Effects of secondary structures of DNA templates on the quantification of qPCR

Abstract

In the current design of qPCR systems, the sequences of primers are the primary concerns. The secondary structures of DNA templates have not been much considered, although they should be also critically important. In this paper, various hairpins with different stem lengths and loop sizes are placed near primer-binding sites, and their effects on the amplification efficiency of qPCR are systematically investigated. When a hairpin is formed either in the inside of the amplicon or in its outside, the amplification is notably suppressed. The magnitudes of suppression increase with the increase of stem length and the decrease of loop size, and are especially significant for the hairpins formed inside the amplicon. With very long stems (e.g., 20-bp), the effect is still more drastic, and no targeted amplification products are formed. On the basis of melting temperature (T) measurements, these suppression effects of hairpins have been mostly ascribed to competitive inhibition of primer binding to the template. It has been concluded that, in order to design precise and reliable qPCR systems, at least 60-bp sequences around primer-binding sites, both inside and outside the amplicons, must be analyzed to confirm that stable secondary structures are not formed in the vicinity of primer-binding sites.

The Elephant in the Lab (and Field): Contamination in Aquatic Environmental DNA Studies

“…If negative controls amplify, both options permit sequencing of the amplicon to distinguish between real target detections in field samples and positive control-derived contamination. However, sequence inserts/modifications could affect tertiary structures of the DNA molecules (e.g., hairpin loops) and alter the melting temperature of and polymerase binding affinity to the template DNA (Fan et al, 2019). Great care must be taken when designing these synthetic genes to validate them in silico.…”

Highly Sensitive EGFRvIII Detection in Circulating Extracellular Vesicle RNA of Glioma Patients

“…Interestingly, we observed that the EGFRvIII mRNA transcript contains several four guanine (4G) repeat sequences, especially close to the junction site (exon1:exon8), which is also the target region of interest. These 4G sequences have a well-documented role in the formation of G-quadruplexes as well as other 3D secondary structures (16,17), which are known to play an inhibitory role in reverse transcription and PCR amplification (18,19). Addition of PCR additives may destabilize secondary structures, as previously reported for the amplification of the highly GC-rich TERT promoter region (20).…”

Relative and Absolute Quantification of Aberrant and Normal Splice Variants in HBBIVSI−110 (G > A) β-Thalassemia

“…This phenomenon may possibly be explained by efficient intramolecular hairpin formation and linear amplification of the vector backbone for linear 2i SC constructs, which would interfere with amplification of the SC amplicons, but this contingency was not investigated further. Taken together, we believe that in multiplex reactions containing both templates within the plasmid backbone we observe reduced efficiencies owing to: (a) the formation of chimeric/recombinant amplicons [24] and consequent secondary structures interfering with primer and probe binding [25] and (b) removal of primer binding sites from initial-round amplicons due to 5-3 exonuclease activity of the Hot Start TaqMan Polymerase and the relatively close proximity of the two cDNA amplicons in linear (4073 bp) and especially the circular (247 bp) 2i construct. Both of the suggested inhibitory factors, in addition to supercoiled circular conformation, were effectively minimized by using fragmented 2i SC.…”