Partially degenerate oligonucleotide standards and methods for generating the same

• US 20060121491A1
• Filed: 12/02/2004
• Published: 06/08/2006
• Est. Priority Date: 12/02/2004
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wolber, Paul K., Dokidis, Anthea
• Application Number:
  US11/001,867
• Publication Number:
  US 20060121491A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction

Abstract

In one embodiment of the present invention, a set of partially degenerate oligonucleotides can be used as standards for monitoring array-feature consistency during manufacturing and for calibrating array experiments. In another embodiment, methods for generating deterministic, fully-characterized sets of partially degenerate sequence standards are provided, in which parameters such as the oligonucleotide-sequence length, the generic sequence string, and the complexity of a set of oligonucleotides can be controlled by a user. Various sets of oligonucleotides with partially degenerate sequences may be combined in order to provide more desirable standards for a variety of array-related uses.

Claims

1. A standard for array calibration and for array quality-control comprising a set of oligonucleotide molecules having partially degenerate sequences and having a complexity, log (S), less than 11.

2. The standard of claim 1 wherein the complexity is less than 15.

3. The standard of claim 1 wherein the set of oligonucleotide molecules includes a combination of two or more subsets of partially degenerate sequences, each subset independently produced.

4. The standard of claim 1 wherein the oligonucleotide molecules are composed of at least one of:

deoxyribonucleic acids (DNA);

ribonucleic acids (RNA);

locked nucleic acids (LNA);

bridged nucleic acids (BNA);

unstructured nucleic acids (UNA);

peptide nucleic acids (PNA); and

derivatives of DNA, RNA, LNA, BNA, UNA, and PNA.

5. The standard of claim 1 wherein the oligonucleotide molecules are labeled by one of:

a chemiluminescent moiety;

a moiety including a mass tag; and

a moiety including a radioisotope.

6. A method for preparing a standard set of oligonucleotides useful for calibrating arrays and for monitoring quality-control of arrays comprising:

selecting one or more sets of oligonucleotides, each set of oligonucleotides specified by a symbol string;

for each symbol string, inputting the symbol string into a synthesizer to produce a set of oligonucleotide molecules having partially degenerate sequences specified by the symbol string; and

when two or more sets are produced as specified by two or more symbol strings, combining the two or more sets of oligonucleotides to produce a standard set of oligonucleotides.

7. The method of claim 6 wherein selecting one or more sets of oligonucleotides further comprises employing computational techniques in order to select a set of oligonucleotide standards that

binds to a majority of different probe molecules of the array;

and

binds with varied affinities to different probe molecules of the array.

8. The method of claim 6 wherein the set of oligonucleotide standards comprises partially degenerate oligonucleotide standard molecules that

minimally cross-hybridize with other partially degenerate oligonucleotide standard molecules of the set;

minimally self-hybridize; and

minimally cross-hybridize with experimentally-derived sample target molecules.

9. A method for evaluating a batch of manufactured arrays comprising:

exposing a subset of test arrays selected from the batch of manufactured arrays to a standard set of oligonucleotides having partially degenerate sequences;

determining a set of signal intensities for each test array;

comparing the determined signal intensities for the test arrays to determine a comparison metric; and

rejecting the batch of manufactured arrays when the determined comparison metric exceeds a threshold value.

10. The method of claim 9 wherein the comparison metric is derived from a statistical variance computed for the signal intensities of the test arrays.

11. The method of claim 9 wherein the comparison metric is derived from a statistical difference between the signal intensities measured for the test arrays and a standard set of signal intensities.