Improved quantitative branched DNA hybridization assay by Mark L. Collins

  1. Simplify manufacturing by limiting the number of capture extenders (CEs) and label extenders (LEs) to 2 in most cases. 4 total probes should provide fairly uniform hybridization efficiency to highly structured targets. This limited number of extender probes would also make the bDNA assay easier to multiplex and to manufacture.
  2. The multiplex assay would optimally be done on beads with different capture sequences, followed by separating beads based on specific capture sequences or tags correlated to specific capture sequences, then specific dehybridizations (optional, but recommended), then detection.
  3. Computer design the CE and LE probes to absolutely minimize cross-hybridization to each other and to the generic sequences.
  4. Screen each CE against each LE probe and choose probes with highest signal {target present}/noise {no target present}. This screen should also remove LE probes with homology to generic capture probe.
  5. Tail the label extenders with 3000-5000 residues of dC. Purify.
  6. Tail oligo(dG)-oligo(dA) with 3000-5000 residues of dA to make a generic preamplifier. Purify
  7. Hybridize the LEs and CEs to the target in the microwells overnight.
  8. Wash stringently.
  9. Hybridize the preamplifier to the targets bound in microwells
  10. Wash well.
  11. Hybridize the generic branched DNA to the generic preamplifier (the bDNA has a dT20 sequence in it, which will hybridize to the poly(dA) tail of the preamplifier.
  12. Wash well.
  13. Bind labeled probe to branched DNA amplifier.
  14. Wash well.
  15. Detect with most sensitive method, e.g. chemiluminescence.
  16. Alternatively, de-hybridize with a sequence complementary to the well-bound capture probe at relatively low stringency in chemiluminescent detection reagent.
  17. Transfer to blank opaque wells and detect (should enhance S/N by leaving a lot of background behind).
  18. The amount of signal amplification per LE could be as much as 500 * 250 * 45 = 5, 600,000. Using dC5000, dG10-dA5000, dT20 with a branched DNA with 45 sites for binding labeled probe.
  19. 1-10 molecule detection should be routine.
  20. Improvement: bind the chemiluminescent enhancers directly to the target via hybridization of another type of LE for a smarter, more target-dependent signal.

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