Bias toward a 1:1 ratio in primer-introduced restriction analysis PCR: mechanism and minimization

Abstract

ABSTRACT. Primer-introduced restriction analysis is widely used in molecular genetics. However, several studies have reported inconsistent data regarding sequencing, mainly among heterozygous samples. This discrepancy may be related to the bias towards a 1:1 ratio typically observed in heterozygous digestion products. In this study, we investigated the mechanism and minimization of this observed bias. Three mismatched polymerase chain reaction (PCR) models were analyzed by testing different PCR conditions and reaction mixtures. For EPHX1 gene rs1051740 single-nucleotide polymorphism PCR, DNA concentration, denaturation and elongation time, annealing temperature, and cycle number significantly influenced product ratios. For SERPINA1 gene PIMmalton deletion (ΔPhe52) and CHRNA3 gene PIRA PCR analysis rs1051730 single-nucleotide polymorphism PCRs, significant bias fluctuations were observed only for the annealing temperature and cycle number conditions. The relevance of these results to the amplification efficiency parameter is discussed. Rather than reducing the observed bias, our data provide evidence of a counterbalance for preferential amplification, depending on cycle number, annealing temperature, and amplification efficiency alteration. Our results are relevant for application to primer-introduced restriction analysis PCR assays.

Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

“…When the annealing temperature lowered to 45 °C, the ratios of the PCR products were similar to that of the specific primer pair. Similar findings were reported by Denden , showing that annealing temperature is a key parameter affecting bias caused by primer mismatches.…”

“…These findings indicate that reducing the PCR cycle number effectively reduced the self‐annealing of the template in the later cycles of the amplification. Some investigators have also reported that the proportion of artifacts decreased at lower cycle numbers , however, the impact was less pronounced in this study. This was probably because of the majority of the artifacts were mismatch which was caused by DNA polymerase randomly (data not shown).…”

“…Suzuki et al used amplified 16S rRNA gene fragment as template, while we used total DNA. The deviation of PCR product proportion from 1:1 was also observed in two species community analysis with total DNA as the template . However, the effect of cycle number on the generation of bias was apparent.…”

“…In the three sets of digestion, the restriction sites of Sau 3A I is more randomly distributed than Bsa 29 I and Hind III + Xba I, which made the simplification more efficient and balanced across the different strains. Denden proposed that the early amplification cycles which were affected more directly by flanking regions in the template were important for amplification efficiency. Accordingly, our results show that digestion of template with restriction enzymes significantly increased the amplification efficiency.…”

“…However, lack of knowledge regarding the contribution of each parameter involved in such analysis does not allow for quantitative conclusions. Each step in community analysis is open to error or bias , including cell lysis, DNA extraction and purification , primer selection, PCR conditions , and cloning. Although, many studies have attempted to reduce the PCR bias and artifacts in artificial communities, it remains difficult to apply these results to environmental samples.…”