Simultaneous sequencing of tagged polynucleotides

• US 5,763,175 A
• Filed: 11/17/1995
• Issued: 06/09/1998
• Est. Priority Date: 11/17/1995
• Status: Expired due to Term

• Current Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Brenner, Sydney
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/560,313
• Time in Patent Office: 935 Days
• Field of Search: 435/6, 536/23.1, 536/24.2, 536/24.3, 935/76-78
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides a method for sequencing each polynucleotide of a population by using an oligonucleotide tag assigned to each such polynucleotide for transfering sequence information to a tag complement located on a spatially addressable array of such complements. That is, a unique tag is attached to each polynucleotide of a population which can be copied and used to shuttle sequence information to its complement at a fixed position on an array of such complements. After a tag hybridizes with its complement, a signal is generated that is indicative of the transferred sequence information. Sequences of the tagged polynucleotides are determined by repeated cycles of information transfer and signal detection at the positions of the corresponding tag complements.

Claims

1. A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of:

(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached;

(b) selectively amplifying tag-polynucleotide conjugates with primers whose 3'"'"' ends form perfectly matched duplexes with one or more terminal nucleotides of the polynucleotides of the population;

(c) labeling each tag of the selectively amplified tag-polynucleotide conjugates according to the identity of the one or more terminal nucleotides of its associated polynucleotide;

(d) cleaving the tags from the selectively amplified tag-polynucleotide conjugates; and

(e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

2. The method of claim 1 further including the steps of (f) cleaving said identified nucleotides from said polynucleotides;

and (g) repeating said steps (b) through (f).

3. The method of claim 2 wherein said step of selectively amplifying includes amplifying said tag-polynucleotide conjugates by a polymerase chain reaction using said primers and a second primer.

6. The method of claim 3 wherein said oligonucleotide tags comprise a plurality of subunits wherein each subunit consists of an oligonucleotide having a length of from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set, wherein a duplex between a subunit of such a set and a complement of any other subunit of the set would have at least two mismatches.

7. The method of claim 6 wherein said oligonucleotide tags and said tag complements are single stranded.

4. A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of:

(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached;

(b) amplifying the tag-polynucleotide conjugates by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3'"'"' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the tag-polynucleotide conjugate, such that a tag-polynucleotide conjugate is amplified only if the defined 3'"'"' terminal nucleotide basepairs with a nucleotide of the tag-polynucleotide conjugate;

(c) cleaving the tags from the tag-polynucleotide conjugates;

(d) labeling the tags according to the identity of the defined 3'"'"' terminal nucleotide of the second primer; and

(e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

5. The method of claim 4 further including the steps of (f) cleaving said identified nucleotides from said polynucleotides;

and (g) repeating said steps (b) through (f).

8. The method of claim 5 wherein said oligonucleotide tags comprise a plurality of subunits wherein each subunit consists of an oligonucleotide having a length of from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set, wherein a duplex between a subunit of such a set and a complement of any other subunit of the set would have at least two mismatches.

9. The method of claim 8 wherein said oligonucleotide tags and said tag complements are single stranded.

10. A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of:

(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population;

(b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached;

(c) amplifying the sampled polynucleotides by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3'"'"' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the polynucleotide, such that a polynucleotide is amplified only if the defined 3'"'"' terminal nucleotide basepairs with a nucleotide of the polynucleotide;

(d) copying the tags from the polynucleotides;

(e) labeling the copied tags according to the identity of the defined 3'"'"' terminal nucleotide of the second primer; and

(f) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

11. The method of claim 10 further including the steps of (g) cleaving said identified nucleotides from said polynucleotides;

and (h) repeating said steps (c) through (g).

12. The method of claim 11 wherein said oligonucleotide tags comprise a plurality of subunits wherein each subunit consists of an oligonucleotide having a length of from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set, wherein a duplex between a subunit of such a set and a complement of any other subunit of the set would have at least two mismatches.

13. The method of claim 12 wherein said oligonucleotide tags and said tag complements are single stranded.