Multiplex polynucleotide capture methods and compositions

• US 6,514,699 B1
• Filed: 06/13/2000
• Issued: 02/04/2003
• Est. Priority Date: 10/04/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Fry, George, Chen, Jer-Kang, OʼNeill, Roger A., Chiesa, Claudia
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/593,312
• Time in Patent Office: 966 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention relates to methods and compositions for simultaneously generating a plurality of polynucleotide sequencing ladders or PCR amplification products. Each sequencing ladder is generated from a recoverable primer, i.e., an oligonucleotide primer comprising a recovery tag. The recovery tag may be an oligonucleotide. Each sequencing ladder has a unique recovery tag. After the generation of the multiple sequencing ladders, the different sequencing ladders are separated from one another, i.e., purified, by binding to recovery tag binding compounds that have been immobilized on one or more solid supports. The recovery tag binding compounds are immobilized on the solid support in an addressable manner, i.e., the recovery tag binding compounds have distinct locations on the solid support. The binding of the sequencing ladders to the recovery tag binding compounds serves to separate the different polynucleotide sequencing ladders present in a given solution. The separated sequencing ladders may then be released from the immobilized recovery tag binding compounds and subsequently resolved into individual components of the sequencing ladders or PCR products. The subject methods of separating sequencing ladders simultaneously generated in the same vessel may readily be adapted to separate a plurality of simultaneously generated polynucleotide amplification products. Other aspects of the invention are kits for the generation or recovery of a plurality of polynucleotide sequencing ladders or amplification products. The kits comprise a plurality of recoverable primers having unique recovery tags. Preferably, the recovery tags are polynucleotides that have substantially the same denaturation temperature when bound to appropriate recovery tag binding compounds, i.e., the primers comprise an integrated set. The kits may further comprise recovery tag binding compounds. The kits may further comprise labeled chain terminators. Other aspects of the invention include polynucleotide recovery devices.

Claims

1. A method of simultaneously generating a plurality of polynucleotide extension products, said method comprising the steps,

mixing a plurality of recoverable primers with a plurality of polynucleotide templates,

generating a plurality of polynucleotide extension products, wherein each of the extension products is derived from one of the recoverable primers and wherein the primer extension products are polynucleotide amplification reaction products from amplification primer pairs, wherein at least one member of each primer pair is a recoverable primer having a recovery tag that is an oligonucleotide and,

binding said extension products to recovery tag binding compounds so as to effect separation of the extension products, wherein the recovery tag binding compounds are immobilized in an addressable manner on one or more solid supports.

2. A method according to claim 1, said method further comprising the step of releasing the recovery tags from the solid supports, whereby a plurality of purified amplification products is produced.

3. A method according to claim 1, wherein at least one of said recovery tags comprises a balancing polynucleotide sequence.

4. A method according to claim 1, wherein the recoverable primers are labeled.

5. A method according to claim 1, wherein the recoverable primers are not labeled.

6. A method according to claim 1, wherein the amplification templates are located on the same polynucleotide.

7. A method according to claim 1, wherein the amplification templates are located on different polynucleotides.

9. A method according to claim 1, wherein the solid support is a plurality of beads.

10. A method according to claim 1, wherein the solid support is a multi-chamber liquid holding device.

11. A method according to claim 1, wherein the solid support comprises a plurality of capillary channels.

12. A method according to claim 1, wherein the recovery tag binding compounds are polynucleotides complementary to polynucleotides synthesized during the nucleic acid amplification.

8. A method of simultaneously generating a plurality of polynucleotide extension products, said method comprising the steps,

mixing a plurality of recoverable primers with a plurality of polynucleotide templates,

generating a plurality of polynucleotide extension products, wherein each of the extension products is derived from one of the recoverable primers,

binding said extension products to recovery tag binding compounds so as to effect separation of the extension products, wherein the recovery tag binding compounds are immobilized in an addressable manner on one or more solid supports, wherein the solid support is a multiple-projection electrophoresis loading device.

13. A method of simultaneously generating a plurality of polynucleotide extension products, said method comprising the steps,

mixing a plurality of recoverable primers with a plurality of polynucleotide templates,

generating a plurality of polynucleotide extension products, wherein each of the extension products is derived from one of the recoverable primers,

binding said extension products to recovery tag binding compounds, wherein the recovery tag binding compounds are immobilized in an addressable manner on one or more solid supports,

releasing the amplification products bound to the recovery tag binding compounds, whereby a plurality of amplification products is released,

mixing a plurality of recoverable sequencing primers with the released amplification products,

generating a plurality of sequencing ladders, wherein each of the sequencing ladders is derived from a released amplification product,

binding the sequencing ladders to second recovery tag binding compounds, wherein the second recovery tag binding compounds are immobilized in an addressable manner on a second solid support.