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Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

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Schematic outline of solid-phase cloning. (A) RE-based: biotinylated PCR products are captured and purified on streptavidin-coated paramagnetic beads (1). Solid-phase digestion of immobilized DNA, followed by washing away of cut pieces and buffer exchange (2). A second restriction enzyme cleaves at a site close to the beads (3). The supernatant is separated from the beads and the restriction enzyme is heat-inactivated. This purified and cleaved PCR product is ligated into a vector (4). (B) Head-to-tail assembly: PCR amplified backbone and inserts are separately immobilized onto streptavidin-coated paramagnetic beads. (1) Non-biotinylated strands are eluted in NaOH. Eluted backbone strands are discarded while the eluted insert strands are kept. (2) The eluted insert strand hybridizes to complementary sequence on the bead-bound fragments. (3) A polymerase is added to extend the two strands. (4) To introduce more inserts, steps 1–3 are repeated. (5) The final construct is released from the beads by one restriction enzyme cutting at both ends, allowing it to be ligated and circularized.
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Figure 1: Schematic outline of solid-phase cloning. (A) RE-based: biotinylated PCR products are captured and purified on streptavidin-coated paramagnetic beads (1). Solid-phase digestion of immobilized DNA, followed by washing away of cut pieces and buffer exchange (2). A second restriction enzyme cleaves at a site close to the beads (3). The supernatant is separated from the beads and the restriction enzyme is heat-inactivated. This purified and cleaved PCR product is ligated into a vector (4). (B) Head-to-tail assembly: PCR amplified backbone and inserts are separately immobilized onto streptavidin-coated paramagnetic beads. (1) Non-biotinylated strands are eluted in NaOH. Eluted backbone strands are discarded while the eluted insert strands are kept. (2) The eluted insert strand hybridizes to complementary sequence on the bead-bound fragments. (3) A polymerase is added to extend the two strands. (4) To introduce more inserts, steps 1–3 are repeated. (5) The final construct is released from the beads by one restriction enzyme cutting at both ends, allowing it to be ligated and circularized.

Mentions: Magnetic solid-phase support enables easy DNA capture, washing and switching to preferred reaction conditions between assembly steps. The procedures of the two methods are outlined in Figure 1. The restriction based method (Figure 1A) uses the paramagnetic beads to purify and sequentially wash, buffer exchange and digest PCR products, thereby providing a very simple and robust workflow for generation of ligated constructs without need for manual gel or spin-column based purification steps. Briefly, RE-based SPC starts by capturing PCR amplified inserts on streptavidin-coated paramagnetic beads, which then are sequentially washed, buffer-exchanged and incubated at appropriate temperatures for serial solid-phase digestions using independent REases in a liquid handler.


Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

Lundqvist M, Edfors F, Sivertsson Å, Hallström BM, Hudson EP, Tegel H, Holmberg A, Uhlén M, Rockberg J - Nucleic Acids Res. (2015)

Schematic outline of solid-phase cloning. (A) RE-based: biotinylated PCR products are captured and purified on streptavidin-coated paramagnetic beads (1). Solid-phase digestion of immobilized DNA, followed by washing away of cut pieces and buffer exchange (2). A second restriction enzyme cleaves at a site close to the beads (3). The supernatant is separated from the beads and the restriction enzyme is heat-inactivated. This purified and cleaved PCR product is ligated into a vector (4). (B) Head-to-tail assembly: PCR amplified backbone and inserts are separately immobilized onto streptavidin-coated paramagnetic beads. (1) Non-biotinylated strands are eluted in NaOH. Eluted backbone strands are discarded while the eluted insert strands are kept. (2) The eluted insert strand hybridizes to complementary sequence on the bead-bound fragments. (3) A polymerase is added to extend the two strands. (4) To introduce more inserts, steps 1–3 are repeated. (5) The final construct is released from the beads by one restriction enzyme cutting at both ends, allowing it to be ligated and circularized.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402512&req=5

Figure 1: Schematic outline of solid-phase cloning. (A) RE-based: biotinylated PCR products are captured and purified on streptavidin-coated paramagnetic beads (1). Solid-phase digestion of immobilized DNA, followed by washing away of cut pieces and buffer exchange (2). A second restriction enzyme cleaves at a site close to the beads (3). The supernatant is separated from the beads and the restriction enzyme is heat-inactivated. This purified and cleaved PCR product is ligated into a vector (4). (B) Head-to-tail assembly: PCR amplified backbone and inserts are separately immobilized onto streptavidin-coated paramagnetic beads. (1) Non-biotinylated strands are eluted in NaOH. Eluted backbone strands are discarded while the eluted insert strands are kept. (2) The eluted insert strand hybridizes to complementary sequence on the bead-bound fragments. (3) A polymerase is added to extend the two strands. (4) To introduce more inserts, steps 1–3 are repeated. (5) The final construct is released from the beads by one restriction enzyme cutting at both ends, allowing it to be ligated and circularized.
Mentions: Magnetic solid-phase support enables easy DNA capture, washing and switching to preferred reaction conditions between assembly steps. The procedures of the two methods are outlined in Figure 1. The restriction based method (Figure 1A) uses the paramagnetic beads to purify and sequentially wash, buffer exchange and digest PCR products, thereby providing a very simple and robust workflow for generation of ligated constructs without need for manual gel or spin-column based purification steps. Briefly, RE-based SPC starts by capturing PCR amplified inserts on streptavidin-coated paramagnetic beads, which then are sequentially washed, buffer-exchanged and incubated at appropriate temperatures for serial solid-phase digestions using independent REases in a liquid handler.

Bottom Line: We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors.In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in.The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%.

View Article: PubMed Central - PubMed

Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.

Show MeSH