Abstract
Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.
Highlights
De novo gene synthesis relies on the chemical synthesis of oligonucleotides that are used as the building blocks for enzymatic assembly, making it possible to synthesize multi-kilobase genes and even whole genomes [1,2,3]
PCR amplification of multiple DNA oligonucleotides containing a wide range of GC content often results in uneven amplification, where a sequence is preferentially amplified over others in a comlplex pool unintentionally, preventing efficient assembly of a complete larger construct
We hypothesized that insertion of a subcycling step during the PCR amplification process would yield a more uniform amplification of all types of oligonucleotides in the mix
Summary
De novo gene synthesis relies on the chemical synthesis of oligonucleotides that are used as the building blocks for enzymatic assembly, making it possible to synthesize multi-kilobase genes and even whole genomes [1,2,3]. This technology is more affordable than cloning genes and it enables the use of existing genome databases to construct any intended target. These sequences can be problematic during PCR amplification as they form hairpins and various other secondary structures leading to premature termination of the polymerase extension yielding incomplete and non-specific products [8].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
