PCR Variant

Question 1

a process that
involves a reverse transcriptase
(RTase),an enzyme that uses RNA as the
template to make complementary DNA
(cDNA)

• This process is the exact opposite of the
  naturally occurring DNA transcription in
  which RNA is synthesized using DNA as
  the template

Answer 1

Reverse transcription

Question 2

used to amplify multiple target sequences in a
single PCR reaction using multiple primer
sets.

• This method is used to detect deletions,
  polymorphisms, mutations, etc.,

-also used to detect
different viral, bacterial and other pathogens in a single tube.

• This method consumes less time and
  effort in obtaining the results

Answer 2

Multiplex PCR

Question 3

• a modification of PCR that
  was designed to improve sensitivity and
  specificity.
• involves the use of two primer
  sets and two successive PCR reactions. External primers and Nested primers.

Answer 3

Nested PCR

Question 4

1. detection of Rickettsia, Bartonella, and similar organisms in blood
   (bacteremia) and tissues,
2. detection of herpesvirus and enterovirus in the CSF
3. detection of M. tuberculosis in sputum sample

Answer 4

Application of Nested PCR

Question 5

It monitors the amplification of a targeted
DNA molecule during the PCR (i.e., in real
time)

Answer 5

qPCR

Question 6

Two common methods for the detection of
PCR products in real-time PCR are:

Answer 6

1)non-specific fluorescent dyes
(2) sequence-specific DNA probes consisting
of oligonucleotides

Question 7

intercalate with any double-stranded DNA

Answer 7

1)non-specific fluorescent dyes

Question 8

are labelled with a
fluorescent reporter, which permits detection
only after hybridization of the probe with its
complementary sequence

Answer 8

sequence-specific DNA probes

Question 9

The_____ of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to
15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or
the “noise” of the reaction

Answer 9

Baseline

Question 10

The _____ of the real-time PCR reaction is the level of signal that reflects a statistically significant increase over
the calculated baseline signal

Answer 10

threshold

Question 11

The ______ at which the amplification plot crosses the threshold (i.e., there is a significant detectable increase in
fluorescence).

CT can be a fractional number and allows calculation of the starting template amount

Answer 11

• Cycle Threshold

Question 12

There are two types of DNA analysis in
qPCR

Answer 12

1. ds DNA binding dye
   chemistry
2. fluorophore-linked probes

Question 13

_____ for both specific and nonspecific
detection of amplified products

Answer 13

ds DNA binding dye
chemistry

Question 14

for specific PCR product detection

Answer 14

fluorophore-linked probes

Question 15

commonly used; unsuitable for high-resolution melt curve analysis (HRM)

Answer 15

SYBR Green

Question 16

less inhibitory to PCR; suitable forqPCR using a fast cycling protocol

Answer 16

EvaGreen

Question 17

This analysis consists of applying heat to the sample (from 50°C to 95°C) and monitoring the fluorescence emission
during the process

Answer 17

Melting Curve Analysis

Question 18

• are small fluorescent molecules that are attached to oligonucleotides in
  order to function as probes

Answer 18

Fluorophores

Question 19

There are two types of fluorophores:
donor/reporter and acceptor/quencher

Answer 19

Types of Fluorophores Used in qPCR

Question 20

• Oligonucleotides that combine a primer and probe in a single molecule.
• They can be classified into three groups: Harpins, Cyclicons and Angler® primerprobes.
• Fluorescence emitted from primer-probes is detected and measured during the
  denaturation or extension phase of the qPCR, depending on the type of primer-probe
  used

Answer 20

Primer probes

Question 21

detected and measured during the denaturation or extension phase of the qPCR, depending on the type of primer-probe
used

Answer 21

Fluorescence emitted from primer-probes

Question 22

include Scorpions, Amplifluor, and Light-Upon-eXtension
(LUX)

Answer 22

Hairpin primer-probes

Question 23

Oligonucleotides with an attached-donor and/or - acceptor fluorophore. There are two
types: hydrolysis and hybridization probes

Answer 23

Probes

Question 24

Their mechanism of action relies on the 5′–3′ exonuclease activity of Taq polymerase, which degrades the bound probe during amplification.

Answer 24

Hydrolysis probes

Question 25

The fluorescence emitted by binding hybridization probes can be measured either during the annealing or the extension phase

Answer 25

Hybridization probes

Question 25

The fluorescence emitted by binding hybridization probes can be measured either during the annealing or the extension phase

Answer 25

Hybridization probes

Question 26

1.During PCR, probe hybridizes to the target
2.Probe is displaced by your primer during extension
3.Probe cleaved by 5’-3; nuclease activity of polymerase
4.Fluorescence signal form your reporter molecule will be detected

Answer 26

TaqMan probe

Question 27

• enables the absolute and
  reproducible quantification of target nucleic acids
• the initial sample mix is
  partitioned into a large number of
  microscopic wells prior to the
  amplification step, resulting in either 1 or
  0 targets being present in each well

Answer 27

dPCR

Question 28

measures the amount of DNA after amplification is complete and then determines
the fraction of replicates.

This is representative of an endpoint measurement as it
requires the observation of the data after the experiment is completed. The dPCR, thus,
allows measurement of absolute quantification of amplicons.

Answer 28

dPCR

Question 29

records the relative fluorescence of the DNA at specific points
during the amplification process, which requires stops in the experimental process.
This “real-time” aspect of qPCR may theoretically affect results due to the stopping of
the experiment

Answer 29

In contrast to qPCR

Question 30

• Nucleic acid sequence-Based Amplification (NASBA)
• Strand Displacement Amplification (SDA)
• Rolling Circle Amplification (RCA)
• Loop-Mediated Isothermal Amplification (LAMP)
• Recombinase Polymerase Amplification (RPA)
• Helicase-Dependent Amplification (HDA)

Answer 30

Isothermal Amplification Techniques

Question 31

• a specific, simple,
  rapid and cost-effective nucleic acid amplification method
• This method relies on the auto-cycling strand displacement deoxyribonucleic acid
  (DNA) synthesis which is carried out at a constant temperature water bath/heat block in the presence of Bacillus
  stearothermophylus(Bst) DNA polymerase

Answer 31

Loop-mediated isothermal
amplification

Question 32

for RNA genomes or
mRNA (gene expression)

Answer 32

RT-PCR (Reverse Transcription PCR)

Question 33

multiple sets of primers

Answer 33

Multiplex PCR

Question 34

PCR x 2, confirms PCR specificity

Answer 34

Nested PCR

Question 35

real-time quantification, relative quantification

Answer 35

qPCR

Question 36

absolute quantification

Answer 36

Digital PCR

Question 37

isothermal

Answer 37

LAMP-PCR