INTRODUCTIONINTRODUCTION

Introduction

  In the past, amplifying (replication) of DNA was done in bacteria and took weeks.

In 1971, paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro

However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis

He was awarded the Nobel Prize in Chemistry in 1983 for his invention

Introduction

Definition:

The polymerase chain reactionThe polymerase chain reaction (PCR) is a technique (widely used in molecular biology) used to amplify (i.e., replicate) a piece of DNA(target DNA) by in vitro enzymatic replication.

Introduction

It derives its name from one of its key components, a DNA polymerase which is imprtant enzyme has essential role into DNA replication

As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified.

PRIMER DESIGNPRIMER DESIGN

Primer design

A basic PCR set up requires several components and reagents. These components include:

1- thermocycler The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step

A very old three-temperature thermal cycler for PCR

Primer design

An old thermal cycler for PCR An new thermal cycler for PCR

Primer design

2- eight PCR tubes, each tube contains 15-100 mcl from DNA sample

Primer design

3- DNA template that contains the DNA region (target) to be amplified.

4- One or more primers, which are complementary to the DNA regions at the 5' (five prime) and 3' (three prime) ends of the DNA region.

5- a heat-stable DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.

A 1976 discovery of Taq polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)

Primer design

6- Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand.

Primer design

7- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

8- Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.

9- Monovalent cation potassium ions.

PRINCIPLEPRINCIPLE

principle

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence.

Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size

The principle of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps.

principle

These different temperature steps are necessary to bring about physical separation of the strands in a DNA double helix (DNA melting), and permit DNA synthesis by the DNA polymerase to selectively amplify the target DNA.

The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used.

PROCEDUREPROCEDURE

procedure

The PCR usually consists of a series of 20 to 35 repeated temperature changes called cycles; each cycle typically consists of three discrete temperature steps, each temperature step called hold .

The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include :

A- the enzyme used for DNA synthesis, B- the concentration of divalent ions and dNTPs in the reaction, C- and the melting temperature (Tm) of the primers

•1- Denaturation step: •This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

procedure

procedure

2- Annealing step:

The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.

•3- Extension/Elongation step: •The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme.

•At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction.

•The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.

procedure

procedure

•3- Extension/Elongation step:

procedure

Annealing stepAnnealing step

•Denaturation stepDenaturation step

•Extension stepExtension step

On

e Cycle

This cycle repeated for 20 to 35 times(cycles).

The amplification of the gene in PCR is Exponential amplification

PCR take several hours, commonly take 72 hrs.

procedure

Types of PCR

It is also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction .

Real-time PCR:

The procedure follows the general principle of polymerase chain reaction

its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle.

Two common methods of quantification are the use of fluorescent dyes )fluorescent reporter) that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.

Types of PCR

Real-time PCR:

1- The PCR reaction is prepared as usual (normal PCR) and the reporter probe is added.

Types of PCR

Real-time PCR:

2- As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.

Types of PCR

Real-time PCR:

3- Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.

Types of PCR

Real-time PCR:

4- Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.

Types of PCR

Real-time PCR:

Types of PCR

Real-time PCR:

Types of PCR

Reverse transcription PCR:

)RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid ) RNA) molecule.

The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction.

Types of PCR

Reverse transcription PCR:

In the first step of RT-PCR, called the "first strand reaction," complementary DNA )cDNA) is made from a messenger RNA template using dNTPs and an RNA-dependent DNA polymerase reverse transcriptase through the process of reverse transcription.

After the reverse transcriptase reaction is complete and the cDNA has been generated from the original single-stranded mRNA, standard polymerase chain reaction, termed the "second strand reaction," is done .

PCR is used in medical and biological research labs

for a variety of applications, which include:

1- DNA cloning.

2- diagnosis of hereditary diseases.

3- identification of genetic fingerprints (used in forensics and paternity testing).

4- detection and diagnosis of infectious diseases.

Application of PCR