Compositions for sorting polynucleotides

a polynucleotide and compound technology, applied in the field of polynucleotides, can solve the problems of incompatibility of presence of purified signals, limited effect of such reagents, and inability to identify, sort, and/or track molecules

Inactive Publication Date: 2007-08-21
SOLEXA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]My invention overcomes a key deficiency of current methods of tagging or labeling molecules with oligonucleotides: By coding the sequences of the tags in accordance with the invention, the stability of any mismatched duplex or triplex between a tag and complement to another tag is far lower than that of any preferably matched duplex between the tag and its own complement. Thus, the problem of incorrect sorting because of mismatch duplexes of GC-rich tags bei

Problems solved by technology

Unfortunately, such spurious signals are not uncommon because base pairing and base stacking free energies vary widely among nucleotides in a duplex or triplex structure.
Even though reagents, such as tetramethylammonium chloride, are available to negate base-specific stability differences of oligonucleotide duplexes, the effect of such reagents is often limited and their pre

Method used

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  • Compositions for sorting polynucleotides
  • Compositions for sorting polynucleotides
  • Compositions for sorting polynucleotides

Examples

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example i

Sorting Multiple Target Polynucleotides Derived from pUC 19

[0099]A mixture of three target polynucleotide-tag conjugates are obtained as follows: First, the following six oligonucleotides are synthesized and combined pairwise to form tag 1, tag 2, and tag 3 (SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:17):

[0100]

5′-pTCGACC(w1)(w2)(w3)(w4)(w5)(w6)(w7)(w8)(w1)A        GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′                         Tag 15′-pTCGACC(w6)(w7)(w8)(w1)(w2)(w6)(w4)(w2)(w1)A        GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′                         Tag 25′-pTCGACC(w3)(w2)(w1)(w1)(w5)(w8)(w8)(w4)(w4)A        GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′                         Tag 3

where “p” indicates a monophosphate, the wi's represent the subunits define in Table I, and the terms “(**)” represent their respective complements. ApUC19 is digested with Sal I and Hind III, the large fragment is purified, and separately ligated with tags 1, 2, and 3, to form pUC19-1, pUC1...

example ii

Parallel Sequencing of SV40 Fragments

[0103]A repertoire of 36-mer tags consisting of nine 4-nucleotide subunits selected from Table I is prepared by separately synthesizing tags and tag complements by a split and mix approach, as described above. The repertoire is synthesized so as to permit ligation into a Sma I / Hind III digested M13mp19. Thus, as in Example I, one set of oligonucleotides begins with the addition of A followed by nine rounds of split and mix synthesis wherein the oligonucleotide is extended subunit-wise by 3′-phosphoramidite derivatized 4-mers corresponding to the subunits of Table I. The synthesis of then completed with the nucleotide-by-nucleotide addition of one half of the Sma I recognition site (GGG), two C's, and a 5′-monophosphate, e.g. via the Phosphate-ON reagent available from Clontech Laboratories (Palo Alto, Calif.). The other set of oligonucleotides begins with the addition of three C's (portion of the Sma I recognition site) and two G's, followed by n...

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Abstract

The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention each consist of a plurality of subunits 3 to 6 nucleotides in length selected from a minimally cross-hybridizing set. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

Description

[0001]This is a continuation of U.S. patent application Ser. No. 08 / 358,810 filed 19 Dec. 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 322,348 filed 13 Oct. 1994, now abandoned, which application is incorporated by reference.FIELD OF THE INVENTION[0002]The invention relates generally to methods for identifying, sorting, and / or tracking molecules, especially polynucleotides, with oligonucleotide labels, and more particularly, to a method of sorting polynucleotides by specific hybridization to oligonucleotide tags.BACKGROUND[0003]Specific hybridization of oligonucleotides and their analogs is a fundamental process that is employed in a wide variety of research, medical, and industrial applications, including the identification of disease-related polynucleotides in diagnostic assays, screening for clones of novel target polynucleotides, identification of specific polynucleotides in blots of mixtures of polynucleotides, amplification of specific target po...

Claims

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Application Information

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IPC IPC(8): C07H19/00C07H21/02C07H21/04C12N15/09C12Q1/68A61K49/00B01J19/00C07HC07H21/00C12NC12N15/10C12N15/63C12P19/34C12QC12Q1/00C40B40/06C40B70/00G01N33/53G01N33/566G01N37/00
CPCB01J19/0046B01J2219/005B01J2219/00572B01J2219/00605B01J2219/00612B01J2219/00626B01J2219/00648B01J2219/00659B01J2219/00722C07H21/00C12N15/10C12N15/1065C12Q1/6809C12Q1/6827C12Q1/6855C12Q1/6874C40B40/06C40B70/00C40B80/00B01J2219/0061B01J2219/0063B01J2219/00637C12N15/1034C12Q1/6834B01J2219/00641C12Q1/6816C12Q1/6837C12Q2565/519C12Q2525/191C12Q2521/501C12Q2523/101C12Q2525/185C12Q2521/313C12Q2525/161C12Q2565/514C12Q2563/131C12Q2561/125C12Q2565/518C12Q2563/107C12Q2563/185C12Q2565/501C12Q1/68C12N15/63
Inventor BRENNER, SYDNEY
Owner SOLEXA
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