Primer extension detection methods on active electronic microarrays

A method of preserving and reusing a nucleic acid library produced from a patient biological sample, the method comprising:

• (a) isolating mRNA from a patient biological sample;
• (b) reverse-transcribing the mRNA from step (a) to produce a cDNA library;
• (c) amplifying the cDNA library from step (b) by a DNA polymerase reaction utilizing at least one chimeric primer comprising a RNA polymerase recognition site upstream of a sequence specific for a mRNA transcript of interest and a fill-in primer for the complementary nucleic acid strand chosen from the group consisting of sequence specific primers and random primers, wherein at least one of the primers used is chosen from the group consisting of 5′ affinity-moiety labeled chimeric primers and 5′ affinity-moiety labeled sequence specific fill-in primers;
• (d) binding the amplification products from step (c) to a solid support coated with an affinity-binding moiety;
• (e) utilizing the bound amplification products from step (d) as a template for an in vitro transcription reaction;
• (f) separating the in vitro transcription products from step (e) from the amplification products bound to the solid support;
• and
• (g) utilizing the bound amplification products from step (f) as a template for at least one additional in vitro transcription reaction, wherein the amount of in vitro transcription product produced is not significantly less than that produced in step (e).

A method of detecting the extent of hybridization of a nucleic acid in a sample to a probe nucleic acid sequence, the method comprising:

• (a) electronically hybridizing the nucleic acid in a sample to a nucleic acid probe bound to a support at a predetermined location;
• (b) utilizing the hybridized nucleic acid as a template in a nucleic acid polymerase reaction to extend the bound probe, whereby a labeled nucleotide is incorporated into the extended probe;
• and
• (c) detecting the labeled nucleotide incorporated into the extended bound probe at the predetermined location.

A method of providing an internal control for an individual test site in a nucleic acid hybridization reaction assay to determine the presence of at least one nucleic acid sequence of interest in at least one nucleic acid containing sample, wherein the nucleic acid hybridization assay is performed on an electronically controlled microarray comprising at least two test sites, the method comprising:

• (a) attaching a mixed nucleic acid probe consisting of a first nucleic acid probe specific for a first nucleic acid sequence known to be present in the sample, and a second nucleic acid probe specific for a second nucleic acid sequence of interest to a first test site on the electronically controlled microarray;
• (b) attaching a mixed nucleic acid probe consisting of the first nucleic acid probe and a third nucleic acid probe specific for a third nucleic acid sequence of interest, wherein the third nucleic acid sequence of interest may be the same as or different than the second nucleic acid sequence of interest, to a second test site on the electronically controlled microarray;
• (c) electronically hybridizing the sample nucleic acids from at least one sample to the nucleic acid probes on the first and second test sites;
• (d) specifically detecting the extent of hybridization of the sample nucleic acids to the first nucleic acid probe at the first and second test sites;
• (e) specifically detecting the extent of hybridization of the sample nucleic acids to the second and third nucleic acid probes at the first and second test sites;
• (f) comparing the hybridization values obtained for the first nucleic acid probe at the first and second test sites to obtain a normalization factor;
• and
• (g) normalizing the hybridization values obtained in (e) for the second and third probes using the normalization factor obtained in (f).