PCR Primer Sets

What do primers do in PCR?

Primers are chemically synthesized oligonucleotides, also called oligos, that are critical for high quality and high specificity PCR. Primers are complementary to a specific region of target DNA, and upon hybridization, provide 3′-hyrdoxyl group to DNA polymerase to initiate DNA synthesis. The length of the PCR products depend upon the position of primers along the length of the target single stranded DNA. Primers designed to produce shorter amplicons (100-200 nucleotides) are more efficient than primers designed to produce large products (3000-5000 bp). However, with the use of specialized “long and accurate” PCR protocols, amplification of fragments with lengths of up to 40 kbp is possible.

Non-specific binding and multiple-site binding on the target DNA are limited when primers are between 18-22 nucleotides in length. Certain short primers, such as hexamers, decamers, and oligo-dT primers are designed to hybridize to multiple sites on the target DNA. Some PCR primers are designed to hybridize to multiple sites in a genomic repeat sequence and used to amplify genomic polymorphic repeat regions to produce “DNA fingerprints.” Specific ribosomal gene sequences can also be amplified by using primers specific to conserved regions of DNA in ribosomal genes. 1, 2

Key points to keep in mind during primer design:
  • Primers should have a length between 17-28 nucleotides
  • Base composition of 50-60% (G+C)
  • The 3′ end should contain a G or C or CG or GC to increase the efficiency of primers
  • Tm (melting temperature) should be between 55-80 °C
  • Primers should be designed in a way to avoid self-complementarity, leading to the formation of hairpin like secondary structures 3

1. van Pelt-Verkuil, Elizabeth, Alex Van Belkum, and John P. Hays. "PCR primers." Springer Netherlands, 2008.
2. White, Bruce A. "PCR Protocols, current methods and applications." Vol. 15. Humana, Totowa, NJ (1993).
3. Chen, Bing-Yuan, and Harry W. Janes, eds. "PCR cloning protocols." Vol. 192. Springer Science & Business Media, 2002.

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