Molecular Diagnostics Nucleic Acid Amplification by PCR

Question 1

Who invented PCR?

Answer 1

Kary Mullis

Question 2

Terminology:
-Produces many copies of DNA
-Most utilized molecular technique

Answer 2

Polymerase Chain Reaction (PCR)

Question 3

Terminology:
One copy of target DNA can yield billions of copies

Answer 3

Amplification

Question 4

Applications of PCR (3)

Answer 4

-Infectious disease testing
-Genetic defects
-Forensics

Question 5

Know A-E

Answer 5

A: Taq DNA polymerase
B: Primers
C: dNTPs (deoxynucleoside triphosphate)
D: Buffer
E: DNA

Question 6

Three steps of standard PCR

Answer 6

1. Denaturation
2. Annealing
3. Elongation/Extension

Question 7

Temperature range for denaturation

Answer 7

94-96C

Question 8

PCR Steps:
-Breaks hydrogen bonds
-Separates target DNA strands

Answer 8

Denaturation

Question 9

Temperature range for annealing

Answer 9

50-65C

Question 10

Elongation/Extension occurs at what temperature?

Answer 10

72C

Question 11

Why does elongation take place at 72C?

Answer 11

It’s the optimal temperature for Taq polymerase to efficiently build new DNA strands

Question 12

PCR Steps:
Primers hybridize to denatured DNA strands

Answer 12

Annealing

Question 13

PCR Steps:
Taq polymerase synthesizes new complementary DNA strands

Answer 13

Elongation/Extension

Question 14

Why does Elongation/Extraction occurs at 72C?

Answer 14

It’s the optimal temperature for Taq polymerase to function

Question 15

Components for PCR (6)

Answer 15

-Sample
-Primers
-Nucleotides
-Taq polymerase
-Mix buffer
-PCR tube

Question 16

What is Taq polymerase?

Answer 16

A DNA polymerase commonly used in PCR

Question 17

Where does Taq polymerase come from?

Answer 17

A heat-tolerant bacterium known as Thermus aquaticus

Question 18

In vivo, what is responsible for denaturation of DNA? (enzyme)

Answer 18

Helicase

Question 19

In vitro, how is DNA denatured?

Answer 19

Heat

Question 20

Why do G-C pairs requires more energy (or heat) for the primer to bond to the target strand when compare to A-T?

Answer 20

G-C pairs have 3 hydrogen bonds whereas A-T pairs only have 2 hydrogen bonds

Question 21

Terminology:
Products of PCR (all the copies of DNA)

Answer 21

Amplicons

Question 22

What are 3 post-PCR analysis methods?

Answer 22

-Gel electrophoresis
-Labeled probes
-DNA sequencing

Question 23

TRUE or FALSE:
Standard PCR is limited to length limitations of up to 1000 base pairs

Answer 23

TRUE

Question 24

3 ways to prevent contamination for standard PCR

Answer 24

-Creation of aerosols
-Physical separation of space if possible
-Unidirectional workflow

Question 25

PCR Variations (4)

Answer 25

-Multiplex PCR
-Reverse transcriptase PCR
-Real Time PCR
-Other PCR

Question 26

2 types of “other PCR” mentioned

Answer 26

-Nested PCR
-Sequence-specific PCR

Question 27

Which PCR variation:
-mRNA used to synthesize cDNA
-Testing for RNA viruses
-RNA is fragile

Answer 27

RT-PCR

Question 28

Which PCR variation:
-TaqMan probe
-Uses FRET
-Sequence-specific
-Allows for quantization of starting material in samples
-Expensive

Answer 28

qPCR

Question 29

2 main types of detector in qPCR

Answer 29

-Non-sequence specific dyes (SYBR Green)
-Sequence-specific probes (TaqMan and Molecular Beacon)

Question 30

Which PCR variation:
-Simultaneous detection of multiple targets in a single tube/well
-SNP genotyping, pathogen detection, forensics
-More information with smaller sample size
-Primer design can be complex

Answer 30

Multiplex PCR

Question 31

What is Multiplex PCR commonly used for?

Answer 31

Panel testing

Question 32

What is Reverse Transcriptase PCR commonly used for?

Answer 32

-Testing RNA viruses
-Detecting gene expression in cancer cells

Question 33

Which qPCR detector uses fluorescence energy resonance technology (FRET)?

Answer 33

Sequence-specific probes:
-TaqMan Probes

Question 34

TaqMan probes used in qPCR utilize FRET, which stands for what?

Answer 34

Fluorescence Resonance Energy Technology

Question 35

On a qPCR amplification plot, what does the Ct value represent?

Answer 35

Cycle threshold