NUCLEIC ACID AMPLIFICATION

Question 1

• Most common nucleic acid amplification method

Answer 1

POLYMERASE CHAIN REACTION

Question 2

What the 3 categories in Nucleic acid amplification?

Answer 2

TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION

Question 2

Advantages of PCR:

Answer 2

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

Question 3

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

Answer 3

AMPLICONS

Question 4

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

Answer 4

PCR

Question 4

• 1st to be amplified

Answer 4

Escherichia coli plasmid pBR322

Question 5

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

Answer 5

DENATURING (90-96C)

Question 6

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

Answer 6

DENATURING

Question 7

DNA TEMPLATE is derived from?

Answer 7

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

Question 8

90-60 CELSIUS

Answer 8

DENATURING

Question 9

50-70 CELSIUS

Answer 9

ANNEALING

Question 10

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

Answer 10

ANNEALING

Question 10

Primers are also known as?

Answer 10

OLIGODEOXYNUCLEOTIDES

Question 11

This step determine the specificity of PCR.

Answer 11

ANNEALING

Question 12

-More specific & hybridize at slower rate

Answer 12

LONG PRIMERS

Question 13

• Less specific & hybridize at faster rate

Answer 13

SHORT PRIMERS

Question 14

2 TYPES OF PRIMERS:

Answer 14

FORWARD , REVERSE

Question 15

• 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

Answer 15

PRIMER DIMERS

Question 15

(68oC - 75oC)

Answer 15

ELONGATION/EXTENSION

Question 15

• Aberrant primer binding
• Primers bind to unintended sequences

Answer 15

MISPRIMING

Question 15

WHAT ARE THE FACTORS THAT MAY AFFECT THE EXACT Tm

Answer 15

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

Question 16

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

Answer 16

EXTENSION/ELONGATION

Question 17

10sec – 60sec

Answer 17

EXTENSION/ELONGATION

Question 18

20-60 secs

Answer 18

DENATURING

Question 19

20-90 secs

Answer 19

APPEALING

Question 20

- Enzyme for the addition of nucleotides to the hybridized primer - High temperature in denaturation step will inactivate the enzyme

Answer 20

DNA POLYMERASE

Question 21

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

Answer 21

Taq POLYMERASE

Question 22

Taq POLYMERASE is obtained from? - THERMOSTABLE

Answer 22

THERMUS AQUATICUS

Question 22

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

Answer 22

Tth POLYMERASE

Question 22

Tth POLYMERASE is obtained from?

Answer 22

Thermus thermophilus

Question 23

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

Answer 23

VENT POLYMERASE

Question 23

VENT POLYMERASE is obtained from?

Answer 23

Thermococcus Litoralis

Question 24

- Incorporates ddNTPS - Chain termination sequencing

Answer 24

THERMOSEQUENASE AND 17 SEQUENASE

Question 24

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

Answer 24

STOFFEL FRAGMENT

Question 25

Why is vigorous agitation of enzyme is not recommended?

Answer 25

- may alter the structures of enzyme and will denature irreversibly and air bubbles may damage it

Question 25

PCR REAGENTS MASTER MIX:

Answer 25

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

Question 26

- It Directs DNA synthesis to the desired region

Answer 26

PRIMERS

Question 26

- Divalent cation, required by the enzyme

Answer 26

1.5 mM MgCl2

Question 27

Building blocks that extend the primers

Answer 27

dATP, dCTP, dGTP, dTTP (dNTPs)

Question 27

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

Answer 27

Tris, pH 8.4

Question 28

- Monovalent cation (salt), for optimal hybridization of primers to template

Answer 28

KCl

Question 29

- 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

Answer 29

DEOXYRIBONUCLEOTIDE BASES

Question 30

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

Answer 30

PCR BUFFERS

Question 31

▪ Lower denaturing temperature

Answer 31

Formamide (1% - 10%)

Question 32

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

Answer 32

WATER BATHS & HEAT BLOCKS

Question 33

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

Answer 33

THERMAL CYCLERS/THERMOCYCLERS

Question 34

PCR products can be analyzed by:

Answer 34

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

Question 35

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

Answer 35

NEGATIVE CONTROLS

Question 36

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

Answer 36

POSITIVE CONTROLS

Question 37

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

Answer 37

NEGATIVE TEMPLATE CONTROLS

Question 37

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

Answer 37

AMPLIFICATION CONTROLS

Question 38

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

Answer 38

PCR CONTAMINATION

Question 39

WHAT IS THE MOST COMMON SOURCE OF PCR CONTAMINATION?

Answer 39

Products of previous amplifications (may aerosolized if uncapped

Question 40

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

Answer 40

CHEMICAL CONTAMINATION CONTROL

Question 41

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)

Answer 41

PHYSICAL CONTAMINATION CONTROL

Question 42

CONTROL METHODS * For decontamination of workspaces

Answer 42

10% bleach (7 mM sodium hypochlorite)

Question 43

CONTROL METHODS * To determine concentration

Answer 43

WIPE TEST