4. NUCLEIC ACID AMPLIFICATION - LAB

Question 1

make many copies of target DNA

Answer 1

NUCLEIC ACID AMPLIFICATION

Question 2

Three Categories of Nucleic acid amplification

Answer 2

• 1. Target amplification systems
• 2. Probe amplification system
• 3. Signal amplification system

Question 3

category of NAA that targets nucleic acid based (PCR)

Answer 3

Target amplification systems

Question 4

category of NAA that is probe specific for target sequence

Answer 4

Probe amplification system

Question 5

is a short region of double-stranded DNA

Answer 5

TARGET

Question 6

Involves making many copies of a specific DNA sequence

Answer 6

TARGET AMPLIFICATION

Question 7

is the first and prototypical method for amplifying target nucleic acid

Answer 7

PCR

Question 8

PCR Discovered by

Answer 8

Kary Mullis in 1983 (Nobel Prize Awardee)

Question 9

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).

Answer 9

Target Amplification

Question 10

PCR

The first successful amplification was a short fragment of the

Answer 10

Escherichia coli plasmid (pBR322

Question 11

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

Answer 11

sickle cell anemia

Question 12

PCR products

Answer 12

Amplicons

Question 13

binds beside the target sequence

Answer 13

Primer

Question 14

binds directly to the target sequence

Answer 14

Probe

Question 15

AMPIFICATION PROGRAM Components:

Answer 15

DNA template
Short oligonucleotide primers
Nucleotides
Polymerase
Buffers

Question 16

Short oligonucleotide primers types

Answer 16

FORWARD AND REVERSE

Question 17

useful in addition of dNTPs to growing strands

Answer 17

Polymerase

Question 18

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

Answer 18

Buffers

Question 19

ELEMENTS OF PCR CYCLE

Answer 19

• 1. Denaturation
• 2. Annealing
• 3. Extension/Elongation

Question 20

Denaturation Temperature (°C)
Time (sec)

Answer 20

90-96 temp
20-60 time

Question 21

Annealing Temperature (°C)
Time (sec)

Answer 21

50-70 temp
20-90 time

Question 22

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

Answer 22

68-75 temp
10-60 time

Question 23

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

Answer 23

Forward Primer

Question 24

Forward Primer formation of amplicons

Answer 24

Forward from 3’ to 5

Question 25

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

Answer 25

Reverse Primer

Question 26

Reverse Primer formation of amplicons

Answer 26

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

Question 27

Tm formula for annealing

Answer 27

Tm = 4x #GC + 2x#AT

Question 28

attachment of primer away from the target sequence

Answer 28

Mispriming

Question 29

Binding of primers onto each other through short (2- to 3-base) homologies at their 3' ends and the copying of each primer sequence

Answer 29

Primer Dimer

Question 30

DNA TEMPLATE Sources:

Answer 30

- Patient's genomic or mitochondrial DNA - Viruses - Bacteria - Fungi - Parasites

Question 31

DNA POLYMERASES used in PCR

Answer 31

Taq polymerase Tth polymerase Vent polymerase

Question 32

- most known polymerase for PCR - Thermostable enzyme (stable even at high temp)

Answer 32

Taq polymerase

Question 33

Taq polymerase Isolated from the thermophilic bacterium

Answer 33

Thermus aquaticus

Question 34

Tth polymerase From

Answer 34

Thermus thermophilus

Question 35

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

Answer 35

Tth polymerase

Question 36

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

Answer 36

Vent polymerase

Question 37

Proofreading enzyme

Answer 37

Vent polymerase

Question 38

- Lacking the 289 N-terminal amino acids of Taq polymerase recommended for allele- - specific PCR and for amplification of regions with high GC content

Answer 38

Stoffel fragment

Question 39

Modified Polymerase Enzymes

Answer 39

Stoffel fragment

Question 40

Accessory components

Answer 40

- 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

Question 41

Accessory components for the stabilization of enzyme

Answer 41

- Bovine serum albumin (10 to 100 µg/mL)

Question 42

Accessory components to enhance the enzyme activity

Answer 42

- Dithiothreitol (0.01 mM)

Question 43

Accessory components lower denaturation temperature to reduce secondary structure

Answer 43

Formamide (1% to 10%)

Question 44

Accessory components for stability of enzyme and reduce secondary structure

Answer 44

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

Question 45

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

Answer 45

THERMAL CYCLER/THERMOCYCLERS

Question 46

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

Answer 46

Real-time PCR

Question 47

Qualitative presence or absence of amplicons

Answer 47

Conventional PCR- End point Analysis

Question 48

CONTROLS FOR PCR

Answer 48

Positive Control Negative control

Question 49

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

Answer 49

Positive Control

Question 50

- 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

Answer 50

Negative control Without DNA

Question 51

- Also called as negative template control - Ensures that the primers are not annealing to nontarget sequences of DNA

Answer 51

Negative control With DNA

Question 52

CONTAMINATION CONTROLS for PCR

Answer 52

Physical Chemical or enzymatic Wipe test

Question 53

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

Answer 53

Physical contamination control

Question 54

- 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

Answer 54

Chemical/Enzymatic contamination control

Question 55

most effective for surface decontamination

Answer 55

10% bleach

Question 56

will intercalate in DNA, resist amplification

Answer 56

Psoralen

Question 57

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

Answer 57

Wipe Test

Question 58

Used to remove the possible mispriming

Answer 58

Hot start

Question 59

Hot start 3 ways

Answer 59

on ice preheated cycler sequestered enzyme