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Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

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Related in: MedlinePlus

Overview of the library construction—combination of Trimera mouse technology, ONCL method and phage-display. Lower part shows a schematic representation of phagemid vector pMid21 used for display of antibody Fab fragments. This vector is derived from pCES1 (8). The polylinker region comprises two signal sequences, Cκ domain, ribosome binding site (rbs), CH1 domain, hexahistidine tag and a c-myc-derived sequence (His/myc tags). Light chain genes can be cloned as ApaLI–AscI (1,2) fragments and variable heavy chain genes as SfiI–NheI (3,4) fragments. The MluI (5) restriction sites flanking both sites of the M13 gene III stump enable the removal of the stump anchor domain, which allows the production of soluble Fab fragments. Expression of the bicistronic operon is under control of the LacZ promoter (pLacZ).
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fig1: Overview of the library construction—combination of Trimera mouse technology, ONCL method and phage-display. Lower part shows a schematic representation of phagemid vector pMid21 used for display of antibody Fab fragments. This vector is derived from pCES1 (8). The polylinker region comprises two signal sequences, Cκ domain, ribosome binding site (rbs), CH1 domain, hexahistidine tag and a c-myc-derived sequence (His/myc tags). Light chain genes can be cloned as ApaLI–AscI (1,2) fragments and variable heavy chain genes as SfiI–NheI (3,4) fragments. The MluI (5) restriction sites flanking both sites of the M13 gene III stump enable the removal of the stump anchor domain, which allows the production of soluble Fab fragments. Expression of the bicistronic operon is under control of the LacZ promoter (pLacZ).

Mentions: For the construction of the primary light chain repertoires, the VκCκ and VλCλ PCR products, appended with the ApaLI and AscI restriction sites, were digested using 50 U/μg of each enzyme and agarose gel purified. Using T4 DNA ligase (NEB), 1.5 μg of each resulting DNA fragment was ligated into 2.5 μg of similarly cut phagemid vector pMID21 (Figure 1). Subsequently, 400 ng of desalted λ-ligation mixture and 500 ng of the κ mixture were electroporated separately into the Escherichia coli strain TG1 using 10 ng of ligation mixture per electroporation event. The Fab library was obtained by cloning of the VH repertoire into each of the light chain sublibraries. The VHCH1 PCR products, appended with the SfiI and NheI restriction sites, were digested using 50 U/μg of each enzyme and agarose gel purified. Using T4 DNA ligase (NEB), 6.7 μg of the VHCH1 DNA fragments were ligated into 5 μg of the similarly cut λ or κ sublibrary DNA. Finally, 1250 ng of desalted λHC-ligation mixture and 1250 ng of the κHC one were electroporated into the E.coli strain TG1 using 25 ng of ligation mixture per electroporation event.


Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

Schoonbroodt S, Frans N, DeSouza M, Eren R, Priel S, Brosh N, Ben-Porath J, Zauberman A, Ilan E, Dagan S, Cohen EH, Hoogenboom HR, Ladner RC, Hoet RM - Nucleic Acids Res. (2005)

Overview of the library construction—combination of Trimera mouse technology, ONCL method and phage-display. Lower part shows a schematic representation of phagemid vector pMid21 used for display of antibody Fab fragments. This vector is derived from pCES1 (8). The polylinker region comprises two signal sequences, Cκ domain, ribosome binding site (rbs), CH1 domain, hexahistidine tag and a c-myc-derived sequence (His/myc tags). Light chain genes can be cloned as ApaLI–AscI (1,2) fragments and variable heavy chain genes as SfiI–NheI (3,4) fragments. The MluI (5) restriction sites flanking both sites of the M13 gene III stump enable the removal of the stump anchor domain, which allows the production of soluble Fab fragments. Expression of the bicistronic operon is under control of the LacZ promoter (pLacZ).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131936&req=5

fig1: Overview of the library construction—combination of Trimera mouse technology, ONCL method and phage-display. Lower part shows a schematic representation of phagemid vector pMid21 used for display of antibody Fab fragments. This vector is derived from pCES1 (8). The polylinker region comprises two signal sequences, Cκ domain, ribosome binding site (rbs), CH1 domain, hexahistidine tag and a c-myc-derived sequence (His/myc tags). Light chain genes can be cloned as ApaLI–AscI (1,2) fragments and variable heavy chain genes as SfiI–NheI (3,4) fragments. The MluI (5) restriction sites flanking both sites of the M13 gene III stump enable the removal of the stump anchor domain, which allows the production of soluble Fab fragments. Expression of the bicistronic operon is under control of the LacZ promoter (pLacZ).
Mentions: For the construction of the primary light chain repertoires, the VκCκ and VλCλ PCR products, appended with the ApaLI and AscI restriction sites, were digested using 50 U/μg of each enzyme and agarose gel purified. Using T4 DNA ligase (NEB), 1.5 μg of each resulting DNA fragment was ligated into 2.5 μg of similarly cut phagemid vector pMID21 (Figure 1). Subsequently, 400 ng of desalted λ-ligation mixture and 500 ng of the κ mixture were electroporated separately into the Escherichia coli strain TG1 using 10 ng of ligation mixture per electroporation event. The Fab library was obtained by cloning of the VH repertoire into each of the light chain sublibraries. The VHCH1 PCR products, appended with the SfiI and NheI restriction sites, were digested using 50 U/μg of each enzyme and agarose gel purified. Using T4 DNA ligase (NEB), 6.7 μg of the VHCH1 DNA fragments were ligated into 5 μg of the similarly cut λ or κ sublibrary DNA. Finally, 1250 ng of desalted λHC-ligation mixture and 1250 ng of the κHC one were electroporated into the E.coli strain TG1 using 25 ng of ligation mixture per electroporation event.

Bottom Line: DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires.Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family.We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

View Article: PubMed Central - PubMed

Affiliation: Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.

ABSTRACT
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Show MeSH
Related in: MedlinePlus