)
  • Dithiothreitol (0.01 mM)
  • Formamide (1% to 10%)
  • Chaotropic agents (detergents) such as Triton X-100, glycerol, and dimethyl sulfoxide
41
Q

Accessory components for the stabilization of enzyme

A
  • Bovine serum albumin (10 to 100 µg/mL)
42
Q

Accessory components to enhance the enzyme activity

A
  • Dithiothreitol (0.01 mM)
43
Q

Accessory components lower denaturation temperature to reduce secondary structure

A

Formamide (1% to 10%)

44
Q

Accessory components for stability of enzyme and reduce secondary structure

A

Chaotropic agents (detergents) such as Triton X-100, glycerol, and dimethyl sulfoxide

45
Q

Designed to rapidly and automatically ramp (change) to the required incubation temperatures, holding at each one for designated periods
- Designed with heated lids that eliminated the requirement for vapor barriers

A

THERMAL CYCLER/THERMOCYCLERS

46
Q

(Quantitative)systems are equipped with fluorescent detectors to measure the PCR product as the reaction proceeds

A

Real-time PCR

47
Q

Qualitative presence or absence of amplicons

A

Conventional PCR- End point Analysis

48
Q

CONTROLS FOR PCR

A

Positive Control
Negative control

49
Q

Ensure that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately

A

Positive Control

50
Q
  • Also called as contamination control or reagent blank
  • Ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run
A

Negative control
Without DNA

51
Q
  • Also called as negative template control
  • Ensures that the primers are not annealing to nontarget sequences of DNA
A

Negative control With DNA

52
Q

CONTAMINATION CONTROLS for PCR

A

Physical
Chemical or enzymatic
Wipe test

53
Q
  • Separation of Pre- PCR and Post- PCR areas
  • Air-locks, positive air flow, one way
  • PCR hoods with UV/ biosafety cabinet
A

Physical contamination control

54
Q
  • dUTP + uracil-N-glycosylase (added to the PCR reaction)- instead of adding dNTP, add dUTP = UNG (Uracil N-glycosylase) will degrade all DNA with uracil
A

Chemical/Enzymatic contamination control

55
Q

most effective for surface decontamination

A

10% bleach

56
Q

will intercalate in DNA, resist amplification

A

Psoralen

57
Q
  • Filter paper is wiped on any exposed or touched/ contaminated surfaces in the pre- PCR setup, extraction, and amplification areas
A

Wipe Test

58
Q

Used to remove the possible mispriming

A

Hot start

59
Q

Hot start 3 ways

A

on ice
preheated cycler
sequestered enzyme

MOLECULAR BIOLOGY (17 decks)

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