POLYMERASE CHAIN REACTION

Question 1

3 General types of amplification technique

Answer 1

• Target amplification
• Signal amplification
• Probe amplification

Question 2

• Invented in 1983 by

Answer 2

Dr. Kary Mullis

Question 3

• An in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence

Answer 3

PCR

Question 4

PCR

• based on using the ability of_______ to synthesize new strand of DNA complementary to the offered template strand. (It needs a_____ to which it can add the first nucleotide.)

Answer 4

DNA polymerase

primer

Question 5

– The DNA sample that contains the specific sequence you want to amplify. It serves as the blueprint for new DNA strands.

Answer 5

Template DNA

Question 6

– A short single-stranded DNA that binds to the 3’ end of the template strand, helping extend the new DNA strand in the 5’ to 3’ direction.

Answer 6

Downstream Oligonucleotide Primer (Reverse Primer)

Question 7

– A short single-stranded DNA that binds to the 3’ end of the complementary strand, initiating DNA synthesis in the opposite direction.

Answer 7

Upstream Oligonucleotide Primer (Forward Primer)

Question 8

: A heat-stable enzyme that synthesizes new DNA strands by adding nucleotides to the primers.

Answer 8

Taq DNA Polymerase

Question 9

– Provides Mg²⁺ ions, which are essential cofactors for Taq polymerase, helping it function properly and improving enzyme efficiency.

Answer 9

25 mM MgCl₂ (Magnesium chloride)

Question 10

– A mixture of deoxynucleotide triphosphates (dATP, dTTP, dGTP, dCTP) that serve as the building blocks for new DNA strand synthesis.

Answer 10

dNTP Mix (10 mM of Each dNTP)

Question 11

PCR

Two Problems

Answer 11

• There are a lot of other sequences in a genome that we are not interested indetecting

• The amount of DNA in samples we are interested in is very small

Question 12

TEMPLATE DNA
• Target DNA
• Contains the region/sequence to be amplified
• Up to____
•____ in a 50 uL total reaction mixture

Answer 12

3Kb

0.1-1 ug

Question 13

PRIMERS
• Specific for ends of the region to
be amplified
• Complementary to the 3’ ends of
target DNA

• Length:____ nucleotides
• GC content:_____
• ______temperature should be
determined
• Concentration: 50 pmol (1uM final
concentration in a 50 uL reaction

Answer 13

15-30 nucleotides

40-60%

Annealing

Question 14

PRIMERS
• Two primers must be designed for PCR:

Answer 14

Forward primer
Reverse primer

Question 15

• the_________ or primer is complimentary to the 3’ end of antisense strand (3’-5’)

Answer 15

forward primer

Question 16

• the______ primer is complimentary to the 3’ end of sense strand (5’-3’)

Answer 16

reverse primer

Question 17

• Stabilizes the DNA polymerase, DNA, and nucleotides

Answer 17

BUFFER

Question 18

• Stabilizes the DNA polymerase, DNA, and nucleotides
• 500 mM_____
• 100 mM_____

Answer 18

KCl

Tris-HCl (8.3)

Question 19

• The medium for all other components

Answer 19

Water

Question 20

• Essential cofactor of DNA polymerase

• Stabilizes the DNA double helix

Answer 20

MAGNESIUM

Question 21

MAGNESIUM (cofactor)
• Too little:_____
• Too much:______

• Used at____ to ____ in the assay

Answer 21

enzyme will not work

non-specific amplifications

0.5 to 3.5 uM

Question 22

Precautions:
• Completely____ magnesium solution
•____ magnesium solution for
several seconds before pipetting
• If DNA samples contain EDTA…

Answer 22

thaw

Vortex

Question 23

• The enzyme that does the extension

• Heat stable

• Approximately _____of Taq DNAPol per 50 uL reaction

Answer 23

DNA polymerase

1.25 U

Question 24

POLYMERASE

Variants:
• Taq –_______
• Pfu –
• KOD DNA Polymerase – a recombinant form of_____

Answer 24

Thermus aquaticus

hyperthermophilic Pyrococcus
furiosus

Thermococcus kodakarensis KOD1 DNA polymerase

Question 25

• Added to the growing chain • Activated NTPs

Answer 25

NUCLEOTIDES

Question 26

NUCLEOTIDES • Stored at 10 mM, pH____ • Add 20-200 uM in assay

Answer 26

7.0

Question 27

PCR “Reaction” Components

Answer 27

• Taq DNA polymerase • Primers • dNTPs • Butfer • DNA

Question 28

Thermal cycler • Switch on the thermal cycler and set program based on the profile shown • The cycle includes (3) basic steps:

Answer 28

1. Denaturation 2. Annealing 3. Extension

Question 29

Temperature Denaturation Anneling Extension

Answer 29

90-96 50-70 68-75

Question 30

Seconds D A E

Answer 30

20-60 20-90 10-60

Question 31

- Reverse transcriptase enzyme converts RNA into complementary DNA (cDNA).-The cDNA is then amplified using conventional PCR techniques.

Answer 31

Conventional PCR

Question 32

- Multiple primer sets specific to different target sequences are included in a single PCR reaction. - Amplification of multiple targets occurs simultaneously during the same PCR cycling conditions. - Various methods such as primer design, annealing temperature optimization, and multiplex PCR kits are used to ensure specificity and efficiency of amplification.

Answer 32

Real time PCR qPCR

Question 33

- Allows amplification and analysis of RNA molecules. - Useful for gene expression studies and detection of RNA viruses.

Answer 33

Conventional PCR

Question 34

- Saves time and resources by amplifying multiple targets in a single reaction. - Reduces the risk of sample depletion and contamination. - Suitable for high-throughput screening and detection of multiple pathogens or genetic markers

Answer 34

Real Time PCR

Question 35

- Sensitivity can be affected by RNA integrity and efficiency of reverse transcription. - Requires careful primer design and optimization for specific RNA targets. - Potential for non-specific amplification due to RNA secondary structures.

Answer 35

Conventional PCR

Question 36

- Requires careful primer design and optimization to avoid cross-reactivity and non-specific amplification. - Increased complexity may lead to reduced amplification efficiency or sensitivity for individual targets. - Optimization can be challenging due to differences in primer efficiencies and target abundances.

Answer 36

Real time PCR

Question 37

Performing PCR 1. Put your tube in the apparatus 2. Let the program run (____cycles) 3. If primers fit, there is amplification of target DNA 4. If primers do not fit, no amplification product => the DNA was not in the sample 5. Detect if there is PCR product

Answer 37

25-40 cycles

Question 38

AVOID CONTAMINATION • DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. • A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing the reaction mixture. • Fresh gloves should be worn for DNA purification and each reaction set-up.