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What is a primer?
A primer is defined as a sequence of short nucleotides that initiate the DNA synthesis.
How to design a primer?
Designing a primer for DNA synthesis involves some important properties and steps. The properties include melting temperature, primer specificity, GC content, primer length, amplicon length, annealing temperature and Primer-self complementarity and primer dimers. The steps involved in this process are defining the target region, choosing the primer length, analysing a primer sequence, refining primers and primer testing.
Properties:
Melting temperature:
It is the temperature at which the dissociation of primers from the complementary strands takes place. All the primers in a reaction should have the same melting temperature.
Primer specificity:
The primers are designed in such a way that they must bind to the specific DNA sequences which are targets and not binding to the DNA sequences which are similar to the target DNA sequence.
GC content:
The GC nucleotides should be about 40-60% in the primers and those primers which have least GC content will not be stable and the ones with high GC content will lead to non-specific binding of the primers.
Primer length:
Usually, the length of the primers range from 18 to 24 base pairs.
Amplicon length:
The amplicon is defined as the DNA sequences that are amplified and its length also plays a major role in primer designing, specifically in qPCR.
Annealing temperature:
The temperature of annealing in PCR and other reactions (where DNA synthesis takes place) should be below the primer melting temperature to enable its efficiency in annealing.
Primer self-complementarity and primer dimers:
The primers are not allowed to have any self-complementarity between them or the primer dimers as it will result in the synthesis of unwanted products of DNA amplification.
Steps:
Defining the target region:
Identify the particular DNA sequences that require to be amplified.
Choosing the primer length:
The choosing of length of the primer as per the requirements of the experiments and the melting temperatures of the primers.
Analysing a primer sequence:
Some sets of software's are used to analyse the primer sequence for their melting temperatures, primer dimers, self-complementarity and the presence of the GC content.
Refining primers:
This involves the refining of primer sequences as per their efficiency and specificity.
Primer testing:
There are some specific cases in PCR where we need to conduct the primer testing prior to its use.
Types of primers:
There are two different types of primers namely DNA and RNA primers. These are further classified into forward and reverse primers.
DNA primers:
They usually take part in the in vitro DNA synthesis and are chemically synthesised.
RNA primers:
They are often involved in the cloning and in vivo DNA replication. RNA primers are synthesised by the enzyme primase.
Forward primers:
The binding of the specific primers to the 3' end of the template DNA strand takes place as they direct the forward synthesising of the DNA.
Reverse primers:
The binding of the specific primers to the 5' end of the template DNA strand occurs as they direct the synthesising of the DNA in reverse direction.
Software's involved in primer designing:
The softwares involved in primer designing include Primer-BLAST, Vector Builder's Primer design tool, SeqBuilder Pro, Primer & Primer 3 Plus, Snapgene, OLIGO primer analysis software, IDT's primer quest tool, NEBase changer, Primer & Premier, Eurofins Genomics' PCR Primer Design Tool and Oligo Perfect designer.
Removal of primers:
It happens in the DNA replication, following the insertion of the Okazaki fragments next to the RNA primers and soon after it is done, the deoxyribonucleotides occupy the gaps left by RNA primers.
Note: The okazaki fragments are those that are formed as a result of discontinuous DNA replication. i.e. The reverse primer discontinuously helps in the DNA replication whereas the forward primer synthesises the DNA strand continuously. Hence, it is collectively called discontinuous replication.
Degenerate primers:
These are primers that are not identical but have the similar genetic information that codes for the same amino acid. For example, if AGJ is the code for a specific amino acid, serine in the case of the degenerate primers, then A is Adenine , G is Guanine and J is either C (Cytosine) or U (Uracil). These primers are used to synthesize amino acids when the target sequence is unknown. They can bind to the target sequences and amplify the genes to produce amino acids and proteins.
