PCR Flashcards
Who invented the polymerase chain reaction (PCR) technique?
Kary B. Mullis
PCR was invented in 1985.
What is the primary function of PCR?
To make millions of copies of a scarce sample of DNA.
What method was primarily used before PCR for producing many copies of a gene?
DNA cloning.
What happens to double stranded DNA during the PCR process?
It ‘melts’ to become single stranded DNA.
What is Taq polymerase used for in PCR?
To make a copy of the DNA sequence.
Fill in the blank: The chemical reaction that happens during PCR relies on several key chemical components, including _______.
[Template DNA, Primers, dNTPs, Taq Polymerase, Buffer, MgCl2, sH2O]
What are the three main steps in a PCR cycle?
Denaturation, Annealing, Extension.
What is the temperature used for denaturation in PCR?
Approximately 95°C.
What occurs during the denaturation step of PCR?
The hydrogen bonds between base pairs break, separating the double stranded DNA.
What is the purpose of the annealing step in PCR?
To allow primers to bind to their complementary sequences on the single stranded DNA.
What temperature range is typically used for the annealing step?
Between 30 and 65°C.
What does the extension step involve in PCR?
Taq polymerase synthesizes a new DNA strand by adding dNTPs to the template strand.
How many times can the three PCR steps repeat?
Up to 35 times.
What is a master mix in PCR?
A mixture of all PCR reagents minus the template DNA.
What is the typical concentration of template DNA added to a PCR reaction?
10ng.
What are the three controls required for PCR?
Reagent control, Negative control, Positive control.
True or False: The negative control in PCR contains the sequence of interest.
False.
What are primers in PCR?
Short ssDNA sequences that bind to template DNA at specific locations.
What is the typical concentration range for primers in a PCR reaction?
0.1–0.5 μM.
What are dNTPs used for in PCR?
They are the raw materials used by Taq polymerase to make copies of the template strand.
What are the four types of dNTPs used in PCR?
ATP, TTP, CTP, GTP.
What is the role of Taq polymerase in PCR?
To synthesize new DNA from the 3’ end of primers bound to template DNA.
From which organism is Taq polymerase isolated?
Thermus aquaticus.
What is the importance of MgCl2 in PCR?
It is important for Taq polymerase function and aids in primer annealing.
What should the concentration of MgCl2 be in a typical PCR reaction?
Between 0.1-0.5 mM.
What is the purpose of using a buffer in PCR?
To maintain pH and appropriate salt concentrations for enzymes to function.
What is the total volume of a typical PCR reaction?
50uL.
What type of instrument is used for performing PCR cycles automatically?
Thermal cyclers.
What temperature is typically used for the extension step in PCR?
Between 60 and 75°C.
What is the key purpose of optimizing a PCR reaction?
To ensure a specific target DNA sequence is amplified without creating non-specific byproducts.
What is a primer dimer in PCR?
A non-specific byproduct that occurs when primers bind to each other instead of the template DNA.
What are some variables that need optimizing in PCR?
Primer design, Template concentration, MgCl2 concentration, Taq concentration, Annealing temperature, Cycle number.
What is the recommended length for primers to ensure specificity?
Between 18-30 bp.
What are the variables that need optimizing in PCR design?
- Primer design
- Template concentration
- MgCl2 concentration
- Taq concentration
- Annealing temperature
- Cycle number
- Inhibitors
- Additives
What is the ideal length for primers in PCR?
18-30bp
What is the ideal melting temperature range for a primer?
55°C - 70°C
How is melting temperature (Tm) roughly calculated?
Tm = 4(G+C) + 2(A+T)
What should the 3’ end of a primer end with for optimal binding?
A G or a C
What concentration range is typical for primers in a PCR reaction?
0.1–0.5 μM
What happens if primer concentration is too high?
Non-specific amplification occurs due to more opportunities for off-target binding
What occurs if primer concentration is too low?
Not enough product is produced
What issues arise from too high template concentration in PCR?
- Running out of reagents
- Inhibiting Taq activity
What problems can occur with too low template concentration?
- Insufficient PCR product
- Non-specific annealing of primers
- Primer dimers
What is the effect of high MgCl2 concentration in PCR?
Non-specific primer binding
What is the effect of low MgCl2 concentration in PCR?
Primers will not bind
What is the maximum number of PCR cycles typically performed?
35 cycles
Name some common chemicals that can inhibit PCR.
- Heparin
- Salt
- Heme
- SDS
- EDTA
- Protein
- Phenol
What are some common additives used in PCR?
- DMSO
- Formamide
- Glycerol
- Polyethylene glycol
- Gelatin
- 7-deaza-dGTP
What does DMSO do in PCR?
Increases specificity and lowers Tm of primer/template hybridization
What is the purpose of formamide in PCR?
Increases specificity and improves amplification of high GC templates
What role does glycerol play in PCR?
Increases specificity and lowers Tm of primer/template hybridization
What is the function of gelatin in PCR?
Neutralizes inhibitors of Taq and increases yield
What is hot-start PCR?
A modification of PCR to prevent non-specific amplification by activating the polymerase only at high temperatures
What is nested PCR?
A technique that uses two sets of primers to increase specificity and sensitivity
What is reverse transcriptase PCR (RT-PCR)?
Uses RNA as a template and reverse transcriptase to synthesize cDNA
What does qPCR stand for?
Quantitative real-time PCR
What is the cycle threshold (CT) value in qPCR?
The cycle at which fluorescence exceeds 10x standard deviation of the background signal
What are some applications of PCR?
- Genetic disorder diagnosis
- Prenatal diagnosis
- Cancer detection
- Forensics
- Ancient DNA amplification
- Sequencing
- Infection testing
What is the purpose of HLA typing in PCR applications?
To determine immune responses and organ transplant compatibility
What is erythrocyte typing?
Identifying blood group antigens to prevent hemolytic reactions
How does PCR aid in human identification?
By creating a unique DNA fingerprint through SNPs and STRs
What is the benefit of using PCR for infection testing?
Allows for accurate identification of microbes without culturing
What is the typical composition of a reverse transcriptase master mix?
- Buffer
- MgCl2
- dNTPs
- Primer
- RNase inhibitor
- Reverse transcriptase
True or False: All bands in a PCR gel indicate successful amplification.
False
What can cause no bands in PCR reactions?
- Taq inhibition
- Pipetting error
- No DNA added
