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Preparation of templates for nucleic acid sequencing

a nucleic acid sequencing and template technology, applied in chemical libraries, sugar derivative preparation, combinational chemistry, etc., can solve the problem that the arrays comprised of such bridged structures provide inefficient templates for nucleic acid sequencing

Inactive Publication Date: 2009-05-07
ILLUMINA CAMBRIDGE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0027]The present invention will now be further described. In the following passages different features of the various aspects of the invention are defined in more detail. Each feature so defined in connection with one aspect of the invention may be combined with features

Problems solved by technology

Arrays comprised of such bridged structures provide inefficient templates for nucleic acid sequencing, since hybridisation of a conventional sequencing primer to one of the immobilised strands is not favoured compared to annealing of this strand to its immobilised complementary strand under standard hybridisation conditions.
A disadvantage of the use of restriction enzymes for linearisation is that it requires the presence of a specific recognition sequence for the enzyme at a suitable location in the bridged template structure.
There is a risk that the same recognition sequence may appear elsewhere in the bridged structure, meaning that the enzyme may cut at one or more further sites, in addition to the intended cleavage site for linearisation.
This may be a particular problem where the bridged structures to be linearised are derived by solid-phase amplification of templates of partially unknown sequence, since it cannot be predicted in advance whether a particular enzyme will cut within the region of unknown sequence.

Method used

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  • Preparation of templates for nucleic acid sequencing
  • Preparation of templates for nucleic acid sequencing
  • Preparation of templates for nucleic acid sequencing

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cleavage Evaluation of Diol Primer in 8 Channel (Baccarat) Chip

Oligonucleotides Used:

[0200]Labelled P5 complementary oligonucleotide (supplied by Eurogentec):

(SEQ ID NO:1)5′-Texas Red-TCGGTGGTCGCCGTATCATT-3′-OH

[0201]Grafting control primer (supplied by Eurogentec):

(SEQ ID NO:2)5′-phosphorothioate-GTAGACTGCATGACCTGTAG-3′-Cy3

[0202]P5 non-cleavable primer (supplied by Eurogentec):

(SEQ ID NO:3)5′-phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCGA-3′OH

[0203]P5 cleavable primer (supplied by Fidelity systems):

(SEQ ID NO:4)5′-phosphorothioate-arm 26-diol22A-AATGATACGGCGACCACCGA-3′OH

[0204]The structures of the arm26 and diol22A components were as follows:

[0205]Grafting was performed according to the procedure described under general methods. Channels 1 and 2 of an 8 channel chip were grafted using the non-cleavable primer, channels 5, 6 and 7 using the cleavable diol linker and channel 8 using the grafting control.

[0206]A first hybridization using the complementary P5 oligonucleotide (SEQ ID NO...

example 2

Nucleic Acid Colony Formation on Acrylamide (SFA) Coated 8 Channel (Baccarat) Chips Using Diol Primer

Oligonucleotides Used:

[0209]PS non-cleavable primer (supplied by Eurogentec):

(SEQ ID NO:3)5′-phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCGA-3′OH

[0210]P7 non-cleavable primer (supplied by Eurogentec):

(SEQ ID NO:5)5′-phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATACGA-3′OH

[0211]P5 cleavable primer (supplied by ATD):

5′-phosphorothioate-TTTTTTTTTT-(diol)X3-AATGATACGGCGACCACCGA-3′OH

(equivalent to SEQ ID NO:3 but including diol linkage)

[0212]The structure of the “diol” linker incorporated into the cleavable primer was as follows:

[0213]It will be noted that the linker unit is incorporated with the diol in a protected OAc form during oligonucleotide synthesis. The free diol is released by ammonia treatment during oligonucleotide cleavage / deprotection. Therefore, the primers used in the grafting reaction contain the free diol.

[0214]Grafting was performed according to the procedure described in ...

example 3

Cleavage and Subsequent Hybridization of Nucleic Acid Colonies Generated on Diol Cleavable Primer

Olionucleotide Used:

[0217]A594 sequencing primer (supplied by Eurogentec):

(SEQ ID NO:6)5′-A594-CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTG-3′-OH

[0218]Channels to be cleaved were treated with a solution of 0.1M of sodium periodate and 0.1M ethanolamine in water for 1 hour at room temperature. All the other channels were washed with milliQ water. All channels were then washed for 30 minutes with milliQ water at room temperature.

[0219]Hybridization was carried out using the sequencing primer (SEQ ID NO:6) labelled with A594 to evaluate the percentage of non-cleaved oligonucleotide. The sequencing primer was hybridised to the linearised clusters prepared as described above at 500 nM, using standard conditions for hybridisation as described under the general methods. The chip was then imaged using an orange filter with an exposure time of 1 s.

Results

[0220]As expected, the channels grafted with the...

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Abstract

The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise:providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end,cleaving one or both strands of the double-stranded nucleic acid molecule, andsubjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction.

Description

FIELD OF THE INVENTION[0001]The invention relates to the preparation of templates for nucleic acid sequencing reactions and to methods of sequencing such templates. In particular, the invention relates to the preparation of template nucleic acid molecules ready for sequencing by cleavage of one or both strands of a double-stranded nucleic acid immobilised on a solid support.BACKGROUND TO THE INVENTION[0002]Nucleic acid sequencing methods have been known in the art for many years. One of the best-known methods is the Sanger “dideoxy” method which relies upon the use of dideoxyribonucleoside triphosphates as chain terminators. The Sanger method has been adapted for use in automated sequencing with the use of chain terminators incorporating fluorescent labels.[0003]There are also known in the art methods of nucleic acid sequencing which are based on successive cycles of incorporation of fluorescently labelled nucleic acid analogues. In such “sequencing by synthesis” or “cycle sequencin...

Claims

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Application Information

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IPC IPC(8): C40B20/00C07H1/00C12P19/34C40B50/06
CPCC12Q1/6806C12Q1/6834C12Q1/6874C12Q2565/543C12Q2535/101C12Q2523/113C12Q2523/107C12Q2521/331C12Q2521/307B01J19/0046B01J2219/00596B01J2219/00675B01J2219/00608B01J2219/00722
Inventor LIU, XIAOHAIMILTON, JOHNSMITH, GEOFFREY PAULBARNES, COLINRASOLONJATOVO, ISABELLE MARIE JULIARIGATTI, ROBERTOWU, XIAOLINOST, TOBIAS WILLIAM BARRWORSLEY, GRAHAM JOHNEARNSHAW, DAVID JAMESTURCATTI, GERARDOROMIEU, ANTHONY
Owner ILLUMINA CAMBRIDGE LTD
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