Methods for synthesizing pools of probes

摘要

Compositions, methods and kits are disclosed for synthesizing and amplifying pools of probes using precursor oligonucleotides. In some aspects the precursor is amplified and nicking enzymes are used to separate the full length probes from the amplification products. The methods enable the preparation of single stranded DNA probes of defined sequence and length that are suitable for use in target detection assays.

主权项

1. A method for synthesizing a pool of probes comprising a plurality of full length probes, the method comprising the steps of:
(a) obtaining a plurality of precursor probe oligonucleotides, each comprising a full length probe from the plurality of full length probes, flanked by a first recognition site for a first nicking enzyme and a second recognition site for a second nicking enzyme; (b) amplifying the precursor probe oligonucleotides by hybridizing a first primer to the 3′ end of the precursor probes and extending the first primer to make a first extension product and then hybridizing a second primer to the 3′ end of the first extension product and extending the second primer to make a double stranded precursor; (c) amplifying the double stranded precursor by PCR using first and second primers labeled with a first hapten; (d) treating the product of step (c) with terminal-transferase and a labeled nucleotide to label the 3′ ends of the product of step (c) with a second hapten; (e) digesting the product of step (d) with the first and second nicking enzymes to generate nicks on either side of each full length probe; and (f) separating the full length probes from the nucleic acids that are labeled with the first or the second hapten by mixing the product of (e) with a solid support coated with the binding partners for the first and second haptens and then separating the solid support phase from the solutions phase, wherein the full length probes remain in the solution phase.