The Polymerase Chain reaction (PCR) is a wet lab strategy used to amplify segments of DNA they find interesting. For this technique, Primers are needed among some other components.

Primers:
These are the two small DNA fragments capable of firmly hybridizing on each side of the gene in a highly specific manner.

Primer3:
It is the primer design program to help scientists decide which portion of the large sequence makes the best primer, this avoiding a series of potential problems.

Follow the following set of instructions to learn the use of this tool;

To use this Tool you first need the FASTA sequence of the gene you need to amplify. For this in this case study it will be of dUTPase of E.coli

Go to GenBank . Select nucleotide form the search bar and enter the accession number of the Gene. In this case it is x01714

Click FASTA to get its FASTA sequence.

Copy and paste this sequence in a note pad. This is the sequence that will later be entered into the Primer3 tool to design Primers for its amplification.

Now point your browser to Biotools.

Click the Primer Selection Tool link in the DNA Sequence Analysis section.

Paste the FASTA sequence you retrieved earlier in the Sequence window.

If you want to add constraints to your search you can change the settings of the form below (discussed later), or leave it by default (preferred if you are new to the tool).

Click the Pick Primers button.

Interpreting the Output:

Output includes,

The position, length, predicted melting temperature, and G+C percentage in the oligonucleotide.

Map of the best oligonecleotide pair (left and right primers) according to the constraints listed in the form.

Four other alternative solutions are also proposed with there position, length, predicted melting temperature and G+C percentage.

To modify default settings:

To modify the settings, the form allows reasonable experimental solutions and constraints to be imposed upon the primer selection process, including

Searching for only left or right primer, or a single hybridization probe

Proposing your own left or right primers

Selecting sequence positions to be flanked or excluded

Selecting a range of product sizes

Imposing a range of oliginucleotide sizes, G+C percentages, or melting points

And many more highly sophisticated options

To understand the terms in the form you can visit the Help Site:

Contributed by:Samia Aslam