External and semi-internal controls for PCR amplification of homologous sequences in mixed templates

Kalle, Elena; Гулевич, А. А.; Rensing, Christopher

doi:10.1016/j.mimet.2013.09.014

Cited by 9 publications

(9 citation statements)

References 69 publications

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“…as therapeutics, for drug discovery, as delivery and targeting units [4] , [5] , diagnostics, e.g. as sensors [6] – [9] or as tools in chemical biology [10] – [12] .…”

Section: Introductionmentioning

confidence: 99%

By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

et al. 2014

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The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments. Based on sequence information and the amplification behaviour of defined enriched nucleic acid molecules we suppose a molecular mechanism through which these amplification by-products are built. Better understanding of these mechanisms might help to find solutions minimizing by-product formation and improving the success rate of aptamer selection.

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“…as therapeutics, for drug discovery, as delivery and targeting units [4] , [5] , diagnostics, e.g. as sensors [6] – [9] or as tools in chemical biology [10] – [12] .…”

Section: Introductionmentioning

confidence: 99%

By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

et al. 2014

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“…To estimate inhibition compatibility of samples with mixed templates, we recently proposed a new approach that is a compromise between external and internal amplification controls. We called this approach a semi-internal control [111] and the idea was to amplify the mixed template with known identity together with an aliquot of a DNAse-treated sample. In a semi-internal control, the native sample DNA is destroyed and thus only DNA from an added model mix can serve as the template.…”

Section: Factors That Can Increase Artifacts Formation In Multi-templmentioning

confidence: 99%

“…[112], [113], [114], [115]. Yet, biased profiles can be highly reproducible depending on the polymerase used for the reaction [111]. Although unsupported by experimental data, criteria such as the maximum yield of a PCR product [116] or the highest variety of amplicon types [117], [118] are often used for multi-template PCR optimization.…”

Section: Factors That Can Increase Artifacts Formation In Multi-templmentioning

confidence: 99%

“…Instead, a mixed sample with known identity can be used as an external control in this case. The same control amplified with an aliquot of the DNAse-treated test sample can be a rational compromise for the internal control [111].…”

Section: Factors That Can Increase Artifacts Formation In Multi-templmentioning

confidence: 99%

“…On the more positive side is the fact that patterns of the mixed template PCR might be highly reproducible [111]. It might therefore be reasonable to abandon the palmistry approach and stop assigning significance to each separate band or phylotype.…”

Section: Multi-template Pcr Measurementsmentioning

confidence: 99%

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Multi-template polymerase chain reaction

Kalle

Kubista

Rensing

2014

Biomolecular Detection and Quantification

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PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

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High-Throughput IgG Conversion of Phage Displayed Fab Antibody Fragments by AmplYFast

Sterner

Zehetmeier

2017

Methods in Molecular Biology

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Phage display of antibody libraries is an invaluable strategy in antibody discovery. Many synthetic antibody library formats utilize monovalent antibody binding fragments (Fab), displayed on filamentous phage and expressed in Escherichia coli for selection and screening procedures, respectively. For most therapeutic applications, however, the final antibody candidate favors a bivalent immunoglobulin G (IgG) format, due to its particular effector function, half-life, and avidity.Here, we present an optimized subcloning method, termed AmplYFast, for the fast and convenient conversion of phage-displayed monovalent Fab fragments into full-length IgG or immunoglobulins of any other isotype. By using biotinylated primers, unique mammalian expression vectors, and multi-well plates, AmplYFast combines the rapid amplification, digestion, and ligation of recombinant Ig heavy and light chain sequences in an easy-to-operate high-throughput manner. Thus, AmplYFast improves quality and efficiency in DNA cloning and significantly minimizes timelines to antibody lead identification.

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External and semi-internal controls for PCR amplification of homologous sequences in mixed templates

Cited by 9 publications

References 69 publications

By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

Multi-template polymerase chain reaction

High-Throughput IgG Conversion of Phage Displayed Fab Antibody Fragments by AmplYFast

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