Abstract
Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5′ tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.
Highlights
PCR for DNA amplification is a foundational, powerful technique with widespread applications in molecular biology, biotechnology, synthetic biology, diagnostics, detection, identification, and forensic analysis (Moore, 2005)
The first principle underlying suppression thermo-interlaced (STI) PCR is the use of the PCR suppression (PS) effect to suppress the amplification of non-specific PCR products, which are usually smaller than target products, to avoid their competitive amplification along with the specific products and to enhance the amplification of the target sequence
We utilized the principles of PCR suppression and thermo-interlaced cycling (TIC) to develop a simple, robust, and powerful method, STI PCR, that enables efficient and specific amplification of long sequences from complex genomes and synthetic DNA pools
Summary
PCR for DNA amplification is a foundational, powerful technique with widespread applications in molecular biology, biotechnology, synthetic biology, diagnostics, detection, identification, and forensic analysis (Moore, 2005). The efficient and specific amplification of long DNA fragments from complex genomes and synthetic DNA pools, which is often required for analyzing the functions of large genes, multigene clusters, and genomic structural variations and for constructing large functional structures, has long been a vexing problem. Synthetic biology is emerging as an important discipline with the potential to impact many academic and industrial applications. The engineering of biological systems in synthetic biology often requires de novo synthesis of long DNA sequences (synthons) that comprise complete functional units, but current technologies are inefficient for building large DNA synthons (Hughes and Ellington, 2017)
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