Methods for monitoring the expression of alternatively spliced genes

• US 20060141506A1
• Filed: 11/23/2005
• Published: 06/29/2006
• Est. Priority Date: 04/25/2000
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Balaban, David
• Application Number:
  US11/287,330
• Publication Number:
  US 20060141506A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2539/105 Involving introns, exons, or splice junctions
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

Methods, probe arrays and computer software products are provided for determining the arrangement of sequence elements. In one embodiment, methods for making and using exon chips are provided. The exon chips may be used to identify and quantify splice variants.

Claims

1-49. -49. (canceled)

50. A method for determining the sequence of a variant splicing product of an initial mRNA transcript composed of exons and introns, the method comprising:

employing different types of positive probes that hybridize with, and produce signals corresponding to, subsequences of the initial mRNA transcript in a sample solution;

employing at least one type of negative control probe corresponding to each type of positive probe to produce negative-control-probe signals;

detecting signals produced from positive probes in order to determine subsequences of the initial mRNA transcript present in a sample solution and construct an initial sequence of the variant splicing product; and

detecting signals produced from the negative control probes to resolve ambiguities in the initial sequence of the variant splicing product.

51. The method of claim 50 wherein the different types of positive probes include:

positive tiling probes complementary to subsequences that span the sequence of the initial mRNA transcript;

positive exonic tiling probes complementary to exon sequences within the initial mRNA transcript; and

positive jump probes, each jump probe complementary to a subsequence including a potential splice point between two exons of the initial mRNA transcript.

52. The method of claim 50 wherein negative control probes include:

intron/exon-negative-control probes, including a subsequence that spans the junction between an exon and an intron in the initial mRNA transcript.

53. The method of claim 52 further including:

comparing a signal detected from an intron/exon-negative-control probe to the signal detected from a corresponding positive jump probe;

when the signal detected from the intron/exon-negative-control probe is greater in signal strength to the signal detected from a corresponding positive jump probe, determining that an exon/exon splice point to which the positive jump probe is complementary is probably not present in the variant splicing product;

when the signal detected from the intron/exon-negative-control probe is smaller in signal strength than the signal detected from a corresponding positive jump probe, determining that an exon/exon splice point to which the positive jump probe is complementary is probably present in the variant splicing product; and

when the signal detected from the intron/exon-negative-control probe is comparable in signal strength to the signal detected from a corresponding positive jump probe, determining that the signal detected from the corresponding positive jump probe was generated by one of;

non-specific association of the corresponding positive jump probe with non-fully-complementary target molecules;

non-specific association of the corresponding positive jump probe with non-complementary target molecules;

experimental error;

instrumental error; and

contamination.

54. The method of claim 50 wherein the positive probes and negative control probes are bound to the surface of a microarray, and wherein signals are detected by scanning the microarray.

55. Computer instructions that implement the method for determining the sequence of a variant splicing product of an initial mRNA transcript composed of exons and introns encoded in a computer readable data-storage medium.

56. A microarray manufactured for use in identifying variant splicing products of an initial mRNA transcript, the microarray comprising:

a substrate; and

an active surface of the substrate onto which features containing probe molecules are deposited, the probe molecules including;

positive probes complementary to expected subsequences of variant splicing products of the initial mRNA transcript; and

two or more different types of negative control probes.

57. The microarray of claim 56 wherein positive probes include:

positive tiling probes complementary to subsequences that span the sequence of the initial mRNA transcript;

positive exonic tiling probes complementary to exon sequences within the initial mRNA transcript; and

positive jump probes, each jump probe complementary to a subsequence including a potential splice point between two exons of the initial mRNA transcript.

58. The microarray of claim 56 wherein negative control probes include:

intron/exon-negative-control probes, including a subsequence that spans the junction between an exon and an intron in the initial mRNA transcript.