Primer designing by Primer-Blast

Primer Designing By Primer Blast

Primers serve a crucial function in identifying the target area of the complex genome [1]. Primers or oligonucleotides are short sequences of DNA or RNA that are complementary to a particular DNA sequence, allowing replication to occur during a PCR cycle. Primers may also be used in various techniques, including gene sequencing, amplified fragment length polymorphisms, single-strand conformation polymorphisms, etc. Designing primers is the most crucial step in any of these procedures since low-quality primers yield little or no PCR output. Occasionally, amplification may also produce undesired DNA fragments. This might impede future uses, such as gene sequencing, etc. Designing effective primers is essential for a successful PCR reaction. In addition to several other online programs such as Gene fisher, DoPrimer, primer 3, web primer, etc. [3], the NCBI “Primer-Blast” tool may be used to design primers. Primer-Blast is an easily accessible and user-friendly design tool for primers. The comprehensive software includes modules for generating primer pairs and evaluating the target specificity of created primer pairs. Primer3 is used to produce potential primer pairs for a given sequence in Primer-Blast. The specificity check is performed by default by the Blast search, which searches for matches between primers and targets. New primer pairs are generated by inputting a DNA template, which may be the template’s raw DNA in FASTA format or an NCBI RefSeq accession number.

A suitable primer amplifies the selected area of the complicated genome that is of interest. There are several fundamental parameters for evaluating primer quality. Due to the specificity of primers, a PCR cycle may amplify an area of interest from a complicated DNA mixture. It is claimed that a primer is specific if it is complementary to the sequence of a target area of the genome and not too many non-target regions. Utilizing primer sequences as queries, BLAST is a standard way to test for non-specific binding. The optimal length for a typical PCR primer is between 18 and 24 nucleotides. This size is particular to the target location, and annealing at 50 to 65 degrees Celsius is often effective. Annealing temperature (Ta) is the temperature at which the greatest quantity of primer binds to its target sequence. Ta is dependent on primer length; if it falls below 50°C, the primer should be excluded. At melting temperature (Tm), double-stranded DNA transforms into single-stranded DNA. Primers having a Tm of 52-60°C provide superior results. The optimal and most acceptable Tm variation between primer pairs is less than 5°C [4] Ta is typically set 2 to 5 degrees Celsius below Tm. The design of overly short primers (10 nucleotides) should also be avoided, since their lower Ta (36 to 40°C) impairs the specificity of the PCR result [5]. Primer dimers are the result of hybridization between complementary primer bases. A G/C ratio between 40 and 60 percent is required for stable primer-template DNA binding. It is the ratio between the number of G and C nucleotides and the total number of nucleotides. The primer should (preferably) terminate with either a ‘G’ or ‘C’ residue preceded by a pyrimidine base, termination with double purines (‘GG’; ‘GC’ or ‘CC’) is acceptable but avoid terminating with ‘T’ residues and > 2 G/C residues in the last 3′ bases.

Fig:-Steps of Primer Designing

References-

1. Beyene D, Mukasa SB, Jansson C, Villiers EP, FergusonM, Rabbi IY, Kyamanywa S, Sebuliba J, Baguma Y. (2013). Patterns of nucleotide diversity in Meisa1 andG3pdh in wild and cultivated cassava. SINET: Ethiop. J.Sci., 36(2):65–72.
2. Rishi I, Halder A, Sharma JB , Jain M , Sharma M (2020). Single Strand Conformation Polymorphism and Sequencing of HS6ST2 Gene in Patients of Idiopathic Premature Ovarian Failure. JCDR;14(2): GC01-GC08.
3. Abd-Elsalam KA (2003). Bioinformatic tools and guideline for PCR primer design. African Journal of Biotechnology;2 (5):91-95.
4. Chen SH, Lin CY, Cho CS, Lo CZ, Hsiung CA (2003). Primer Design Assistant (PDA): a web-based primer design tool. Nucleic Acids Res., 31: 3751–3754.
5. Liu ZJ, Cordes JF (2004). DNA marker technologies and their applications in aquaculture genetics. Aquaculture; 238: 1–37.

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