Linear double stranded DNA coupled to single support or tag and methods for producing said linear double stranded DNA
A support and labeling technology, applied in biochemical equipment and methods, microbial measurement/testing, fermentation, etc., can solve problems such as error-prone DNA template preparation
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[0248] In the following, further preferred embodiments of the present invention are described.
[0249] 1. A linear double-stranded DNA comprising a coding strand and a non-coding strand, wherein said DNA comprises (i) a coding sequence element encoded by said coding strand in the 5' to 3' direction of the coding strand, and (ii) an RNA polymerase promoter sequence element upstream of the coding sequence element, wherein the non-coding strand is coupled to a support or tag at its 3' end, and wherein the support or tag is coupled to the The only support or tag for the DNA.
[0250] 2. The linear double-stranded DNA according to embodiment 1, wherein the tag is biotin, preferably with streptavidin, preferably streptavidin-coated microbeads, most preferably streptavidin-coated associated with magnetic microbeads.
[0251] 3. A method for preparing linear double-stranded DNA comprising a coding strand and a non-coding strand, wherein the non-coding strand is coupled to a support...
Embodiment 1
[0370] Example 1: Coupling of linearized DNA to CnBr-activated agarose
[0371] The purpose of this example was to find out whether linearized DNA could be coupled to CnBR-activated sepharose, and if so, whether the DNA was still accessible for enzymatic reactions.
[0372] 1 μg of plasmid DNA (SEQ ID NO: 1 ) was linearized using 10 U XbaI to generate cohesive DNA ends or 10 U Pvull to generate DNA blunt ends. The digestion reaction was carried out in 20 μl of 1× digestion buffer at 37° C. for 1 hour. Subsequently, the reactions were analyzed by agarose gel electrophoresis on a 0.8% agarose gel to ensure complete linearization. Linear DNA was purified using AMPure XP microbeads (Beckman coulter) according to the manufacturer's instructions.
[0373] Coupling of Xbal- or Pvull-linearized DNA onto CnBr-activated 4B or 6MB agarose sepharose (GE Healthcare) was performed according to the manufacturer's instructions. Briefly, 4B or 6MB agarose sepharose was suspended in 1 mM HCl...
Embodiment 2
[0379] Example 2: Reconciliation of linearized DNA with NH using EDC / sulfo-NHS 2 bead coupling
[0380] The purpose of this example is to ascertain the use of EDC sulfo-NHS coupling to NH 2 Whether the linearized DNA of the beads is still accessible to the enzymatic reaction.
[0381] 1 mg of plasmid DNA (SEQ ID NO: 1 ) was linearized using 500 U of Pvull to generate DNA blunt ends. The digestion reaction was carried out in 5 ml 1× digestion buffer at 37°C for 2 hours. The resulting DNA blunt ends were dephosphorylated using 300 U alkaline phosphatase for 30 minutes at 37°C to prevent religation. Phosphatase reactions were stopped by adding 0.1% SDS for 10 minutes at 65°C. Then, the linearized and dephosphorylated DNA was washed with 1 ml of ice-cold propanol and centrifuged at 20,000 g for 20 min at room temperature. Dry the precipitate for 10-30 minutes. DNA was then redigested with Asel for 2 hours at 37°C to generate carboxylic acid groups, which are essential for ...
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