Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences.
A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (approximately 0.4-0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (approximately 0.4-0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4-0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.
Gao
Xinxin
X
Department of Chemistry, University of Miami, PO Box 249118, Coral Gables, FL, USA.
Yo
Peggy
P
Keith
Andrew
A
Ragan
Timothy J
TJ
Harris
Thomas K
TK
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005813:Genes, Synthetic / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt; D015203:Reproducibility of Results; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics; D013816:Thermodynamics
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D004247:DNA; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa