THERMODYNAMICALLY BALANCED INSIDE-OUT (TBIO) PCR-BASED GENE SYNTHESIS: A NOVEL METHOD OF PRIMER DESIGN FOR HIGH-FIDELITY ASSEMBLY OF LONGER GENE SEQUENCES

Xinxin Gao¹, Peggy Yo², Andrew Keith¹, Timothy J. Ragan², and Thomas K. Harris^1,2,*

Department of Chemistry¹, University of Miami, and Department of Biochemistry and Molecular Biology², University of Miami School of Medicine, P. O. Box 016129 (R-629), Miami, Florida 33101-6129, USA, *tkharris@miami.edu

INTRODUCTION. To date, the most convenient and commonly used method of gene synthesis involves generating short oligonucleotide primers that contiguously code for a complete sense-strand and an antisense-strand of DNA; and the 5'- and 3'-termini of each primer overlaps with two flanking complementary primers (1,2). The double-stranded DNA gene sequence is first assembled by combining all of the sense- and antisense-strand primers and performing PCR.  Then, the fully assembled gene sequence is further PCR-amplified by using the two outer-most 5'-sense- and 5'-antisense-strand primers. It has been noted that larger DNA gene sequences (³0.5 kb) become increasingly difficult to synthesize, due to the increased possibility of mis-priming as the number of primers in the pool is increased.  In such cases, smaller sequential DNA fragments (£0.5 kb) must be synthesized and joined together in order to generate the complete gene sequence. Typically, a unique restriction site is engineered into the 5'- and 3'-terminal ends of each pair of sequential fragments to be joined.  Thus, synthesis of longer gene sequences can become problematic as the requirement for restriction enzyme digestion and ligation of numerous sequential fragments increases. In this work, two alternative PCR-based methods were compared for synthesizing codon-optimized gene sequences for the human protein kinases PKB2 (1494 bp), S6K1 (1622 bp), and PDK1 (1712 bp) without having to engineer and carry out restriction enzyme digestion and ligation of multiple fragments. 

METHOD. The novel TBIO method of PCR-based gene synthesis is distinguished from the thermodynamically-balanced conventional (TBC) method (1,2) in that TBIO synthesis requires only sense-strand primers for the amino-terminal half and only antisense-strand primers for the carboxy-terminal half of a gene sequence. Although the TBC and TBIO primer design strategies are distinguishingly different, a procedure was developed in which TBC and TBIO primer sets were generated for one gene sequence using identical overlapping regions with optimized T_m values, which provided an unbiased comparison of the effectiveness of the two techniques. A TBC primer set consisting of 60-mer oligonucleotides is converted to a TBIO primer set (i) by retaining the amino-terminal sense-strand primers and the carboxy-terminal antisense-strand primers and (ii) by synthesizing the reverse complement strands for the amino-terminal antisense-strand primers and the carboxy-terminal sense-strand primers.

RESULTS.  For the TBC method of PCR-based gene synthesis (1,2), an array of PCR conditions (e.g., number of primers, concentrations of primers, and number of PCR cycles) was performed with temperature-optimized TBC primer sets. For all three gene sequences tested, only the quarter-length gene fragments ranging from 332 to 465 bp could be synthesized, while the full-length (1494 to 1712 bp) and half-length fragments (722 to 894 bp) could not be synthesized. In addition, an array of PCR conditions (e.g., number of fragments, concentrations of fragments, and number of PCR cycles) was carried out in order to assess the ability to join all possible combinations of the sequential overlapping four quarter-sized fragments. For all three gene sequences, the quarter-sized fragments could be joined either to another quarter-sized fragment or to a half-sized fragment. In no case could two half-sized fragments be joined together or a three quarter-sized fragment be joined to a quarter-sized fragment to yield a full-length gene sequence. Thus, TBC synthesis of the full-length codon-optimized PKB2, S6K1, and PDK1 gene sequences requires at least one round of restriction enzyme digestion and ligation.

Due to the unpredictable nature of generating defined gene fragments with site-specific restriction sites, a novel TBIO method of PCR-based gene synthesis was developed and optimized to yield the full-length error-free synthetic genes of PKB2, S6K1, and PDK1. First, an array of PCR conditions (e.g., number of primers, concentrations of primers, and number of PCR cycles) was performed with temperature-optimized TBIO primer sets. The TBIO method could reliably provide bidirectional elongations of up to 480 bp by utilizing an optimized concentration gradient of four to six pair of TBIO primers. In contrast to the TBC method, the central fragment generated by the TBIO method could be repeatedly elongated until the full-length gene sequence was obtained. Although, the maximum number of elongation reactions has not been verified by synthesizing longer sequences (>2,000 bp), the evidence presented in this report suggests that the novel TBIO method of primer design and gene synthesis provides a significant advantage to conventional PCR-based synthesis of longer gene sequences, due to the decreased requirement of numerous restriction enzyme digestions and ligations.   

DISCUSSION. The underlying strategy for the novel TBIO method of gene synthesis is fundamentally distinct from that of the conventional or TBC method. For the TBC method, each primer is complementary to two other primers in the assembly reaction so that many different primer extension reactions occur simultaneously during assembly. As PCR assembly progresses, any number of combinations of fragments can form, which may contain nucleotide regions that can anneal with complementary nucleotide regions on other fragments, which is the premise of the TBC method of assembly. However, there exists the possibility that a single-stranded DNA molecule could simultaneously re-anneal to more than one complementary partner, resulting in formation of complex that inhibits primer extension. Thus, the assembly reaction can produce numerous random fragments for regions throughout the gene sequence. In contrast to the TBC method, the TBIO method involves complementation between the next pair of outside primers with the termini of a fully synthesized inside fragment. TBIO bidirectional elongation must be completed for a given outside primer pair before the next round of bidirectional elongation can take place. Thus, TBIO synthesis yields a well-defined and narrower range of products in contrast to the numerous and unpredictable products that can result from TBC synthesis. The TBIO method of PCR-based gene synthesis provides a robust alternative approach for engineering custom DNA sequences. 

ACKNOWLEDGEMENT. This work was supported by Florida Biomedical Research Program Grant BM026 to T.K.H.

REFERENCES.

1. Stemmer,W.P.C., Crameri,A., Ha,K.D., Brennan,T.M., and Heyneker,H.L. (1995) Gene 164, 49-53.
2. Hoover,D.M., and Lubkowski,J. (2002) Nuc. Acids Res. 30, e43.