PCR

Question 1

Polymerase Chain Reaction (PCR) was invented by__________, an American
biochemist.

Answer 1

Kary B. MulliS

Question 2

Critical component of the reaction because they determine the
specificity of the PCR.

Answer 2

Primers

Question 3

They are designed to contain sequences complementary to sites
flanking the region to be analyzed.

Answer 3

Primers

Question 4

HOW MANY BASES IN LENGHT ARE PRIMERS

Answer 4

20 to 30 bases

Question 5

• designed to hybridize to the complementary
  strand of the target DNA just upstream of the region to be amplified.

Answer 5

Forward primer

Question 6

• designed to hybridize to the complementary
  strand of the target DNA just downstream of the region to be
  amplified.

Answer 6

Reverse primeR

Question 7

• serves as a starting point for
  setting the optimal annealing temperature.

Answer 7

Primer melting temperature (Tm)

Question 8

Primers should be designed such that the ____________
have similar Tm so that both will hybridize optimally at the same
annealing temperature.

Answer 8

forward and reverse primers

Question 9

determines the accuracy of binding to its
complementary sequence and not to other sequences.

Answer 9

Primer sequence

Question 10

The template may be derived from the patient ’s genomic or mitochondrial DNA or from
viruses, bacteria, fungi, or parasites that might be infecting the patient.

Answer 10

DNA Template

Question 11

nucleotides containing triphosphate groups

Answer 11

Deoxyribonucleotide triphosphates (dNTPs)

Question 12

the building blocks from which the DNA polymerase synthesizes a new DNA strand

Answer 12

(dNTPs)

Question 13

isolated from the thermophilic bacterium Thermus aquaticus

Answer 13

Taq polymerase

Question 13

Standard procedures require concentrations of each nucleotide OF DNTP

Answer 13

0.1 to 0.5 mM

Question 14

heat-resistant enzyme
remains intact during the high-temperature DNA denaturation process

Answer 14

Taq polymerase

Question 15

Taq polymerase

Answer 15

Tth polymerase

Question 16

has reverse-transcriptase activity, so it can be used in reverse-transcriptase PCR
(RT-PCR)

Answer 16

Tth polymerase

Question 17

Provide the optimal conditions for enzyme activity.

Answer 17

PCR Buffers

Question 18

This causes DNA melting, or denaturation, of the double-stranded DNA template by
breaking the hydrogen bonds between complementary bases, yielding two single-
stranded DNA molecules.

Answer 18

1.Denaturation

Question 19

Most critical for the specificity of the PCR.

Answer 19

Annealing

Question 20

Determine the specificity of the amplification.

Answer 20

Annealing

Question 21

two
oligonucleotide primers that will prime the synthesis of
DNA anneal (hybridize) to complementary sequences
on the template.

Answer 21

Annealing

Question 22

This is where DNA synthesis occurs.

Answer 22

Extension

Question 23

In this step, the polymerase synthesizes a copy of
the template DNA by adding nucleotides to the
hybridized primers.

Answer 23

In this step, the polymerase synthesizes a copy of
the template DNA by adding nucleotides to the
hybridized primers.

Question 24

This step occurs at the optimal temperature of the enzyme, 68°C to 75°C

Answer 24

Extension

Question 25

temperatures will range from 50°C to 70°C

Answer 25

Annealing

Question 26

DENATURATION This step is the first regular cycling event and consists of heating the reaction chamber to

Answer 26

90–96°C for 20–30 seconds.

Question 27

At the end of the three steps, or one cycle (denaturation, primer annealing, and primer extension),

Answer 27

one copy of double-stranded DNA has been replicated into two double- stranded copies.

Question 28

Returning to the denaturing temperature starts the second cycle with the end result being a

Answer 28

doubling in the number of double- stranded DNA molecules again.

Question 28

Conventional PCR Also known as

Answer 28

endpoint PCR

Question 29

It involves repeated cycles of denaturation, annealing, and extension, resulting in exponential amplification of the target DNA sequence.

Answer 29

Conventional PCR

Question 30

amplify RNA targets

Answer 30

Reverse- Transcriptase Polymerase Chain reaction

Question 31

is first produced from RNA targets by reverse transcription, and then the cDNA is amplified by PCR.

Answer 31

Complementary DNA (cDNA)

Question 32

Enzyme isolated from RNA virus

Answer 32

Reverse transcriptase

Question 33

These primers can generate cDNA from all RNA in the specimen.

Answer 33

Reverse transcriptase

Question 34

This technique uses two pairs of amplification primers and two rounds of PCR.

Answer 34

Nested Polymerase Chain Reaction

Question 35

Typically, one primer pair is used in the first round of the amplification of PCR of

Answer 35

15–30 cycles.

Question 36

IN THE NESTLED PCR The increased sensitivity arises from the ______ and the increased specificity arises from the ___________ set to sequences produced by the first round.

Answer 36

The increased sensitivity arises from the high total cycle number, and the increased specificity arises from the annealing of the second primer set to sequences produced by the first round.

Question 37

The major concern for this method is the contamination that occurs during the transfer of the first-round product to the second tube for the second round of amplification.

Answer 37

Nested Polymerase Chain Reaction this can be avoided by physically separating the first- and

Question 38

PCR allows simultaneous amplification of multiple target DNA sequences within a single reaction tube.

Answer 38

Multiplex Polymerase Chain Reaction

Question 39

primers should have similar annealing temperatures and must be not complementary to each other.

Answer 39

Multiplex Polymerase Chain Reaction

Question 40

xis a technique that quantifies nucleic acid molecules by partitioning a sample into many individual reactions, allowing for absolute quantification of target sequences.

Answer 40

Digital Polymerase Chain Reaction

Question 41

doesn 't rely on standard curves, making it highly sensitive and accurate, especially for detecting rare events or mutations.

Answer 41

Digital Polymerase Chain Reaction

Question 42

This method is a way of quantifying the amplification of a DNA as it occurs, which means it detects the accumulation of amplicon after each thermal cycle in real time.

Answer 42

Real-time PCR (qPCR)

Question 43

Quantitative PCR

Answer 43

Real-time PCR (qPCR)

Question 44

implies that data collection and analysis occur as a reaction proceeds.

Answer 44

“Real time”

Question 45

the fluorometric probes are used to bind target DNA during the annealing phase of PCR.

Answer 45

qPCR

Question 46

The dye can ultimately be detected in order to determine the success of the amplification reaction, and the initial quantity of nucleic acid present in the sample is depicted in graphical form

Answer 46

Real-time PCR (qPCR)

Question 47

The qPCR instrument consists of:

Answer 47

Thermal cycler with an integrated excitation light source (a lamp, laser, or LED [light-emitting diode]) Fluorescence detection system or fluorimeter Software that displays the recorded fluorescence data as a DNA amplification curve.

Question 48

involves the use of fluorescent dyes that bind to double-stranded DNA (dsDNA). These dyes emit fluorescence when bound to dsDNA,

Answer 48

dsDNA Binding Dye Chemistry

Question 49

It binds to double-stranded DNA and exhibits increased fluorescence upon binding.

Answer 49

SYBR Green

Question 50

Similar to SYBR Green, it fluoresces upon binding to dsDNA and is compatible with various PCR instruments and protocols.

Answer 50

EvaGreen

Question 51

Better peak resolution in multiplex real-time PCR.

Answer 51

EvaGreen

Question 52

involves the use of sequence-specific fluorescent probes that hybridize to a complementary sequence within the PCR amplicon.

Answer 52

Fluorophore-Linked Probes

Question 53

Two main types of detection chemistries used in qPCR:

Answer 53

Fluorophore-Linked Probes

Question 54

fluorophore-linked probes used for specific detection of PCR products in real-time PCR. These probes are typically oligonucleotides labeled with a fluorescent reporter dye (e.g., FAM) at one end and a quencher dye at the other end.

Answer 54

TaqMan Probes

Question 55

The fluorescence of the reporter dye is quenched in the intact probe, but upon cleavage by the DNA polymerase during amplification, the fluorescence is released, allowing for real-time detection of the PCR product.

Answer 55

TaqMan Probes

Question 56

hairpin-shaped oligonucleotide probes labeled with a fluorescent reporter dye (e.g., FAM) at one end and a quencher dye at the other end.

Answer 56

Molecular Beacons

Question 57

Consist of an oligonucleotide labeled with a fluorescent reporter dye (e.g., FAM) at one end and a quencher dye at the other end.

Answer 57

Hydrolysis Probes (e.g., Dual-Labeled Probes)

Question 58

These probes are designed to hybridize specifically to the target sequence, and upon cleavage by the DNA polymerase during amplification, the fluorescence of the reporter dye is released, allowing for real-time detection of the PCR product.

Answer 58

Hydrolysis Probes (e.g., Dual-Labeled Probes)

Question 59

One of the most commonly used fluorescent dyes, emitting a green fluorescence. It is often used in real-time PCR and other nucleic acid detection assays.

Answer 59

FAM (6-Carboxyfluorescein):

Question 60

Similar to FAM, VIC emits fluorescence in the green spectrum. It is often used as an alternative to FAM for multiplex PCR assays.

Answer 60

VIC (or TET):

Question 61

Another green-emitting fluorescent dye, commonly used in genotyping and SNP analysis.

Answer 61

HEX (Hexachloro-fluorescein):

Question 62

This dye emits fluorescence in the orange/red spectrum. It is commonly used as a passive reference dye in real-time PCR to normalize fluorescence signals.

Answer 62

ROX (6-Carboxy-X-rhodamine):

Question 63

Emitting fluorescence in the red spectrum, Cy5 is often

Answer 63

Cy5 (Cyanine 5):

Question 64

Similar to Cy5, but emitting fluorescence in the green spectrum. It is also used in DNA microarray analysis and other labeling techniques.

Answer 64

Cy3 (Cyanine 3):

Question 65

emitting fluorescence in the near-infrared spectrum, Cy7 is used in applications requiring deep tissue penetration, such as in vivo imaging and multiplex assays.

Answer 65

Cy7 (Cyanine 7): E