US 2006/0141501 A1 · Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays

Abstract

Methods and compositions are provided that are useful for detecting and reporting a plurality of different target polynucleotide sequences in a sample, such as polynucleotides corresponding to a plurality of different genes expressed by a cell or cells. In particular, the invention provides methods for screening a plurality of candidate polynucleotide probes to evaluate both the sensitivity and the specificity with which each candidate probe hybridizes to a target polynucleoide sequence. Candidate polynucleotide probes can then be ranked according to both their sensitivity and specificity, and probes that have optimal sensitivity and specificity for a target polynucleotide sequence can be selected. In one embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “screening chips” wherein a large number of target polynucleotide sequences are detected using a single microarray have a few (e.g., 1-5) probes for each target polynucleotide sequence. In a particularly preferred embodiment, the invention provides a screening chip that can detect genetic transcripts from the entire genome of an organism. In an alternative embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “signature chips” to more accurately detect certain selected “signature genes” using several polynucleotide probes (e:g., 10-20) for each signature gene. The invention additionally provides microarrays containing polynucleotide probes for a large number of genes expressed by a cell or organism. Further, methods for detecting a plurality of polynucleotide molecules, including a large number of genes expressed by a cell or organism, are also provided.

Claims21

Dependent claims

1-212. (canceled)

A method for selecting one or more different polynucleotide probes for detecting a target polynucleotide,

said target polynucleotide comprising a nucleotide sequence of a gene or gene product expressed by a cell or organism,
said one or more different polynucleotide probes being selected from a plurality of different candidate polynucleotide probes, each different candidate polynucleotide probe comprising a different sequence that is complementary and hybridizable to a nucleotide sequence of said target polynucleotide,
said method comprising:
(a) selecting from said plurality of different candidate polynucleotide probes a plurality of polynucleotide probes that have a magnitude of binding energy for hybridization to said target polynucleotide that is above a selected threshold;
(b) ranking the plurality of polynucleotide probes selected in step (a) according to
(b1) probe length, and
(b2) distance of the complementary sequence for each probe in the target polynucleotide from one end of said target polynucleotide;
(c) de-overlapping said ranked polynucleotide probes by a method which comprises
(c1) selecting the top ranked polynucleotide probe from said ranked polynucleotide probes, and
(c2) repeatedly selecting the next polynucleotide probe from said ranked polynucleotide probes which comprises a nucleotide sequence complementary to a sequence in said target polynucleotide that overlaps the complementary sequence of the previously selected polynucleotide probe in said target polynucleotide by no more than a selected number of nucleotide bases;
and
(d) ranking said de-overlapped polynucleotide probes according to the most negative binding energy with which each of said de-overlapped polynucleotide probes cross-hybridizes to one or more other polynucleotide sequences expressed by said cell or organism,
wherein said one or more different polynucleotide probes for detecting said target polynucleotide are selected from said ranked, de-overlapped polynucleotide probes.

10 more elements

A method for selecting one or more different polynucleotide probes for detecting a target polynucleotide,

said target polynucleotide comprising a nucleotide sequence of a gene or gene product expressed by a cell or organism,
said one or more different polynucleotide probes being selected from a plurality of different candidate polynucleotide probes, each different candidate polynucleotide probe comprising a different sequence that is complementary and hybridizable to a nucleotide sequence of said target polynucleotide,
said method comprising:
(a) selecting from said plurality of different candidate polynucleotide probes a plurality of polynucleotide probes that have a magnitude of binding energy for hybridization to said target polynucleotide that is above a selected threshold;
(b) ranking said plurality of polynucleotide probes selected in step (a) according to a minimax score, said minimax score for each said selected polynucleotide probe (b1) being the largest magnitude of binding energy with which said selected polynucleotide probes cross-hybridizes to one or more other polynucleotide sequences expressed by said cell or organism;
or (b2) being determined using the product of the binding energy with which said selected polynucleotide probes cross-hybridizes to each of said one or more other polynucleotide sequences expressed by said cell or organism and a weighting factor;
and
(c) selecting one or more different polynucleotide probes from said plurality of polynucleotide probes according to their ranks,
thereby selecting one or more different polynucleotide probes for detecting said target polynucleotide.

6 more elements

Dependencies are inferred from the claim text.

Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays

Life of this patent

Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays

Life of this patent

Abstract

Claims21