UNIT 7. POLYMERASE CHAIN REACTION

false negative detection.

In diagnotics using NA, a common problem is the limited concentrations of NA available, which causes _________

1. Target Amplification

2. Signal Amplification

3. Probe Amplification

3 General types of amplification techniques

Target Amplification

3 General types of amplification techniques

• a target sequence is well defined and is copied many times in vitro

• Anything outside of the defined target is not amplified.

• PCR

• Transcription-Mediated Amplification (TMA)

• Loop-Mediated amplification (LAMP)

• whole genome amplification (WGA)

Target Amplification examples

Signal Amplification

3 General types of amplification techniques

• a technique of multiplying or amplifying the signal produced while maintaining the amount of the target.

• can be achieved by the sequential hybridization of branching NA structures and continuous enzyme action on the substrate.

• branched DNA (bDNA)

• serial invasive signal amplification

Signal Amplification examples

Probe Amplification

3 General types of amplification techniques

• the probe or a product of a probe that is attached to the target is amplified.

• rolling circle amplification (RCA)

• ligase displacement amplification

Probe Amplification examples

Polymerase-catalyzed chain reaction

original name of PCR

Kary Mullis (1983)

who discovered PCR

Escherichia coli plasmid, pBR322

first successful amplification was made in a short fragment of ___________

amplification of beta globin for the diagnosis of sickle cell anemia

first clinical application of PCR

PCR

described as the in vitro replication of DNA

1-2 hours

PCR allows the production of millions of copies of DNA within ______

• template,

• DNA polymerase,

• deoxyribonucleotide triphosphate (dNTPs or the bases), and

• primers

PCR components

amplicons

The copies produced in PCR is referred to as ______

1. Denaturation

2. Annealing/ Attachment

3. Extension

3 Basic Steps of PCR

Denaturation (94-96°C)

3 Basic Steps of PCR

• allow the separation the double-stranded DNA template into two single-strands.

Annealing (50-70°C)

3 Basic Steps of PCR

• the primers hybridizes or attaches to a specific part of the template that will be amplified

primers

The temperature need in annealing step depends on the _______

optimization;

melting temperature (Tm)

The starting point of _________ can be determined using the _____________________ of the primer sequences

Extension (68-72°C)

3 Basic Steps of PCR

• DNA polymerase synthesizes a copy of the DNA template by adding nucleotide bases to the hybridized primers.

phosphodiester bond

In extension, the addition of bases happens upon formation of ______________ between an incoming base and the template.

two-step amplification;

annealing;

extension

The usual three-step PCR can be shortened to a ________________ if the _________step requires a temperature that is high and close enough to the required __________ temperature

DNA template

COMPONENTS OF PCR

• obtained from the specimen submitted

• genomic or mitochondrial DNA

• from a human patient, or from microorganisms such as viruses, bacteria, fungi, or parasites

DNA isolation techniques

Through various ________________ , high quality and quantity DNA should be obtained to allow successful amplification and detection steps.

100 ng to 1 µg

In routine experiments, _____________ of DNA can be used

Primers

COMPONENTS OF PCR

• are oligonucleotides

• short, single-stranded DNA sequence

• used to hybridize with the sample DNA and define the region of the DNA that will be amplified

18-25 base pairs long;

not be complementary

Primers should be _______________ each, and should ________________ to each other

Tm = 55-65°C

G/C = 50-50%

Melting temperature and G/C content of primers

3’ end;

5' end

Moreover, the primer should have a _____ that hybridizes very well to the template, and the _____ can be initially less complementary to template.

primer dimers & hairpins

Primers should not form

• 0.1-0.5 M in optimal reactions

• 5 pmol of each primer/25 µl PCR reaction

Primer concentrations should be

(0.5 µM);

nonspecific products

Higher primer concentrations _______ may cause the accumulation of _____________ due to priming at nonspecific sites on the template

Taq DNA

The most common replication enzyme used for PCR amplification is

Thermus aquaticus;

heat-resistant enzyme;

half-life of 40 minutes at 95°C (optimal temperature is 72°C)

Taq DNA is isolated from the extremophile _______________, and a _______________ with a ____________________

recombinant protein

Taq DNA polymerase, nowadays, is mostly supplied as a _________________

• Pwo DNA polymerase (hyperthermophilic bacterium Pyrococcus woesi)

• Tth DNA polymerase (Thermus thermophilus species)

Other PCR enzymes

2-4 kilobases per minute

Taq Pol can perform replication at a rate of __________________ at its optimal temperature (~72 °C)

• Independent 5′→3′ exonuclease activity (N-terminal domain, residues 1–290)

• No No 3′→5′ exonuclease activity

Taq polymerase (2) acitivities

Independent 5′→3′ exonuclease activity (N-terminal domain, residues 1–290)

Taq polymerase (2) acitivities

• It can remove nucleotides ahead of it while synthesizing DNA. This allows it to degrade RNA primers or DNA probes during extension (used in techniques like TaqMan assays).

No 3′→5′ exonuclease activity

Taq polymerase (2) acitivities

• It lacks proofreading ability.

• This means it cannot remove incorrectly added bases from the growing DNA strand.

832-amino acid protein

This DNA-dependent DNA polymerase has _________________

75°C-80°C;

-50% sequence homology with E. coli DNA Pol I

optimal polymerization activity is achieved at _________ and has ___________________________________

within the C-terminal-400 residues (the polymerase domain).

N = Met

C = Glu

No cys

The N and C terminal residues of Taq DNA polymerase, with no _____

1-2.5 units per 100 µl reaction volume (most often supplied as 5 U/µL).

he recommended polymerase concentration is usually _______________________________

dNTPs

In the annealing step at 550C, the 5 3 polymerase activity of the DNA polymerase incorporates ______ and synthesizes the daughter strands.

0.2 mM

Each dNTP is usually ________ concentration in the PCR-mix.

0.010-0.015 mM

free dNTPs should be present in the reaction at a concentration not less than _______________

lowering dNTP concentrations (0.01-0.05 mM) and reducing Mg

If using nonproofreading DNA polymerases, fidelity can be improved by ________________________________

pH (8.0–9.5) ;

Tris-HCl

PCR buffer keeps the reaction at the right _______ so DNA polymerase works properly. It is usually stabilized by _______, which maintains a stable chemical environment during heating cycles.

potassium ion (K+);

KCl;

primer annealing.

The PCR buffer also contains ___________ from ____, which promotes ______________

ammonium sulfate (NH₄)₂SO₄;

At times, __________________, may replace KCI in the buffer.

NH₄⁺ ion from ammonium sulfate

The ________ destabilizes weak or mismatched primer-template bindings, which reduces nonspecific binding and increases specificity.

Mg²⁺

_____ is essential because DNA polymerase cannot function without it. It stabilizes primer-template interaction and forms complexes with dNTPs, making them usable substrates for the enzyme.

• With KCl buffer → lower MgCl₂ (around 1.5 mM)

• With (NH₄)₂SO₄ buffer → higher MgCl₂ (around 2.0 mM)

Mg²⁺ concentration in KCl vs (NH₄)₂SO₄

• With KCl buffer →

• With (NH₄)₂SO₄ buffer →

Agarose gel

________ is an easy and cost-effective medium that allows DNA molecules to separate in an electrical field based on size and conformation

polyacrylamide gelelectrophoresis (PAGE).

• matrix formed by the polymers in the gel is much finer than that of agarose, thus, offering superior separation of DNA fragments.

• much more difficult and time consuming than agarose electrophoresis

vertical setup

The PAGE system is typically a __________

ethidium bromide;

UV trans-illuminator

Staining with ____________ and visualizing the gel on a ________________ is the most intercalates with the DNA and this agent will transmit UV light as longer wave fluorescence that is easy to see and photograph

Gelred® stain and uView® dye

An alternative to ethidium bromide is the use of commercially available gel stains that can stain DNA such as ________________________

Gel loading buffer (GLB)

This buffer is added to the sample of the PCR reaction that will be electrophoresed.

bromophenol blue

Loading buffer contains a colourant, like ______________, that will allow you to visualize the sample when loading the gel and track the migrating material in the gel during electrophoresis

1. Nested polymerase chain reaction (Nested PCR)

2. Quantitative polymerase chain reaction (qPCR) or real-time polymerase chain reaction (Real-time PCR)

3. Multiplex polymerase chain reaction (Multiplex PCR)

4. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR

5. Rapid Amplification of cDNA Ends (RACE)

6. Inverse polymerase chain reaction (Inverse PCR)

7. Thymidine-linker polymerase chain reaction (T-linker PCR)

TYPES OF PCR

Nested polymerase chain reaction (Nested PCR)

TYPES OF PCR

• uses two primer sets and two successive PCR reactions for added sensitivity and specificity. In this Nested PCR, the PCR product of the first PCR reaction is used as a template for the second set of primers and the second amplification step.

Quantitative polymerase chain reaction (qPCR) or real-time polymerase chain reaction (Real-time PCR)

TYPES OF PCR

• an detect the amplified gene real time and, thus, can quantify gene expression and confirm differential expression of genes. It uses fluorescence dyes in detecting the nucleic-acid sequences.

Multiplex polymerase chain reaction (Multiplex PCR)

TYPES OF PCR

• is used to amplify two or more genes in one PCR run. In Multiplex PCR, two or more primer sets are used for amplification of different targets to save time and effort.

Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)

TYPES OF PCR

• is a modified PCR in which oligonucleotides of partially degenerate sequence are used in amplification. Oligonucleotides of partially degenerate sequence are generated in primer-design software based on the amino acid sequence.

Rapid Amplification of cDNA Ends (RACE)

TYPES OF PCR

• is a modified PCR technique in which amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and unknown sequences at either the 3' or the 5'-end of the mRNA is performed.

Inverse polymerase chain reaction (Inverse PCR)

TYPES OF PCR

• is used to amplify unknown sequences to be cloned by PCR, provided that they are located near a known sequence.

Thymidine-linker polymerase chain reaction (T-linker PCR)

TYPES OF PCR

• is a chromosome walking technique that uses specific T/A ligation (not random ligation). It takes advantage of the fact that Taq DNA polymerase naturally adds a single A (adenine) at the 3′ end of newly synthesized DNA.

3' overhang enzymes;

specific primer;

Taq DNA polymerase;

single A tail;

3' overhang-T linker (named T-linker);

nested PCR;

specific primers and T-linker primers.

In T-linker PCR, genomic DNA is digested with _____________ and primed by a __________, a strand of the target molecule is replicated by______________ and a ___________ is generated on the 3' unknown end of the target molecule, and then, a _____________ is specifically ligated onto the target. Then, the target is amplified by two rounds of ___________ with _________________

1) diagnosis of pathogen,

2) typing genetic markers,

3) DNA template cloning,

4) detecting point mutation,

5) for CDNA cloning,

6) genomic DNA cloning,

7) genome walking, and in vitro mutagenesis

Applications of PCR

PCR

Nested PCR

qPCR or Real-time PCR

Multiplex PCR

PCR utilized in Diagnosing pathogens

Allele specific amplification

PCR utilized in Typing genetic markers

Allele specific amplification

PCR utilized in Detecting point mutations

DOP-PCR

RACE

PCR utilized in DNA cloning

DOP-PCR

whole genome or sub genomic amplification

T-Linker PCR

PCR utilized in Genomic DNA cloning

Inverse PCR

T-Linker PCR

PCR utilized in Genome walking