PCR: Polymerase Chain Reaction Primers Design Practical Exercise

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What should be avoided at the 3′ end of a primer?

Runs of 2 or more bases

Runs of 8 or more bases

Runs of 4 or more bases (correct)

Runs of 6 or more bases

What is the recommended range of GC content for maximum product stability?

60-80%

80-100%

40-60% (correct)

20-40%

What is the recommended range of melting temperature (Tm) for PCR primers?

50°C and 60°C

45°C and 55°C

55°C and 65°C (correct)

60°C and 70°C

Why is it important to avoid self-complementarity in primer design?

To decrease the possibility of primer-dimer formation Signup and view all the answers

What is used to analyze the efficiency and specificity of tested primers?

All of the above Signup and view all the answers

What is the purpose of testing the quality of synthesized primers experimentally?

To evaluate the efficiency and specificity of the primers Signup and view all the answers

What is the purpose of the Polymerase Chain Reaction (PCR) technique?

To duplicate a DNA molecule Signup and view all the answers

What is the temperature at which the denaturation step of PCR takes place?

94°C Signup and view all the answers

What is the function of primers in the PCR technique?

To anneal to the target DNA sequence Signup and view all the answers

Who invented the Polymerase Chain Reaction (PCR) technique?

Kary B. Mullis Signup and view all the answers

What is the name of the thermostable enzyme used in the PCR technique?

Thermus Aquaticus DNA polymerase Signup and view all the answers

What is the benefit of using the PCR technique?

It is a simple, powerful, sensitive, and reliable technique Signup and view all the answers

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 Signup and view all the answers

What is the result of each cycle in PCR?

The number of double stranded DNA pieces is doubled Signup and view all the answers

What is the role of the polymerase during the extension step of PCR?

To read the template from 5' to 3' and add dNTPs Signup and view all the answers

What is the purpose of NCBI Primer-BLAST?

To design primers based on a query sequence Signup and view all the answers

What is the ideal working temperature for the polymerase?

72°C Signup and view all the answers

What is the result of a successful PCR amplification?

Only one band is formed Signup and view all the answers

What is the purpose of downloading the mRNA sequence in fasta format?

To design primers specific to the ACTB actin beta Signup and view all the answers

What is the accession number of the ACTB actin beta used in this exercise?

NM_001101.4 Signup and view all the answers

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 Signup and view all the answers

What is the purpose of the Primer-BLAST tool?

To design primers specific to a target sequence Signup and view all the answers

What format is required for the target sequence in the Primer-BLAST form?

FASTA format or an accession number Signup and view all the answers

What is the dataset selected on the NCBI homepage for this exercise?

Gene Signup and view all the answers

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

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

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.