A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactions Article Swipe
Eugenia Y. Xu
,
Lisa Schneper
,
Daniel A. Notterman
·
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.1371/journal.pone.0292559
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.1371/journal.pone.0292559
In quantitative polymerase chain reaction (qPCR) experiments, primers containing mismatches with respect to the template are widely used in measuring repetitive DNA elements. Primer-template mismatches may lead to underestimation of the input sample quantity due to inefficient annealing and amplification. But how primer-template mismatches affect quantification accuracy has not been rigorously investigated. In this study, we performed a series of qPCR experiments in which we tested three pairs of mismatched telomere primers (tel1/tel2, tel1b/tel2b and telg/telc) and two pairs of perfect-match reference gene primers (36B4-F/-R and IFNB1-F/-R) at three different primer concentrations under four cycling conditions. Templates used were genomic DNA from two human cell lines and oligo duplexes which contained telomere sequences, reference gene sequences, or both. We demonstrated that the underestimation of input sample quantity from reactions containing mismatched primers was not due to lower amplification efficiency ( E ), but due to ineffective usage of the input sample. We defined a novel concept of amplification efficacy (f) which quantifies the effectiveness of input sample amplification by primers. We have modified the conventional qPCR kinetic formula to include f , which corrects the effects of primer mismatches. We demonstrated that reactions containing mismatched telomere primer pairs had similar efficiency ( E ), but varying degrees of reduced efficacy ( f ) in comparison to those with the perfect-match gene primer pairs. Using the quantitative parameter f , underestimation of initial target by telomere primers can be adjusted to provide a more accurate measurement. Additionally, we found that the tel1b/tel2b primer set at concentration of 500 nM and 900 nM exhibited the best amplification efficacy f . This study provides a novel way to incorporate an evaluation of amplification efficacy into qPCR analysis. In turn, it improves mismatched primer selection and quantification accuracy in amplifying DNA repeats using qPCR methods.
Related Topics
Concepts
Primer (cosmetics)
Polymerase chain reaction
Telomere
Biology
Primer dimer
DNA
Computational biology
Genetics
genomic DNA
Molecular biology
Template
Real-time polymerase chain reaction
Gene
Chemistry
Multiplex polymerase chain reaction
Nanotechnology
Materials science
Organic chemistry
Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1371/journal.pone.0292559
- https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0292559&type=printable
- OA Status
- gold
- References
- 60
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4387450373
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4387450373Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1371/journal.pone.0292559Digital Object Identifier
- Title
-
A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactionsWork title
- Type
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articleOpenAlex work type
- Language
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enPrimary language
- Publication year
-
2023Year of publication
- Publication date
-
2023-10-09Full publication date if available
- Authors
-
Eugenia Y. Xu, Lisa Schneper, Daniel A. NottermanList of authors in order
- Landing page
-
https://doi.org/10.1371/journal.pone.0292559Publisher landing page
- PDF URL
-
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0292559&type=printableDirect link to full text PDF
- Open access
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YesWhether a free full text is available
- OA status
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goldOpen access status per OpenAlex
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0292559&type=printableDirect OA link when available
- Concepts
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Primer (cosmetics), Polymerase chain reaction, Telomere, Biology, Primer dimer, DNA, Computational biology, Genetics, genomic DNA, Molecular biology, Template, Real-time polymerase chain reaction, Gene, Chemistry, Multiplex polymerase chain reaction, Nanotechnology, Materials science, Organic chemistryTop concepts (fields/topics) attached by OpenAlex
- Cited by
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0Total citation count in OpenAlex
- References (count)
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60Number of works referenced by this work
- Related works (count)
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10Other works algorithmically related by OpenAlex
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