PCR-based assembly of gene sequences by Thermodynamically Balanced Inside-Out (TBIO) gene synthesis

Timothy J. Ragan, Helen A. Vincent

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review


The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension gene synthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Tms) are designed so that the 5′ half of the DNA is encoded by sense primers and the 3′ half of the DNA molecule is encoded by antisense primers. Primer extension is initiated at the center of the DNA and continues bidirectionally to progressively elongate the DNA molecule. Here we provide the protocols necessary for performing TBIO gene synthesis to generate a DNA molecule of interest.
Original languageEnglish
Title of host publicationDNA Manipulation and Analysis
EditorsGarry Scarlett
Place of PublicationNew York
PublisherHumana Press
Number of pages15
ISBN (Electronic)9781071630044
ISBN (Print)9781071630037
Publication statusPublished - 1 Mar 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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