Quantitative effects of position and type of single mismatch on single base primer extension

Wu, Jer Horng; Hong, Pei‐Ying; Liu, Wen Tso

doi:10.1016/j.mimet.2009.03.001

Cited by 127 publications

(135 citation statements)

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“…For example, an A-to-G mismatch combined with a C-to-T mismatch at the 3Ј terminus would not change the primer's overall GC content and would have only a minor effect on its T m , but such a change would inhibit the amplification reaction substantially. Our results point to a smaller dCq as a single mismatch is moved farther from the 3Ј end, a finding in accord with previous studies (2,4 ). The effect of position was greatest for mismatches at the 3Ј terminus, and steric hindrance due to the purine/purine or pyrimidine/pyrimidine context of these mismatches might play some role in impeding qPCR amplification.…”

Section: Discussionsupporting

confidence: 91%

Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays

et al. 2013

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BACKGROUND Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility. METHODS We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield. RESULTS The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3′ end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased. CONCLUSIONS Single mismatches located >5 bp from the 3′ end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.

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Section: Discussionsupporting

confidence: 91%

Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays

et al. 2013

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“…In the IGF1Ea assay, the sense primer was placed over the insertion point ensuring that the last two bases fitted downstream of the insertion point. This creates a single base mismatch between IGF1Ea and IGF1Ec in the 3 0 end (.GGA-3 0 in IGF1Ea versus.GTA-3 0 in IGF1Ec), which should inhibit amplification from IGF1Ec cDNA (30). Indeed, there was no amplification of the 49 bp larger IGF1Ec isoform in the IGF1Ea assay, based on agarose gel and melting curve analysis.…”

Section: Muscle Mrna Measurementsmentioning

confidence: 97%

GH and IGF1 levels are positively associated with musculotendinous collagen expression: experiments in acromegalic and GH deficiency patients

Doessing¹,

Holm²,

Heinemeier³

et al. 2010

European Journal of Endocrinology

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Objective: Disproportionate growth of musculoskeletal tissue is a major cause of morbidity in both acromegalic (ACRO) and GH-deficient (GHD) patients. GH/IGF1 is likely to play an important role in the regulation of tendon and muscle collagen. We hypothesized that the local production of collagen is associated with the level of GH/IGF1. Design and methods: As primary outcomes, collagen mRNA expression and collagen protein fractional synthesis rate (FSR) were determined locally in skeletal muscle and tendon in nine ACRO and nine GHD patients. Moreover, muscle myofibrillar protein synthesis and tendon collagen morphology were determined. Results and conclusions: Muscle collagen I and III mRNA expression was higher in ACRO patients versus GHD patients (P!0.05), whereas collagen protein FSR did not differ significantly between ACRO and GHD patients in muscle (PZ0.21) and tendon (PZ0.15). IGF1Ea and IGF1Ec mRNA expression in muscle was higher in ACRO patients versus GHD patients (P!0.01). Muscle IGF1Ea mRNA expression correlated positively with collagen I mRNA expression (P!0.01). Tendon collagen fibrillar area tended to be higher in GHD patients relative to ACRO patients (PZ0.07). Thus, we observed a higher expression for collagen and IGF1 mRNA in local musculotendinous tissue in ACRO patients relative to GHD patients. Moreover, there was a tendency towards a higher collagen protein FSR and a smaller collagen fibril diameter in ACRO patients relative to GHD patients. The results indicate a collagenstimulating role of local IGF1 in human connective tissue and add to the understanding of musculoskeletal pathology in patients with either high or low GH/IGF1 axis activity.

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“…All of the HOPE experiments for each sample were performed in triplicate. The parameters of the HOPE analysis, such as the specificity of group-specific primers, working annealing temperature of the single-base primer extension reaction, and multiplexing analysis, were experimentally determined as described previously (19,20).…”

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confidence: 99%

Community and Proteomic Analysis of Methanogenic Consortia Degrading Terephthalate

Chuang

et al. 2013

Appl Environ Microbiol

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c Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122-130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H 2 /CO 2 , and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.T erephthalate (TA) is produced in large quantities in plastic, textile, and petroleum-related industries, and wastewater generated by the TA manufacturing process is often treated with anaerobic methanogenic processes (1). In these processes, TA is converted to CH 4 and CO 2 through the microbial syntrophy established among the enriched microbial populations. It is hypothesized that the hydrogen-producing syntrophic bacteria degrade TA to acetate and H 2 /CO 2 , which are immediately used by the methanogenic archaea adjacent to final gaseous products (2, 3). To validate the syntrophic interaction, a full-cycle 16S rRNA approach was used to successfully identify the identities of the syntrophic bacteria and methanogenic archaea inside the granules and biofilms of different laboratory-scale TA-degrading methanogenic reactors (3, 4). A recent study further used a metagenomic approach to reveal the diversity and physiological traits of the syntrophs and methanogens involved in the TA degradation under moderately thermophilic conditions (46 to 50°C) (5). All of these studies have provided strong evidence to elucidate the degradation of TA based on the microbial populations and the metabolic pathways involved.Furthermore, to directly observe the global expression of microbial functions within the TA-degrading consortium, community-level proteomics can be used (6). To do so, total proteins recovered from an environmental sample are first separated and enzymatically fragmented. The resulting peptide mixture is analyzed using advanced liquid chromatography-mass spectrometry (LC-MS) technology, and information related to the key proteins is identified using in silico analysis of the protein sequence database. ...

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Quantitative effects of position and type of single mismatch on single base primer extension

Cited by 127 publications

References 31 publications

Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays

Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays

GH and IGF1 levels are positively associated with musculotendinous collagen expression: experiments in acromegalic and GH deficiency patients

Community and Proteomic Analysis of Methanogenic Consortia Degrading Terephthalate

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