Assembly of highly diverse genes using degenerate oligonucleotides by temperature cascade

Loh, Qiuting; Bahara, Nur Hidayah Hairul; Choong, Yee Siew; Lim, Theam Soon

doi:10.1016/j.ab.2012.08.025

Cited by 2 publications

(5 citation statements)

References 16 publications

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“…The proposed strategy adapted the temperature cascade method to use a set of varying annealing temperatures to allow highly diverse genes to find their complementary strand at their own specific annealing temperature and hybridize with greater accuracy in a single-pot gene assembly process. 10 The concept of temperature cascade was based on the temperature kinetics theory, in which higher temperature allows the single-stranded degenerate oligonucleotide genes to have great mobility. As the temperature decreases gradually, the oligonucleotide mobility decreases gradually and eventually allows the complementary strands to edge closer to one another at their respective thermal equilibria and hybridize at higher accuracy.…”

Section: Discussionmentioning

confidence: 99%

“…The semisynthetic VH DAb repertoire assembly process was carried out using the temperature cascade assembly method. 10 Seven oligonucleotides consisting of four frameworks and three CDRs were assembled together in a single reaction to generate a full VH DAb region. Each fragment was mixed at equimolar concentrations and was subjected to heating at 95 °C before the temperature was slowly cascaded down to 25 °C at a temperature ramp rate of 0.1 °C/s.…”

Section: Methodsmentioning

confidence: 99%

“…pLABEL is the phagemid vector derived from pFAB1 phagemid. 10 Optimization of the cloning process was carried out to improve the transformation efficiency. The optimum condition for cloning was 50 ng of vector to 500 ng of insert at a ratio of 1:10 vector to insert.…”

Section: Methodsmentioning

confidence: 99%

“…10 The concept of temperature cascade was based on the temperature kinetics theory, in which higher temperature allows the single-stranded degenerate oligonucleotide genes to have great mobility. As the temperature decreases gradually, the oligonucleotide mobility decreases gradually and eventually allows the complementary strands to edge closer to one another at their respective thermal equilibria and hybridize at higher accuracy.…”

Section: Vh Gene Assemblymentioning

confidence: 99%

“…The method takes advantage of the DNA reannealing kinetics to locate the most accurate sequence pair for hybridization before synthesis to double-stranded DNA (dsDNA). 10 The collection of assembled genes will form the combinatorial repertoire to be used in the library generation.…”

Section: Introductionmentioning

confidence: 99%

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Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

et al. 2016

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The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Vh Gene Assemblymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

et al. 2016

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Principles and application of antibody libraries for infectious diseases

et al. 2014

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Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

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Assembly of highly diverse genes using degenerate oligonucleotides by temperature cascade

Cited by 2 publications

References 16 publications

Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

Principles and application of antibody libraries for infectious diseases

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