24 Questions
What should be avoided at the 3′ end of a primer?
Runs of 4 or more bases
What is the recommended range of GC content for maximum product stability?
40-60%
What is the recommended range of melting temperature (Tm) for PCR primers?
55°C and 65°C
Why is it important to avoid self-complementarity in primer design?
To decrease the possibility of primer-dimer formation
What is used to analyze the efficiency and specificity of tested primers?
All of the above
What is the purpose of testing the quality of synthesized primers experimentally?
To evaluate the efficiency and specificity of the primers
What is the purpose of the Polymerase Chain Reaction (PCR) technique?
To duplicate a DNA molecule
What is the temperature at which the denaturation step of PCR takes place?
94°C
What is the function of primers in the PCR technique?
To anneal to the target DNA sequence
Who invented the Polymerase Chain Reaction (PCR) technique?
Kary B. Mullis
What is the name of the thermostable enzyme used in the PCR technique?
Thermus Aquaticus DNA polymerase
What is the benefit of using the PCR technique?
It is a simple, powerful, sensitive, and reliable technique
What is the primary reason why primers that do not have an exact match with the template do not give an extension of the fragment?
The higher temperature breaks the ionic attraction between the primer and template
What is the result of each cycle in PCR?
The number of double stranded DNA pieces is doubled
What is the role of the polymerase during the extension step of PCR?
To read the template from 5' to 3' and add dNTPs
What is the purpose of NCBI Primer-BLAST?
To design primers based on a query sequence
What is the ideal working temperature for the polymerase?
72°C
What is the result of a successful PCR amplification?
Only one band is formed
What is the purpose of downloading the mRNA sequence in fasta format?
To design primers specific to the ACTB actin beta
What is the accession number of the ACTB actin beta used in this exercise?
NM_001101.4
What is the next step after selecting the ACTB of human (gene ID 60) on the NCBI homepage?
Scrolling down to the graphical view of the gene
What is the purpose of the Primer-BLAST tool?
To design primers specific to a target sequence
What format is required for the target sequence in the Primer-BLAST form?
FASTA format or an accession number
What is the dataset selected on the NCBI homepage for this exercise?
Gene
Study Notes
PCR Product Size and Selection
- PCR product size ranges from 100-400 base pairs
- Select primers with a GC content of 40-60% for maximum product stability
- Ensure the 3' end of the primer ends in C or G to promote binding
- Aim for at least 2 G or C bases in the last 5 bases of the primer
- Avoid runs of 4 or more identical bases to prevent primer mispriming
- Optimize melting temperature (Tm) between 55°C and 65°C
- Avoid self-complementarity to decrease primer-dimer formation
Primer Design using NCBI Primer-BLAST
- NCBI provides Primer-BLAST for automatically designing primers based on a query sequence
- Follow steps to design primers specific to the ACTB actin beta gene
- Use RefSeq Accession Number NM_001101.4 for the ACTB gene
- Download the mRNA sequence in fasta format from the NCBI homepage
- Paste the sequence into the Primer-BLAST homepage to design primers
PCR Basics
- PCR (Polymerase Chain Reaction) is a technique that allows for the amplification of specific DNA molecules in vitro
- Invented by Kary B. Mullis in 1985
- PCR is simple, powerful, sensitive, specific, and reliable
- Allows for the duplication of DNA molecules through cycles of enzymatic DNA synthesis
Steps in PCR
- Denaturation at 94°C: Double-stranded DNA template melts, and all enzymatic reactions stop
- Annealing at 54°C: Primers bind to the template
- Extension at 72°C: DNA polymerase adds nucleotides to the primer, synthesizing new DNA strands
PCR Ingredients
- Template DNA
- Primers
- DNA polymerase
- dNTPs (deoxyribonucleoside triphosphates)
- Thermostable enzyme (e.g. Thermus Aquaticus DNA polymerase)
PCR Application
- Amplification of specific DNA molecules in vitro
- Allows for the analysis of DNA sequences
- Widely used in various fields of biological studies
This quiz covers the basics of PCR, including its principle, components, and applications in biochemistry. It's a practical exercise to test your knowledge on primer design and PCR technique.
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