An omega primer consists of 3 functional sections:
- A 3p arm functions as an initiator in a polymerase extension reaction.
- A 5p arm functions as an anchor attaching to a DNA template.
- A loop separates the two arms. In a relay PCR the loop is used as a priming section for monoplex PCR.
This design has fundamental advantages over conventional primers in the following aspects:
Specificity – A viable omega primer requires specific bindings of both 3p arm and 5p arm. This requirement results in a significant increase of specificity as compared to conventional primers.
Ability to tolerate certain template variations – In some applications, the 5p arm is intentionally made long so that it stays hybridized to a corresponding template even when a mismatch (e.g. a SNP) is present in the template. This is a useful feature considering that there are 6 million high allele frequency (>1%) SNPs in human genome of 3 billion bp long, an average of 1 SNP in every 50 bp.
Reduced primer-primer dimer formation – Amplifiable primer-primer dimers are formed only when 3p arms of two primers are cross-hybridized with at least one primer having its 3’ end complementarily bind to the other primer. Omega primers generally have much shorter 3p arms (16 nt) than that of regular primers (35 nt) therefore have statistically a much less probability of forming the amplifiable primer-primer dimers.
Native sequence in an amplicon – An amplicon sequencing read consists of native and primer sequence sections. Primer 3p arms are incorporated into amplicons becoming the primer sections. Since omega primers have much shorter 3p arms (16 nt) than that of regular primers (35 nt), omega primer produced amplicons have higher percentage of native sequence sections.
Specificity Enhancement
An omega primer is designed to hybridize with corresponding template forming a stable looped structure. Mismatches between 3p arm and a template destabilize the looped structure and cause the dissociation of the 3p arm from the template and reduces the probability of polymerase extension of the primer. Additionally, the 3p arm is short and by itself does not provide a sufficient inter-molecular binding with a template and does not sustain any significant polymerase extension reactions.
An extension reaction of an omega primer requires hybridization of both 5p arm and 3p arm. In a typical design, the binding energy of the 5p arm to a template is the same as that of the priming segment of a regular primer. The specificity enhancment of the omega primer is attributed to the requirement of additional recognition site for the 3p arm. In a typical design, median 3p arm length is 16. Assume that a minimum 12 out of 16 template nucleotides must match the 3p arm in order to achieve a significant binding. We have the probability of significant but wrong binding in a complete random sequence:
This means more than 26,000 times specificity enhancement.
