DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis

Abstract

In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. For this study, we compared the polymerase (PCA) and ligase-based (LCR) methods for construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification. Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications. Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application.

Figure 1. Agarose gel images showing the effects of DMSO and betaine during the PCA assembly (A and C) and amplification (B and D) of IGF2R and BRAF gene fragments.

Based on a 20 µl reaction volume, both additives were introduced with increasing percentage (%) for DMSO, and molarity (M) for betaine; ‘No Additive’ lanes correspond to the control samples. A 1 kb DNA ladder in the outermost lanes marked at 400, 500 and 700 bp indicates the area of highest product band population. IGF2R and BRAF full-length gene fragments are shown at 517 bp and 512 bp, respectively.

Figure 2. Agarose gel images showing the effects of DMSO and betaine during LCR assembly (A and C) and amplification (B and D) of IGF2R and BRAF gene fragments.

Based on a 20 µl reaction volume, both additives were introduced with increasing percentage (%) for DMSO, and molarity (M) for betaine; ‘No Additive’ lanes correspond to the control samples. A 1 kb DNA ladder in the outermost lanes marked at 400, 500 and 700 bp indicates the area of highest product band population. IGF2R and BRAF full-length gene fragments are shown at 517 bp and 512 bp, respectively.