Primers that contain portions noncomplementary to the target (often called overhang, flap, or tail) are usually used to add to the PCR product a utility sequence such as a restriction site (1) or a universal detection site (2,3). Stemloop primers make PCR amplification of short templates such as microRNA possible (4). Flap primers are used to reduce the number of sequencing errors in short PCR products (5). We have noticed that primers with short 5′ AT-rich flaps increase realtime PCR fluorescent signal, and this improvement is particularly significant for sequences that are difficult to amplify, such as bisulfite-treated DNA or highly variable viral sequences. The real-time PCR experimental data presented here (see (Table 1) for oligonucleotide sequences) was generated by 5′ minor groove binder (MGB) fluorescent hybridization probes (6), although we have observed similar effects for other platforms such as TaqMan probes (BioSearch Technologies, Novato, CA, USA) (data not shown). An example of the typical benefits of primers containing 5′ AT-rich flaps is shown in (Figure 1), in which they are used to amplify sodium bisulfite-treated DNA. The bisulfite treatment leads to a conversion of all unmethylated cytosines to uracils, leaving methylated cytosines unchanged (7), which constrains primer design. Human genomic DNA was treated with sodium bisulfite, and a portion of the differentially methylated region of the H19 gene was amplified using primers with or without 12-mer 5′ AT-rich flaps ((Table 1)). Fluorescent signal is increased, and PCR yield is higher when bisulfitetreated DNA is amplified with flap primers and fluorescence is generated by a hybridization probe ((Figure 1)A). This increase in signal intensity is also observed when fluorescence is generated by a free intercalating dye such as SYBR Green ((Figure 1)B), and the bands on ethidium bromide-stained agarose gel are also more pronounced ((Figure 1)C).

Figure 1.

We have determined that a 12-bp 5′ AT-rich flap sequence is optimal. Addition of flap sequences longer than 12 bp did not show any significant improvement, while shorter sequences had a lesser impact on the fluorescent signal (see Supplementary Table S1, available online at www.BioTechniques.com). The addition of a flap to either one or the other primer provides a boost to the fluorescent signal, but is not as beneficial as having both primers with flaps. 5′ GC-rich flaps overall proved not as useful as 5′ AT-rich flaps, because they are more prone to form stable secondary structures and negatively impact the PCR (data not shown). The positive effect of the 12-bp 5′ AT-rich flap is more pronounced for shorter primers (Supplementary Table S2). To study the effect of flap addition to primers of decreasing length on cycle threshold (C_T) and fluorescence, we chose a primer pair optimized for the cycling conditions recommended by the PCR Master Mix manufacturer (8) and designed several shorter primer pairs. As expected, shorter primers without flaps had higher C_T values and lower fluorescence gain. The addition of a 12-bp 5′ AT-rich flap nullified the difference in performance. Fourteen-to fifteen-mer primers with these flaps had approximately the same C_T value and fluorescence as the original 22- to 25-mer primers without flaps (Supplementary Table S2). This is useful information if primer design is constrained to short stretches of sequence for any reason.

(Figure 2) demonstrates the effect of 5′ flap primers in real-time PCR with varicella-zoster virus (VZV) DNA template and one-step reverse transcription PCR (RT-PCR) with enterovirus RNA template. The region chosen for the VZV design [open reading frame (ORF) 38 gene] is conservative and does not impose any restrictions on primer design. An increase in total fluorescence gain of approximately 50% due to 5′ primer flaps ((Figure 2)A), and no significant shift in C_T is typical for such unconstrained primer designs. In contrast to VZV, the sequences of the enterovirus genus (Taxonomy ID 12059; www.ncbi.nlm.nih.gov/Taxonomy) are highly variable. Therefore, to enable detection of all known isolates, the primer design is constrained to a short conservative region in the 5′ untranslated region (UTR). The primers also include degenerate and modified bases to account for unavoidable single nucleotide polymorphisms (SNPs) and to boost primer stability (9). As a result, the PCR primers perform suboptimally, and PCR efficiency is compromised. When the flap is added to the suboptimal (non-flap) enterovirus primers, there is a significant increase in performance for both fluorescence and C_T values ((Figure 2)B).

Figure 2.

It is not clear why PCR primers containing the 5′ AT-rich flaps are better, but the benefits are obvious. This innovation could be of benefit to every real-time PCR laboratory, especially when sequence choice for primer design is constrained.