L6 PCR

一、Basics about PCR

Main steps, Cycling condition, Buffer composition

1. Main Steps

1. Initial denaturation
2. Denaturation
3. Anneling
4. Extension
5. Final Extension
6. Hold

the components in the PCR reaction mix

The chemicals in the 10X PCR reaction buffer

1. Tris-HCL pH 8.0-pH9.0
2. KCL - potassium ion promotes primer annealing
3. magnesium ions (Mg2+ from MgCl2) , is a cofactor for Taq polymerase
   • magnesium ions also facilitate primers-DNA binding by stabilizing negative charges on phosphate backbones. (stabilize the pairing)
   • Often around 1.5mM final concentration
   • What would happen if you raise Mg2+ concentration ?
     • For difficult templetes, increase the Taq activity
     • For 较多杂带，Increase the specificity of PCR
   • What else can be included into the buffer ?
     • DMSO: Help denaturation (For high G-C content)

Fidelity of DNA polymerase in PCR

1. Thermo stable DNA polymerase

1. Taq
2. ExTaq, LATaq
3. Pfu
4. KOD
5. Q5
6. Phusion

Concerns in choosing different PCR polymerases

• The fidelity of polymerase – Proof reading activity
  • How accurate the polymerase in copying the DNA template
• The processivity of polymerase
  • How fast of the elongation rate
  • Whether the polymerase can copy DNA from “difficult” template, long template or template that has secondary structure.

Proof-reading activity

Error-prone PCR – a example to manipulate fidelity of polymerase

Comparison of fidelity among different DNA polymerase

2. Different DNA end generated by different type of DNA polymerase

• 3’ end with A overhang – Taq polymerase and its varieties.
• Blunt end – most of proof reading polymerases
• Sticky end – not by PCR

PCR Primer Design

• Primer Length, can be anything more than 15nt, practically between 18-25nt
• Primer Melting Temperature, typically between 50-60 degree
• GC Content: Typically should be 40-60%
• 3’ end stability
• Primer Secondary Structures
• Repeats and Runs

A few tips to get your PCR to work

• PCR is also short for ” Please Check you Result”
• Repeat, repeat and repeat
• A decent amount of full length template
• Primer optimization
• Gradient PCR /Touch up/Touch Down/ Hot start
• Mg2+ concentration

二、Basics about PCR

Applications of PCR

Basic Biomedical Research

• Amplifying specific DNA for downstream applications like
• Cloning and expression of recombinant proteins
• Gene expression analysis

Diseases diagnosis

• Mycobacterium tuberculosis – infection/drug resistance
• HIV – RT-PCR
• Sickle cell anemia (normal, carrier, diseased)

Forensics

• Crime Scene Investigation
• Paternity testing

RT-PCR

1. Synthesis of first strand cDNA
2. PCR components added, the second strand cDNA synthesized
3. amplify cDNA

1. Reverse transcriptase reaction – Priming strategies

Three strategies:

1. Converts all RNA to cDNA
2. Converts mRNA to cDNA
3. Converts specific RNA to cDNA

Realtime Quantitative PCR

How could we monitor the PCR reaction in real-time ?

• dsDNA specific Dye and plate reading enables monitoring of PCR in real-time

Different chemistry for quantification- SYBR Green 1

• Unbound SYBRE Green 1 DNA- binding dye in solution exhibits very little fluorescence. During primer extension and polymerization SYBR Green 1 molecules become intercalated within the double stranded DNA product, resulting in an increase in detected fluorescence

Different chemistry for quantification - TaqMan Probe

1. When the Taqman probe is intact, the reporter and quencher stay close to each other, which prevents the emission of any fluorescence
2. The primer and Taqman probe anneal to the complementary DNA strand following denaturation
3. After hybridization and during the extension phase, the 5 endonuclease activity of the Taq DNA polymerase cleaves the probe, which separates reporter and quencher dyes and fluorescence is detected

What are the assumptions behind “quantitative” ?

• The short answer is “exponential amplification”

Different phase of PCR amplification

1. Exponential:
   • Exact doubling of product
   • Reaction is very precise and specific
2. Linear:
   • The reaction components are becoming limited
   • The reaction efficiency is dropping
3. Plateau:
   • The reaction has stopped
   • No more products are being made

A exponential curve looks “linear” if you put Y-axis in log scale

What are the assumptions behind “quantitative” ?

• PCR gives exponential amplification of target DNA, so the amount of target DNA can be calculated based on cycler number. – Key word: exponential.

1. The idea of Ct(Cq) value and threshold

$\Delta$Rn

• Fluorescence emission of the product at each time point fluorescence emission of the baseline

Ct values

• cycle number at which threshold fluorescence is achieved

Relative quantification: The principle of $\Delta$Ct method

Within exponential phase, the difference of Ct value between samples reflect the difference of initial target concentration between samples.
$$
2^{-(Ct_{SampleA} – Ct_{sampleB})} = \text{Concentration A/B}
$$

Relative quantification: The idea of internal control

Internal Control is needed in order to achieve accurate relative quantification

Delta delta Ct method

• The delta-delta Ct method, also known as the 2^–∆∆Ct^ method, is a simple formula used in order to calculate the relative fold gene expression of samples when performing real-time PCR.

That means, to compare the relative fold gene expression, we should calculate the relative expression level compared to the control group, that is, suppose we want to compare gene A and the Wild type:
$$
\frac{\text{A}}{\text{wild}} = \frac{\text{Mutant_A/Mutant_Control}}{\text{WT_A/WT_Control}} =
$$

$$
2^{-[(Ct_{mutant_A} - Ct_{mutant_Control}) - (Ct_{WT_A} - Ct_{WT_Control})]}
$$

Absolute quantification

• Series dilution of target DNA with Known concentration
• Calculate the DNA concentration in your sample based on standard curve

Melting Curve: An indicator, not a diagnosis

• Melting curve analysis is an assessment of the dissociation characteristics of doublestranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature.

What are the assumptions behind “relative quantification”?

• PCR is exponential – the efficiency of your PCR is two, within a certain range of target DNA concentration.
• The PCR efficiency of both target DNA and internal control are close to 2.
• The Ct between internal control and target are relatively close.
• The PCR efficiency between samples are the same
• The reverse transcription efficiency between samples are the same.

How to make sure such assumptions are true in your experiment?

1. Always check the efficiency of your qPCR
2. Try to include serious dilutions of samples – PCR efficiency and good dynamic range
3. Always start with same batch of samples and same amount of samples
4. A good internal control