Parallel polynucleotide sequencing method using tagged primers

• US 5,935,793 A
• Filed: 09/26/1997
• Issued: 08/10/1999
• Est. Priority Date: 09/27/1996
• Status: Expired due to Term

• Current Assignee: Chinese University of Hong Kong
• Original Assignee: Chinese University of Hong Kong
• Inventors: Wong, Wing H.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/938,565
• Time in Patent Office: 683 Days
• Field of Search: 435/6, 435/810, 435/91.2, 435/91.1, 536/23.1, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within 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

Abstract

The invention is directed to a method for sequencing multiple target polynucleotide segments in parallel, and to compositions and kits therefor. In the method, a plurality of sample polynucleotide fragments are used to form a mixture of different-length sequencing fragments. The sequencing fragments are complementary to at least two different sample fragments, wherein (1) each sequencing fragment terminates at a predefined end with a known base or bases, and (2) each sequencing fragment contains an identifier tag sequence that identifies the sample fragment to which the sequencing fragment corresponds. The sequencing fragments are then separated on the basis of size to produce a plurality of resolved, size-separated bands. Resolved bands are collected in separate aliquots, which, in a preferred embodiment, are then subjected to an amplification step to amplify the complements of the tag sequences in each aliquot, and preferably, the tag sequences too. Amplification is preferably by PCR. The (amplified) aliquots are then separately hybridized with an array of immobilized different-sequence tag probes under conditions effective to provide specific hybridization of tag sequences, or of tag sequence complements, with the corresponding immobilized tag probes, to form a hybridization pattern on the array, from which sequence information of one or more sample fragments are determined.

Claims

1. A method of sequencing in parallel a plurality of polynucleotide sample fragments, the method comprising:

(a) providing a plurality of sample polynucleotide fragments,

(b) from said sample fragments, forming a mixture of different length sequencing fragments that are complementary to at least two different sample fragments, wherein (1) each sequencing fragment terminates at a predefined end with a known base or bases, and (2) each sequencing fragment contains an identifier tag sequence which identifies the sample fragment to which the sequencing fragment corresponds and optionally, the terminating base-type of the fragment, wherein said forming includes the steps of

(1) inserting said sample polynucleotide fragments into a plurality of identical vectors, to form a mixture of sequencing vectors,

(2) isolating a plurality of unique-sequence clones from said sequencing vector mixture,

(3) hybridizing to each unique-sequence clone, a tagged primer containing (i) an identifier tag sequence, and (ii) a first primer sequence located on the 3'"'"'-side of the tag sequence, to form a primer-vector hybrid, where a different identifier tag sequence is used to identify each unique-sequence clone,

(4) performing one or more chain extension reactions on each hybrid to form different-length sequencing fragments each terminating with a known base or bases, and

(5) combining the different-length sequencing fragments generated from the hybrids, to form said sequencing fragment mixture,

(c) separating said sequencing fragments on the basis of fragment length under conditions effective to resolve fragments differing in length by a single base, to produce a plurality of resolved size-separated fragments,

(d) collecting the size-separated fragments in separate aliquots,

(e) amplifying the identifier tag sequences in each aliquot to form multiple copies of oligonucleotides complementary to the identifier tag sequences, and optionally, multiple copies of the identifier tag sequences also,

(f) contacting each amplified aliquot with an array of immobilized different-sequence tag probes, each tag probe (1) being capable of hybridizing specifically with one of said identifier tag sequences or a tag sequence complement thereof, and (2) having an addressable location in said array, where said contacting is conducted under conditions effective to provide specific hybridization of the identifier tag sequences, or tag sequence complements, with the corresponding immobilized tag probes, to form a hybridization pattern on said array,

(g) from the hybridization pattern formed, determining a nucleotide sequence for at least one sample fragment.

2. The method of claim 1, wherein said amplifying includes repeated cycles of binding and extending a second primer which is complementary to the first primer sequence in the sequencing fragments, to generate multiple copies of a sequence complementary to the identifier tag sequence.

3. The method of claim 1, wherein each tagged primer additionally includes a second primer sequence which is located on the 5'"'"'-side of the tag sequence in the tagged primer, and said amplifying includes repeated cycles of binding and extending third and fourth primers which correspond to said first and second primer sequences, to amplify the identifier tag sequences and their complements.

4. The method of claim 1, wherein

step (b)(1) is performed on a plurality of separate, different-sequence tag-vectors, each different-sequence tag-vector having (i) a cloning site, (ii) located on the 3'"'"'-side of the cloning site, a first vector primer sequence which contains a vector-identifier tag region which is unique for each different-sequence tag-vector, to form a plurality of libraries of different-sequence tag-vectors,

step (b)(2) includes isolating at least one clone from each different-sequence tag-vector clone library, and

step (b)(3) includes mixing together a clone isolated from each said different-sequence tag-vector library before said hybridizing, to form a clone mixture, and the tagged primers in said hybridizing step additionally contain sequences complementary to each different vector-tag identifier sequence.

5. The method of claim 4, wherein said different-sequence tag-vectors additionally contain a second vector primer sequence that is the same for all said vectors, and which is located on the 3'"'"'-side of said first vector primer sequence in each said vector.

6. The method of claim 1, wherein sequencing fragments are labeled with a fluorescent label.

7. The method of claim 6, wherein said fluorescent label is attached to the 5'"'"'-end of a sequencing fragment.

8. The method of claim 6, wherein said fluorescent label is attached to the 3'"'"'-end of a sequencing fragment.

9. The method of claim 6, wherein a different fluorescent label is used to identify each different terminating base-type.

10. The method of claim 1, wherein sequencing fragments are labeled with a radioactive label.

11. The method of claim 1, wherein detectable labels are incorporated into said copies during said amplifying.

12. The method of claim 1, wherein said separating is effected by capillary electrophoresis.

13. The method of claim 1, wherein said separating is effected by slab gel electrophoresis.

14. A polynucleotide mixture comprising

a plurality of primer-tag-primer polynucleotides each comprising a first primer sequence, an identifier tag sequence linked to the 3'"'"'-side of the first primer sequence, and a second primer sequence linked to the 3'"'"'-side of the tag sequence,

wherein the first primer sequences are identical to each other, the identifier tag sequence in each primer-tag-primer polynucleotides differs from the tag sequence in every other primer-tag-primer polynucleotide, and the second primer sequences are identical to each other.

15. A sequencing fragment mixture comprising

a plurality of different-sequence sequencing fragments derived from a plurality of different sample polynucleotide templates, each different-sequence sequencing fragment containing

(1) a template-complement region derived from a selected sample template fragment and having a pre-determined base-type located at the 3'"'"'-end of the associated fragment, and

(2) at the 5'"'"'-end of the fragment, a primer-tag-primer region containing (i) a first primer sequence, (ii) an identifier tag sequence linked to the 3'"'"'-side of the first primer sequence, and (iii) a second primer sequence linked to the 3'"'"'-side of the tag sequence,

wherein the first primer sequences in the sequencing fragments are identical to each other, the second primer sequences in said sequencing fragments are identical to each other, and the identifier tag sequence in each primer-tag-primer region uniquely identifies the sample fragment from which the sequencing fragment was derived, and the sequencing fragment'"'"'s 3'"'"'-terminal base type.

16. A kit for sequencing a plurality of polynucleotide fragments, said kit comprising

a plurality of primer-tag-primer polynucleotides each comprising a first primer sequence, an identifier tag sequence linked to the 3'"'"'-side of the first primer sequence, and a second linked to the 3'"'"'-side of the tag sequence, wherein the first primer sequences are identical to each other, the tag sequence in each primer-tag-primer polynucleotides differs from the tag sequence in every other primer-tag-primer polynucleotide, and the second primer sequences are identical to each other, and

an array of immobilized different-sequence tag probes, each tag probe (1) being capable of hybridizing specifically with one of said identifier tag sequences or a tag sequence complement, and (2) having an addressable location in said array.