CHAPTER 1

Basic Principles and Components of PCR

Sandeep Chapagain
Graduate Student
Plant Genomics Lab
Division of Bioresources sciences
Kangwon National University
 Polymerase Chain Reaction (PCR)

• PCR The polymerase chain reaction (PCR) is a scientific technique in

molecular biology to amplify a single or a few copies of a piece of DNA
across several orders of magnitude, generating thousands to millions of
copies of a particular DNA sequence.

• PCR can make billions of copies of a target sequence of DNA in a few hours.

• PCR was invented in the 1984 as a way to make numerous copies of DNA
fragments in the laboratory.

• Its applications are vast and PCR is now an integral part of Molecular
Biology.
 Why named PCR?

 Polymerase: It is called “polymerase” because the only enzyme used in this

reaction is DNA polymerase.

 Chain: It is called “chain” because the products of the first reaction become
substrates of the following one, and so on

 Reaction: Use of different reaction enzymes and buffers eg. dNTP, buffers
 A basic PCR components and reagents
1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - a short segment of DNA that are complementary to the

3' ends of each of the sense and anti-sense strand of the DNA target, they
are needed to get DNA synthesis started.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Taq DNA Polymerase - enzyme that catalyzes the reaction/ manufacture

DNA copies

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the reaction

solution suitable for the activity of the enzyme
 Two Oligonucleotide primers are needed
for PCR reactions

Region between the two primers will be synthesized

• One primer is complementary to one strand of DNA at one end of the

target region
• The other primer is complementary to the other strand of DNA at the
other end of the target region
 How PCR Works
• Protocol

– Put all reagents into a PCR tube

• Melting or heat denaturation

– Bind each primer to its appropriate strand

• 5’ primer to the 5’ to 3’ strand

• 3’ primer to the 3’ to 5’ strand

– Annealing The reaction

– Copy each strand

• DNA polymerase
– Extending TUBE
THERMOCYCLER
(PCR machine)
 Principle of PCR

 Purpose: To amplify a lot of double-stranded DNA molecules (fragments)

with same (identical) size and sequence by enzymatic method and cycling
condition

Condition:
1. Denaturation of ds DNA template

2. Annealing of primers

3. Extension of ds DNA molecules

 The three main steps of PCR
• The basis of PCR is temperature changes and the effect that these temperature
changes have on the DNA.
• In a PCR reaction, the following series of steps is repeated 20-40 times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA fragment
of interest 100,000 fold)

Step 1: Denature DNA (Denaturation)

At 95C, the DNA is denatured (i.e. the two strands are separated)

Step 2: Primers Anneal (Annealing)

At 40C- 65C, the primers anneal (or bind to) their complementary sequences
on the single strands of DNA

Step 3: DNA polymerase Extends the DNA chain (Extension)

At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the
3’ ends of the primers.
 PCR condition; temperature Protocol
• Initial Melt: 94ºC for 2 minutes
• Melt: 95ºC for 20-30 seconds
• Anneal: 55-65ºC for 20-30 seconds
• Extend: 72ºC for 30-60 seconds 30-35
• Final Extension: 72ºC for 3 minutes cycles
• Hold: 4ºC
PCR consist of 3 repeated cycles of DNA synthethsis

Source: Hartwell et al., 4th ed., Chapter 9

 Three basic steps in PCR

(3) Polymerization
(1) Denature (2) Base pairing of from primers
strands primers along templates
DNA template

Primer

Source: Hartwell et al., 4th ed., Chapter 9

Exponential increase in the amount of
target DNA during PCR
 Applications of PCR

• Classification of organisms • Detection of pathogens

• Genotyping • DNA fingerprinting

• Mutagenesis • Drug discovery

• Mutation detection • Genetic matching

• Sequencing • Genetic engineering

• Cancer research • Pre-natal diagnosis

The Nobel Prize in Chemistry 1993
Kary B. Mullis

Kary B. Mullis
Prize motivation:
"for his invention of the
polymerase chain reaction (PCR)
method"
 How to observe PCR product?
Preparing an agarose gel for electrophoresis
Distinguishes DNA fragments by Gel electrophoresis
 place gel in buffered aqueous solution,
 Load DNA samples into wells in gel and apply electric current
• Electrophoresis (movement of charged particles in an electric field)
• DNA has negative charge, so moves toward positive charge
Distinguishes DNA fragments by Gel electrophoresis

Determine size of unknown fragments

by comparison to migration of DNA
markers of known size
 Gene expression/ quantification
Introduction to quantitative real-time
PCR
 What is Real-Time qRT-PCR?
 An in vitro method for enzymatically amplifying defined sequences of RNA
 From all the available quantification techniques it has the highest
sensitivity, reproducibility, simplicity and dynamic range

 Real-Time PCR has become a cornerstone of molecular biology:

 Gene expression analysis
– Cancer research
– Drug research
 Disease diagnosis and management
– Viral quantification
 Food testing
– Percent GMO food
 Animal and plant breeding
– Gene copy number
 Real Time
• signals (generally fluorescent) are monitored as they are generated and are
tracked throughout the program
 Quantitative
• Quantitatively measures the amplification of template

 Reverse Transcription
• Refers to the reverse transcription of the RNA starting material into cDNA
• This step can be conducted in a one-step or more traditionally two-step
method

First generate cDNA

then perform PCR

 Polymerase Chain Reaction

• Method dependent on thermo cycling and enzymes allowing for amplification
of small starting material of DNA
 qRT-PCR – The Basics
1. Isolate RNA from samples
2. Reverse Transcription
3. Pick Reference Gene
4. Design Primers
5. Run qRT-PCR
1. Fluorescent signal (eg. Taqman,
SYBERGreen)
2. Acquire signal at end of each cycle
6. Analyze
1. Set Threshold
2. Obtain CT values
The assay will be tested in triplicate (three technical replicates)
Understanding the Output…

PCR has three phases:

• Exponential
• Earliest segment in the PCR
• Product increases exponentially
• Reagents are not limited

• Linear
• Linear increase in product
• PCR reagents become limited

• Plateau
• Later cycles of PCR
• Reagents become depleted
• Amplification not equal
 Normalisation
• Housekeeping gene(s)
• Accounts for pipetting error and differences in RT efficiencies between
samples
• Constantly expressed at a stable level
• Put the same starting RNA in all RT-reactions when samples are to be
compared
• IT IS NOT THERE TO ACCOUNT FOR MASSIVE DIFFERENCES IN
STARTING MATERIAL!

• Calibrator sample
• Sample run on every assay
• T0 / Untreated
• Compare everything to this sample
 Controls

• NTC (No template control)

• PCR contamination detection
• Primer dimers

• no-RT control
• Detection of genomic contamination and/or non-specific product
• Especially important in transient over-expression experiments to
check for carry over of plasmid DNA
 Dissociation or Melt curve analysis
 To check for single specific product in SYBR green based assays
 Compare to product on gel in early stages of set-up
 Detection of primer dimers and non-specific products

Examples of Melt curve

NTC
Primer dimers Specific product

Specific Non-specific product

product

In this case NTC’s showed no product, so no primer dimers, large non-specific product observed on gel
 What is a standard curve and why is it necessary?

 Real time PCR conducted across a series of serially diluted template/samples

 Tell you what the dynamic (linear) range of the assay is
 Allows calculation of efficiency

Cycle number Ct or take-off

Efficiency
 Efficiency = 10(-1/gradient)-1
 Gradient of -3.32 = 100%
 efficient90-110% considered ok
(-3.1 to -3.6)

Log10 template concentration

Quantification methods- Absolute Quantification
• Samples calculated against a standard curve of known concentration

• Allows quantification as copy number

• Can be used when one condition contains no expression

• Takes some variability into account

• Requires more reagents

• Needs a known standard

• Plasmid DNA containing product – no RT step so efficiencies may be

different
• In-vitro transcription to generate specific mRNA – RT, costly
 Quantification methods- ΔCt/ΔΔCt

Comparative quantification
Threshold cycle number – Ct
Slightly subjective
Calibrator – i.e. T0 or untreated
Normalized to housekeeper (HK)
ΔCt = CtGOI-CtHK
Δ ΔCt = ΔCtsample- Δctcalibrator
Fold change = 2- Δ ΔCt
Assumes efficiency 100% for both GOI and HK
Can substitute 2 for your calc efficiency but must be the same
for GOI and HK
THANK YOU FOR YOUR ATTENTION..