A simple modification increases specificity and efficiency of asymmetric PCR

Tolnai, Z.; Harkai, Ákos; Szeitner, Zsuzsanna; Scholz, Éva Nagyné; Percze, Krisztina; Gyurkovics, Anna; Mészáros, Tamás

doi:10.1016/j.aca.2018.10.017

Cited by 30 publications

(28 citation statements)

References 22 publications

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“…Moreover, since the same adaptor sequences were attached to genomic DNA fragments on both ends, self-looping of the template strand may happen, which could lead to PCR amplification failure in the GC-rich regions. To solve this problem, we designed an asymmetric PCR amplification to enrich ssDNA [21]. Meanwhile, we replaced beads purification with stop buffer to stop fragmentation reaction to reduce the operation time and DNA loss.…”

Section: Discussionmentioning

confidence: 99%

Accurate CNV identification from only a few cells with low GC bias in a single-molecule sequencing platform

Lin²,

Xie

et al. 2020

Preprint

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A technical problem of characterizing copy number variation of several cells with next-generation sequencing is the whole genome amplification induced bias. The result of CNVs and mosaicism detection is affected by the GC bias. Here, we report a rapid non-WGA sample preparation strategy for a single-molecule sequencing platform GenoCare1600. This approach, combined with a single-molecule sequencing platform that avoids the use of WGA and bridge PCR processes, can provide higher reliability with its lower GC bias. By combining our optimized Tn5-based transposon insertion approach with GenoCare, we successfully detected CNVs as small as 1.29M and mosaicism as small as 20%, which is consistent with next-generation sequencing (NGS) data. Moreover, our GenoCare-TTI protocol showed less GC bias and less Mad of Diff. These results suggest that the optimized TTI approach, together with the GenoCare1600 sequencing platform, is a promising option for CNV characterization from maybe one single cell.

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Section: Discussionmentioning

confidence: 99%

Accurate CNV identification from only a few cells with low GC bias in a single-molecule sequencing platform

Lin²,

Xie

et al. 2020

Preprint

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“…Optimization of the conditions for asymmetric PCR is recommended for each SELEX cycle individually [120]. The use of special blocking oligonucleotides is advised to reduce the formation of PCR byproducts [121]. Removal of ineluctable dsDNA product is also desirable because the anti-sense strand can occasionally hybridize and mute possible aptamers [122]; therefore, asymmetric PCR is often combined with other ssDNA regeneration techniques, such as gel purification [118] or enzymatic digestion [108].…”

Section: Pool Conditioningmentioning

confidence: 99%

Inside the Black Box: What Makes SELEX Better?

2019

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Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.

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“…Asymmetric polymerase chain reaction (aPCR) is the simplest method for effective production of ssDNA with on-demand labeling [72]. In theory, it is a straightforward ssDNA production protocol, and appeared after the publication of the PCR technique (Figure 2c) [73].…”

Section: Asymmetric Polymerase Chain Reactionmentioning

confidence: 99%

“…The first one involves dsDNA templates' exponential amplification, and the second one of linear amplification is used for producing ssDNA [74]. While this may seem simple, aPCR is prone to producing nonspecific amplification and therefore generally requires extensive experimentation to optimize the yield of the desired ssDNA [72]. Numerous research groups have attempted to improve aPCR by identifying the appropriate ratio of primers, polymerases, number of amplification cycles, and purification methods [56,75].…”

Section: Asymmetric Polymerase Chain Reactionmentioning

confidence: 99%

“…Numerous research groups have attempted to improve aPCR by identifying the appropriate ratio of primers, polymerases, number of amplification cycles, and purification methods [56,75]. This approach has previously been applied in the synthesis of short ssDNAs [72,73,76], and more recently for DNA origami scaffolds of up to the kb scale [56,77]. High purity ssDNA can be produced by combining aPCR with gel purification or enzymatic degradation of residual chains.…”

Section: Asymmetric Polymerase Chain Reactionmentioning

confidence: 99%

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Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Hao

Qiao

2020

Genes

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Methods for synthesizing arbitrary single-strand DNA (ssDNA) fragments are rapidly becoming fundamental tools for gene editing, DNA origami, DNA storage, and other applications. To meet the rising application requirements, numerous methods have been developed to produce ssDNA. Some approaches allow the synthesis of freely chosen user-defined ssDNA sequences to overcome the restrictions and limitations of different length, purity, and yield. In this perspective, we provide an overview of the representative ssDNA production strategies and their most significant challenges to enable the readers to make informed choices of synthesis methods and enhance the availability of increasingly inexpensive synthetic ssDNA. We also aim to stimulate a broader interest in the continued development of efficient ssDNA synthesis techniques and improve their applications in future research.

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A simple modification increases specificity and efficiency of asymmetric PCR

Cited by 30 publications

References 22 publications

Accurate CNV identification from only a few cells with low GC bias in a single-molecule sequencing platform

Accurate CNV identification from only a few cells with low GC bias in a single-molecule sequencing platform

Inside the Black Box: What Makes SELEX Better?

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

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