Primer Design For Polymerase Chain Reaction-Tips & Tricks

Name: Primer Design For Polymerase Chain Reaction-Tips & Tricks
Rating: 4.3 (206 reviews)

DNA primer design, what your mentor never taught you

Created byShahd S

Last updated 11/2020

English

What you'll learn

Understand how different parameters can affect your primer specificity and sensitivity
Understand the basics of Polymerase chain reaction in detail
Learn how to avoid non-specific bands in your gel by following a specific guideline for DNA primer design
Real example on how to design DNA primer using the required program
Understand how to analyse gel electrophoresis results.

Course content

4 sections • 24 lectures • 1h 13m total length

Introduction2:14
Lecture 1. PCR Cycling3:06
This video provides an introduction to the Polymerase Chain Reaction (PCR) technique.

The video explains:

Components of the PCR reaction: DNA template, DNA polymerase, dNTPs, forward primer and reverse primer.
Steps involved in the PCR (PCR cycling: denaturation, annealing, and extension.
Lecture 2. Standard Amplification Program + Exponential Amplification3:13
This video explains:
The standard PCR amplification program.
The exponential amplification.
Lecture 3. The size difference between the PCR amplification products of the 1st2:57
In PCR amplification, how many dsDNA products we get, where the two strands of the dsDNA, are with the same length of the target sequence?

Let's find out in this lecture!
Quiz

Lecture 4. List of the PCR Primer Design Criteria + Two Resources1:44
Why we need to learn how to design a DNA primer?

Have you ever got a non-specific band in your gel electrophoresis runs? Or got a band with a different size than your target DNA? Well, I bet the answer is yes. How a primer sequence can lead to this?

In this session, we are going to learn how to design a DNA primer that really work.

First, let's watch this video that:

Lists the primer design criteria.
Define the primer specificity and sensitivity.
Lecture 5. The DNA Primer Length2:54
How does the primer length play a role in the designing criteria?

Well, let's find out in this lecture.
Lecture 6. Primer Melting Temperature (Tm)-Wallace equation, Marmmur & Doty equa3:56
There is 4 approximation methods used to calculate the primer melting temperature (Tm), each one gives a reasonable result for a primer with a certain length.

In this lecture, we are going to explain in details, how to calculate the Tm using 3 calculation methods:

Wallace equation.
Marmur and Doty equation.
Salt-adjusted method.

Keep your full attention.
Lecture 7. How to Calculate Enthalpy, Entropy, and Free Energy5:25
Enthalpy, entropy, and free energy are somehow difficult for the beginners to understand instantly. This lecture explains them in a very easy way...
Lecture 8. Primer Tm-The Nearest Neighbor Method2:54
The nearest-neighbor method is more complicated than the other melting temperature calculation methods. In this lecture we simplified the complicated parts for you.
Lecture 9. Annealing Temperature (Ta) of the DNA Primer3:02
The Annealing temperature (Ta), is at which the primers anneal (or bind) to the single stranded DNA and form a hydrogen bond.

This lecture will explain:

How do we determine the annealing temperature empirically?
What happens if the Ta is too low?
What happens if the Ta is too high?
What is the required Ta, for the forward primer and the reverse primer, to attach to the ssDNA at the same time?
Lecture 10. GC-Content of the DNA Primer2:30
This lecture explain

The importance of the GC-content of the primer.
How do we calculate the GC-content percentage?
What is the ideal GC-content percentage of the primer?
Lecture 11. GC Clamp at the 3’ End of the DNA Primer4:01
In this lecture, you will understand:

GC Clamp definition.
The importance of the GC Clamp at the 3' end of the DNA primer.
Designing criteria to be considered at the 3' end of the DNA primer.
Lecture 12. Primer Secondary Structure5:25
This lecture explains:

The primer secondary structure definition.
Types of the primer secondary structure (primer self-dimer, primer cross-dimer, primer hairpin).
Guidelines must be considered to avoid the primer secondary structure.
When do we consider the primer secondary structure as harmless.
Lecture 13. Runs & Repeats in the primer2:54
This lecture explains:

What do we mean by a primer with runs?
What do we mean by a primer with repeats?
Lecture 14. Cross homology0:43
Lecture 15. Primer product length0:37
Learn how to measure the primer product length.
Quiz- 2
Quiz- 3

Lecture 16. Retrieve a Gene Sequence form NCBI, and Determine the Exact Location6:13
lecture 17. Compare Different mRNA Transcripts and Select One to Evaluate a Gene4:56
Lecture 18. Understand Primer3 Setting + Primer Self-complementrity score4:39
Lecture 19. Calculate Primer Self-Complementarity Score0:39
Lecture 20. Check for Primer Cross Homology Using BLAT2:28
Lecture 21. Evaluate Primers Depending on Delta G4:09

Requirements

Interest in learning
Open mind

Description

In this course you will learn:

The basic concepts of the standard polymerase chain reaction (PCR) technique.
The criteria required to design a DNA primer for PCR.
The online programs we usually use to design a DNA primer.
Tips for troubleshooting gel electrophoresis results.

Polymerase chain reaction (PCR)

You will learn in this section:

Steps involved in the PCR (PCR cycling): denaturation, annealing, extension
Components of the PCR reaction: DNA template, DNA polymerase, dNTPs, forward primer and reverse primer.
PCR amplification program.
Exponential amplification.
The size difference between the PCR amplification products of the first, second, and third cycle.

Criteria for PCR primer design

You will learn in this section a detailed explanation of the PCR primer design criteria and how they affect the primer sensitivity and stability including; primer length, primer melting temperature, primer annealing temperature, GC% of the primer, GC-clamp, cross homology and primer secondary structure.

Tools and methods

In this section you will learn how to:

Retrieve a Gene Sequence form NCBI, and Determine the Exact Location for Each Exon on the Chromosome Using Graphics.
Compare Different mRNA Transcripts and Select One to Evaluate a Gene Expression in Novel Cells.
Understand Primer3 Setting.
Calculate Primer Self-Complementarity Score.
Check for Primer Cross Homology Using BLAT.
Evaluate Primers Depending on Delta G.

Gel Electrophoresis Troubleshooting

In this section you will find tips for successful gel electrophoresis. It includes all the possible problems you may encounter, and suggest you a solution for each problem.

Who this course is for:

Biology students can take this course

Primer Design For Polymerase Chain Reaction-Tips & Tricks

What you'll learn

Explore related topics

Course content

Polymerase Chain Reaction4 lectures • 12min

PCR Primer Design Criteria12 lectures • 36min

Tools and Methods (How to...)6 lectures • 23min

Gel Electrophoresis Troubleshooting2 lectures • 3min

Requirements

Description

Who this course is for: