POLYMERASE CHAIN REACTION Flashcards
3 General types of amplification technique
• Target amplification
• Signal amplification
• Probe amplification
• Invented in 1983 by
Dr. Kary Mullis
• An in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence
PCR
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.)
DNA polymerase
primer
– The DNA sample that contains the specific sequence you want to amplify. It serves as the blueprint for new DNA strands.
Template DNA
– 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.
Downstream Oligonucleotide Primer (Reverse Primer)
– A short single-stranded DNA that binds to the 3’ end of the complementary strand, initiating DNA synthesis in the opposite direction.
Upstream Oligonucleotide Primer (Forward Primer)
: A heat-stable enzyme that synthesizes new DNA strands by adding nucleotides to the primers.
Taq DNA Polymerase
– Provides Mg²⁺ ions, which are essential cofactors for Taq polymerase, helping it function properly and improving enzyme efficiency.
25 mM MgCl₂ (Magnesium chloride)
– A mixture of deoxynucleotide triphosphates (dATP, dTTP, dGTP, dCTP) that serve as the building blocks for new DNA strand synthesis.
dNTP Mix (10 mM of Each dNTP)
PCR
Two Problems
• 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
TEMPLATE DNA
• Target DNA
• Contains the region/sequence to be amplified
• Up to____
•____ in a 50 uL total reaction mixture
3Kb
0.1-1 ug
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
15-30 nucleotides
40-60%
Annealing
PRIMERS
• Two primers must be designed for PCR:
Forward primer
Reverse primer
• the_________ or primer is complimentary to the 3’ end of antisense strand (3’-5’)
forward primer
• the______ primer is complimentary to the 3’ end of sense strand (5’-3’)
reverse primer
• Stabilizes the DNA polymerase, DNA, and nucleotides
BUFFER
• Stabilizes the DNA polymerase, DNA, and nucleotides
• 500 mM_____
• 100 mM_____
KCl
Tris-HCl (8.3)
• The medium for all other components
Water
• Essential cofactor of DNA polymerase
• Stabilizes the DNA double helix
MAGNESIUM
MAGNESIUM (cofactor)
• Too little:_____
• Too much:______
• Used at____ to ____ in the assay
enzyme will not work
non-specific amplifications
0.5 to 3.5 uM
Precautions:
• Completely____ magnesium solution
•____ magnesium solution for
several seconds before pipetting
• If DNA samples contain EDTA…
thaw
Vortex
• The enzyme that does the extension
• Heat stable
• Approximately _____of Taq DNAPol per 50 uL reaction
DNA polymerase
1.25 U
POLYMERASE
Variants:
• Taq –_______
• Pfu –
• KOD DNA Polymerase – a recombinant form of_____
Thermus aquaticus
hyperthermophilic Pyrococcus
furiosus
Thermococcus kodakarensis KOD1 DNA polymerase
• Added to the growing chain
• Activated NTPs
NUCLEOTIDES
NUCLEOTIDES
• Stored at 10 mM, pH____
• Add 20-200 uM in assay
7.0
PCR “Reaction” Components
• Taq DNA polymerase
• Primers
• dNTPs
• Butfer
• DNA
Thermal cycler
• Switch on the thermal cycler and
set program based on the profile
shown
• The cycle includes (3) basic steps:
- Denaturation
- Annealing
- Extension
Temperature
Denaturation
Anneling
Extension
90-96
50-70
68-75
Seconds
D
A
E
20-60
20-90
10-60
- Reverse transcriptase enzyme converts RNA into complementary DNA (cDNA).-The cDNA is then amplified using conventional PCR techniques.
Conventional PCR
- 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.
Real time PCR
qPCR
- Allows amplification and analysis of RNA molecules.
- Useful for gene expression studies and detection of RNA viruses.
Conventional PCR
- 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
Real Time PCR
- 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.
Conventional PCR
- 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.
Real time PCR
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
25-40 cycles
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.
