Comparing two conventional methods of emulsion PCR and optimizing of Tegosoft‐based emulsion PCR

Abstract

The selection of aptamers represents a promising route in the development of high affinity ligands. In these processes the formation of by-products is a common problem during the PCR-based amplification of complex oligonucleotide libraries. One approach to overcome this drawback is to separate each template oligonucleotide into an individual reaction compartment provided by a droplet. This method, termed emulsion PCR (ePCR), has already emerged to a standard method in sample preparation for 2nd generation sequencing. In this work, we compare different literature protocols that have been developed to generate stable emulsions for ePCR. We investigate different emulsification methods and evaluate the importance of the initial template concentration. We demonstrate that emulsion stability is of utmost importance for the successful inhibition of by-product formation and give an optimized protocol for generation of an emulsified PCR.

Optimization of On-Bead Emulsion Polymerase Chain Reaction Based on Single Particle Analysis

“…Our results therefore increase the accessibility of ePCR to newcomers and facilitate the integration of ePCR into applications such as liquid biopsy and cancer research, [37,38] metagenomic studies, [39,40] and aptamer discovery. [5,15,18,22,26] Author contributions…”

“…We then performed emulsion PCR under the following cycling conditions: 95 °C for 7 min, followed by 50 cycles of 95 °C for 30 sec, 65 °C for 30 sec and 72 °C for 75 sec and a post-cycling extension at 72 °C for 5 min. [18] A detailed protocol on how the beads are cleaned post-ePCR is in the supporting information.…”

“…However, previous studies using end-point, ensemble-level analyses of the purified PCR products failed to elucidate these characteristics. [18][19][20][21] Thus, optimization of ePCR would be difficult According to Poisson statistics, there is a 'sweet spot' where the number of single-template drops is maximized with minimal number of multi-template droplets. This condition results in only ~20% of emulsion drops containing a template molecule.…”

Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates

“…It is important to note that the protocol which we describe here was optimized using water-in-oil emulsions generated with an oil-surfactant mix comprised of mineral oil, Tegosoft, and Abil WE 09; it is possible that certain aspects of this protocol would have to be adjusted for use in recovery of DNA products from ePCR reactions constructed using other oil-surfactant mixes. Due to their ease of assembly and superior stability [8], Tegosoft-based emulsions are being used with increased frequency [10,[13][14][15][16]. However, oil surfactant mixes composed of various combinations of mineral oil, Triton X-100, Span-80, and Tween-80 are still used in many laboratories.…”

“…Before PCR products generated via ePCR can be used in downstream workflows, they need to be recovered from the water-in-oil emulsion. Often, emulsions are broken following amplification using volatile and highly flammable organic solvents such as diethyl ether, and product is subsequently isolated via precipitation [4][5][6]8,9]. However, the use of such solvents requires the implementation of special environmental controls, and the yield and purity of DNA isolated by precipitation can be highly variable.…”