Parallel primer extension approach to nucleic acid sequence analysis

• US 7,001,722 B1
• Filed: 11/13/2000
• Issued: 02/21/2006
• Est. Priority Date: 06/22/1993
• Status: Expired due to Fees

• Current Assignee: GE Healthcare Bio-Sciences AB (General Electric Company)
• Original Assignee: Baylor College of Medicine (Baylor College), Pharmacia Biotech AB (Danaher Corporation)
• Inventors: Metspalu, Andres, Shumaker, John, Caskey, C. Thomas
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/711,476
• Time in Patent Office: 1,926 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

A method of analyzing a polynucleotide of interest, comprising providing one or more sets of consecutive oligonucleotide primers differing within each set by one base at the growing end therof; annealing a single strand of the polynucleotide or a fragment of the polynucleotide to the oligonucleotide primers under hybridization conditions; subjecting the primers to single base extension reactions with a polymerase and terminating nucleotides, the terminating nucleotides being mutually distinguishable; and observing the location and identity of each terminating nucleotide to thereby analyze the sequence or a part of the nucleotide sequence of the polynucleotide of interest, is disclosed. An apparatus comprising a solid support to which is attached at defined locations thereon one or more sets of consecutive oligonucleotide primers differing within each set by one base at the growing end thereof is also described.

Claims

1. A method for determining nucleotide identity of at least one nucleotide position of a polynucleotide of interest, comprising the steps of;

a) contacting said polynucleotide of interest with a population of single-stranded primers, wherein said single-stranded primers comprise an array of one or more sets of one or more oligonucleotides, wherein at least one set comprises at least two oligonucleotides that are substantially homologous but differ from each other by one base at their 3′

termini, wherein the oligonucleotides of the array have known sequence and wherein each oligonucleotide is attached to a solid support at a known location, to form the array, wherein at least one oligonucleotide of the array hybridizes to said polynucleotide of interest immediately adjacent to each nucleotide position to be identified, generating template-single-stranded primer complexes;

b) subjecting said complexes to a single base extension reaction to extend each annealed primer by a terminating nucleotide, generating extended primers;

c) identifying each terminating nucleotide that has been added to each primer;

thereby determining the identity of at least one nucleotide position of a polynucleotide of interest; and

d) removing the terminating nucleotides from the annealed primers after completed analysis to prepare the solid support for reuse.

2. A method of analyzing a polynucleotide of interest for the presence or absence of an altered region, comprising the steps of:

a) annealing a single sample of the polynucleotide of interest to an array of n primers of N nucleotides in length, wherein each primer is immobilized to a solid support at a known location, and has known sequence, wherein a first primer of the array comprises 1 to N nucleotides, a second primer of the array comprises nucleotides 2 to N of the first primer plus nucleotide N+1, and the n^th primer of the array comprises nucleotides 2 to the last nucleotide of primer n−

1 plus nucleotides (N+(n−

1)) such that each primer differs from the previous primer in the array by one base at the growing end, and wherein primers of the array are capable of hybridizing successively along the polynucleotide of interest, generating a plurality of annealed primers;

b) subjecting the plurality of annealed primers to a single base extension reaction to extend each annealed primer by addition of a terminating nucleotide to form a plurality of extended primers; and

c) observing the identity of each terminating nucleotide added in step b),

thereby analyzing the polynucleotide of interest for the presence or absence of an altered region.

3. The method of claim 2, wherein the single base extension reaction comprises subjecting the plurality of annealed primers to a reaction mixture comprising a polymerase and nucleotides corresponding to each of the four bases.

4. The method of claim 3, wherein the nucleotides corresponding to each of the four bases are mutually distinguishable.

5. The method of claims 3, wherein three of the four nucleotides are differently labeled.

6. The method of claim 5, wherein the three differently labeled nucleotides are fluorescently labeled.

7. The method of claim 2, further comprising analyzing a polynucleotide that is complementary to the polynucleotide of interest.

8. The method of claim 2, wherein the terminating nucleotides are dideoxynucleotides.

9. The method of claim 2, wherein N is between 7 and 30 inclusive.

10. The method of claim 2, wherein N is between 20 and 24 inclusive.

11. The method of claim 2, wherein observing the identity and location of the terminating nucleotides comprises use of a charge coupled device or a photomultiplier tube.

12. The method of claim 2, wherein the terminating nucleotides are dinucleotides.