Composition for the detection of blood cell and immunological response gene expression

Abstract

The present invention relates to a composition comprising a plurality of polynucleotide probes. The composition can be used as hybridizable array elements in a microarray. The present invention also relates to a method for selecting polynucleotide probes for the composition.

Description

[0001] This application is a continuation of U.S. application Ser. No. 09 / 023,655, filed Feb. 9, 1998, the entire contents of which is hereby incorporated by reference.[0002] The present invention relates to a composition comprising a plurality of polynucleotide probes for use in research and diagnostic applications.[0003] DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer. The array has over 50,000 DNA probes to analyze more than 400 distinct mutations of p53. A cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drug metabolism, drug resistance or d...

AI Technical Summary

Benefits of technology

[0080] It is also advantageous to include quantitation controls within the sample to assure that amplification and labeling procedures do not change the true distribution of target polynucleotides in a sample. For this purpose, a sample is spiked with a known amount of a control target polynucleotide and the composition of polynucleotide probes includes reference polynucleotide probes which specifically hybridize with the control target polynucleotides. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.

[0081] Prior to hybridization, it may be desirable to fragment the nucleic acid target polynucleotides. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other nucleic acid target polynucleotides in the sample or noncomplementary polynucleotide probes. Fragmentation can be performed by mechanical or chemical means.

[0089] Hybridization can be performed at low stringency with buffers, such as 6.times. SSPE with 0.005% Triton X-100 at 37.degree. C., which permits hybridization between target and polynucleotide probes that contain some mismatches to form target polynucleotide / probe complexes. Subsequent washes are performed at higher stringency with buffers, such as 0.5.times.SSPE with 0.005% Triton X-100 at 50.degree. C., to retain hybridization of only those target / probe complexes that contain exactly complementary sequences. Alternatively, hybridization can be performed with buffers, such as 5.times.SSC / 0.2% SDS at 60.degree. C. and washes are performed in 2.times.SSC / 0.2% SDS and then in 0.1.times.SSC. Stringency can also be increased by adding agents such as formamide. Background signals can be reduced by the use of detergent, such as sodium dodecyl sulfate, Sarcosyl or Triton X-100, or a blocking agent, such as sperm DNA.

Problems solved by technology

Current cDNA-based arrays suffer from a variety of limitations.

In other instances, there may be a 10% mismatch.

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