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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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The ONCL technology. (A) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. (B) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.
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fig2: The ONCL technology. (A) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. (B) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.

Mentions: A Fab phage antibody library was built from the spleen RNA of 12 responding mice. The 12 RNA samples were processed separately. The capture of V genes was performed in two main steps. First, decapped full-length mRNA extracted from spleen cells of Trimera mice was tailed at the 5′ end with a synthetic RNA oligonucleotide before the reverse transcription was performed. For specific capture and amplification of antibody genes, 3′ end primers complementary to the constant regions of the antibody genes and 5′-biotinylated primers complementary to the attached synthetic sequence (Figure 2A) were used. Priming at this synthetic 5′ sequence avoids the use of primers within the variable regions of the antibody genes that could generate biases against rare subclasses of V genes or ones mutated at the priming sites. Only IgG-derived VH segments were amplified from newly synthesized cDNAs using a primer located in the CH1 of IgG1-4. From this step onwards, the 12 samples, previously processed separately, were pooled in equal amounts.


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)

The ONCL technology. (A) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. (B) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.
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Related In: Results  -  Collection

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fig2: The ONCL technology. (A) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. (B) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.
Mentions: A Fab phage antibody library was built from the spleen RNA of 12 responding mice. The 12 RNA samples were processed separately. The capture of V genes was performed in two main steps. First, decapped full-length mRNA extracted from spleen cells of Trimera mice was tailed at the 5′ end with a synthetic RNA oligonucleotide before the reverse transcription was performed. For specific capture and amplification of antibody genes, 3′ end primers complementary to the constant regions of the antibody genes and 5′-biotinylated primers complementary to the attached synthetic sequence (Figure 2A) were used. Priming at this synthetic 5′ sequence avoids the use of primers within the variable regions of the antibody genes that could generate biases against rare subclasses of V genes or ones mutated at the priming sites. Only IgG-derived VH segments were amplified from newly synthesized cDNAs using a primer located in the CH1 of IgG1-4. From this step onwards, the 12 samples, previously processed separately, were pooled in equal amounts.

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