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MOL. BIO LAB > PRE FI: NUCLEIC ACID AMPLIFICATION > Flashcards

PRE FI: NUCLEIC ACID AMPLIFICATION Flashcards

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1
Q

3 CATEGORIES OF AMPLIFICATION METHODS

A

TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION

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2
Q

POLYMERASE CHAIN REACTION
(PCR)
- DISCOVERED IN 1983 BY?

A

KARY MULLIS

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3
Q

WHAT CATEGORY OF NA AMPLIFICATION?

 POLYMERASE CHAIN REACTION (PCR)
- DISCOVERED IN 1983 BY KARY MULLIS
o Escherichia coli plasmid pBR322
o 1ST TO BE AMPLIFIED USER FRIENDLY, MORE
AUTOMATED AND MORE AMENABLE TO USE.
o ANY REGION OF DNA CAN BE CHOSEN

A

TARGET AMPLIFICATION

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4
Q

COPY OF THE
TARGET DNA

A

AMPLICONS

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5
Q

PROCEDURE OF PCR

A
  1. DENATURATION
  2. ANNEALING
  3. EXTENDING
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6
Q

DENATURATION
- SAMPLE DSDNA IS DENATURED INTO TWO SINGLE STRANDS
- HEATING AT _______ FOR
SEVERAL SECONDS TO
SEVERAL MINUTES, DEPENDING
ON THE TEMPLATE
- 100 ng to 1ug OF DNA IS USED
- FREE FROM CONTAMINATING
PROTEINS
- NO NICKS/BREAKS

A

94°C TO 96°C

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7
Q

DNA TEMPLATE CAN BE DERIVED FROM?

A
  • PATIENT’S GENOMIC DNA
  • PATIENT’S MITOCHONDRIAL DNA
  • DNA FROM VIRUSES, BACTERIA OR PARASITES
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8
Q

BASIC PROCEDURE OF PCR

  • 2 PRIMERS WILL PRIME THE SYNTHESIS OF DNA ANNEAL (HYBRIDIZE) TO COMPLEMENTARY SEQUENCES OF THE TEMPLATE.
  • MOST CRUCIAL STEP
  • 50°C to 70°C
A

ANNEALING

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9
Q

2 primers in annealing
- hybridize in 5’

A

FORWARD

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10
Q

2 primers in annealing
- hybridize at 3’

A

REVERSE

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11
Q

AKA OLIGODEOXYNUCLEOTIDES
- 20-30 BASES
- DIRECTS DNA SYNTHESIS TO THE DESIRED REGION
- DETERMINE THE SPECIFICITY OF PCR
- ANALOGOUS TO PROBES
- CHEMICALLY SYNTHESIZED

A

PRIMERS

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12
Q

MORE SPECIFIC AND HYBRIDIZE AT SLOWER RATE

A

LONG RPIMERS

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13
Q

LESS SPECIFIC AND HYBRIDIZE AT
FASTER RATE

A

SHORT PRIMERS

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14
Q

STARTING POINT OF ANNEALING WILL BE DETERMINED USING THE ___ OF THE PRIMER

A

Tm (MELTING POINT)

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15
Q

FACTORS THAT MAY AFFECT THE
EXACT Tm :

A
  • REACTION CONDITIONS
  • SALT CONCENTRATION
  • TEMPLATE CONDITIONS
  • SECONDARY STRUCTURE ( INTERNAL FOLDING AND HYBRIDIZATION BETWEEN STRANDS)
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16
Q
  • ABERRANT PRIMER BINDING
  • PRIMERS BIND TO UNINTENDED SEQUENCES
  • 22-25C AND LEADS TO FORMATION OF MISPRIMES
  • PREVENTED BY HOT-START PCR
A

MISPRIMING

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17
Q
  • ARTIFACT COMPOSED OF TWO PRIMERS BINDING ONTO EACH OTHER
  • DOUBLE THE SIZE OF THE UTILIZED PRIME
A

PRIMER DIMERS

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18
Q

PROCEDURE OF PCR:
- DNA SYNTHESIS OCCURS
- ADDITION OF NUCLEOTIDES
(PRIMER EXTENSION)
- 68°C to 72°C (OPTIMAL TEMP OF THE ENZYME)
- POLYMERASE SYNTHESIZES A
COPY OF THE TEMPLATE DNA

A

EXTENSION/ELONGATION

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19
Q

EXTENSION/ELONGATIION

  • OPTIMAL TEMP OF
    THE ENZYME
A

68°C to 72°C

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20
Q
  • ENZYME RESPONSIBLE FOR THE ADDITION OF NUCLEOTIDES TO THE HYBRIDIZED PRIMERS
  • HIGH TEMPERATURES IN DENATURATION STEP WILL INACTIVATE THE ENZYME
A

DNA POLYMERASE

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21
Q
  • MAINLY UTILIZED IN PCR
  • FROM Thermus aquaticus
  • THERMOSTABLE (not easily destroyed during denaturation)
  • BY RANDALL SAIKI
A

Taq POLYMERASE

22
Q
  • UTILIZED IN REVERSE-TRANSCRIPTASE PCR (RNA)
  • FROM Thermus thermophilus
A

Tth POLYMERASE

23
Q
  • supporter
  • ALLOWS Taq o Tth to generate large products (30,000 bases)
A

VENT POLYMERASE

24
Q

PCR REAGENTS

  • TYPICALLY IN A PREFORMED SOLUTION CALLED?
A

MASTER MATRIX (BUFFER + TEMPLATE + DNA POLYMERASE + dNTPs + KCl + MgCI + PRIMERS)

25
Q

REAGENT COMPONENTS:
- DIRECTS DNA SYNTHESIS
TO THE DESIRED REGION

A

PRIMER (oligodeoxynucleotides)

26
Q

REAGENTS COMPONENTS:
- BUILDING BLOCKS THAT EXTEND THE PRIMERS

A

dATP, dGTP, dCTP, dTTP

27
Q

REAGENTS COMPONENTS:
- MONOVALENT CATION (SALT), FOR OPTIMAL HYBRIDIZATION OF PRIMERS TO TEMPLATE

A

KCL

28
Q

REAGENTS COMPONENTS;
- BUFFER TO MAINTAIN OPTIMAL PH FOR THE
ENZYME REACTION

A

Tris, pH 8.4

29
Q

REAGENTS COMPONENTS:
- DIVALENT CATION, REQUIRED BY THE ENZYME

A

1.5 mM MgCl2

30
Q

REAGENTS COMPONENTS:
- POLYMERASE ENZYME THAT EXTENDS THE PRIMERS (ADDS DNTPS)

A

POLYMERASE

31
Q
  • A MIXTURE OF FOUR EQUIMOLAR DEOXYNUCLEOTIDE
    TRIPHOSPHATES
     ADENINE (dATP)
     THYMINE (dTTP)
     GUANINE (dGTP)
     CYTOSINE (dCTP)
  • PURITY MAY AFFECT PCR RESULTS
A

DEOXYNULCEOTIDE BASES

32
Q

dNDP AND dMNP CAN ALSO BE A
RESULT OF:

A
  • POOR MANUFACTURING
  • CONTAMINATION OF HEAVY METALS
33
Q
  • PROVIDE OPTIMAL CONDITIONS FOR ENZYME ACTIVITY (pH 8 -
    9.5)
  • INCREASED SALT CONCENTRATION MAY DENATURE LONG DNAS SLOWER THAN SHORTER DNAs
  • CONDITIONS VARY WITH PRIMERS AND TEMPLATES
A

PCR BUFFERS

34
Q

PCR BUFFERS
- MAY CHELATE Mg THAT WILL RESULT TO LOWER ENZYME EFFICIENCY

A

EDTA

35
Q
  • for optimizing reactions
A

ACCESSORY COMPONENTS FOR
BUFFERS

36
Q

ACCESSORY COMPONENTS FOR
BUFFERS
- LOWER DENATURING TEMPERATURE

A

FORMAMIDE

37
Q

ACCESSORY COMPONENTS FOR
BUFFERS
- REDUCER
- ENHANCE ENZYME ACTIVITY

A

DITHIOTHREITOL

38
Q

ACCESSORY COMPONENTES FOR
BUFFERS
-BINDS INHIBITORS
-ENZYME STABILIZER

A

BOVINE SERUM ALBUMIN

39
Q

ACCESSORY COMPONENTES FOR
BUFFERS
- ALLOWS POLYMERASE TO ACCESS DIFFICULT AREAS

A

CHAOTROPIC AGENTS

  • TRITON X 100
  • GLYCEROL
  • DIMETHYL SULFOXIDE
40
Q

PCR EQUIPMENTS:
- FIRST PCR SYSTEM CONDUCTED
- REPLACED BY AUTOMATED METHODS AND THERMOSTABLE ENZYMES

A

WATER BATHS AND HEAT BLOCKS

41
Q

PCR EQUIPMENTS:
- FOR RAPID AND AUTOMATICALLY RAMP TO THE REQUIRED TEMPERATURES
- EARLY VERSION:
 WAX/OIL: (VAPOR BARRIERS) TO PREVENT CONDENSATION OF SAMPLE TO THE TUBE TOPS
- RT PCR TYPES: - WITH FLUORESCENT DETECTORS FOR MEASUREMENT

A

THERMAL CYCLERS/ THERMOCYCLERS

42
Q
  • REAGENTS CAN BE MIXED EITHER SEPARATELY OR MASTER MIX
  • PRE-PCR WORK SHOULD BE PERFORMED IN A CLEAN DESIGNATED AREA
  • NUMBER OF CYCLES IS USUALLY 30-50
    o PCR PRODUCTS CAN BE
    FOR:
  • GEL/CAPILLARY ELECTROPHORESIS
  • NUCLEIC ACID ANALYSIS
  • OTHER MOLECULAR BIOLOGY TESTS
A

PCR REACTIONS

43
Q

CONTROLS:
- ENSURES:
 ENZYME IS ACTIVE
 BUFFER IS OPTIMAL
 PRIMERS ARE PRIMING RIGHT SEQUENCES
 THERMAL CYCLERS ARE CYCLING PROPERLY

A

POSITIVE CONTROLS

44
Q

CONTROLS:
- AKA. CONTAMINATION CONTROL OR REAGENT BLANK
- Negative control without DNA
- Ensures that the reaction mix is not contaminated with template DNA or amplified products from previous run.

A

NEGATIVE CONTROL

45
Q

CONTROLS:
- WITH A DNA THAT HAS NO TARGET SEQUENCE THERE SHOULD BE:
o NO ANNEALING
- Ensures the primers are not annealing to nontarget sequences of DNA.

A

NEGATIVE TEMPLATE CONTROLS

46
Q

CONTROLS:
- CONTAINS A PRIMER BINDING SITE
- DETERMINE FALSE NEGATIVES (AMPLIFICATION FAILURE)

A

AMPLIFICATION CONTROLS

47
Q
  • COMMON IN OPEN TUBE METHODS
  • MOST COMMON SOURCES:
    o PRODUCTS PREVIOUS AMPLIFICATIONS (MAY
    AEROSOLIZED IF UNCAPPED)
A

PCR CONTAMINATION

48
Q

PCR CONTAMINATION CONTROL METHODS:
- SEPARATE PRE AND POST PCR AREAS
- POSITIVE AIRFLOW, AIRLOCKS
- CHANGE OF GLOVES
- UV FOR DECONTAMINATION

A

PHYSICAL CONTAMINATION CONTROL

49
Q

ATTACH T, U, C, IN DNA UNDER UV (PREVENT
DENATURATION AND AMPLIFICATION)

A

PSORALENS

50
Q

PCR CONTAMINATION CONTROL METHODS:
- FOR DECONTAMINATION OF WORKSPACES
o 10% BLEACH
- ENZYMATIC: DTP-UNG FOR PREVIOUS AMPLIFICATIONS
- WIPE TESTS TO DETERMINE CONTAMINATION

A

CHEMICAL CONTAMINATION CONTROL

MOL. BIO LAB (9 decks)

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