Reagents, methods, and libraries for gel-free bead-based sequencing

A substrate and sequence technology, applied in the field provided by the invention, can solve the problems of restricting wide application and errors in highly similar sequences

Inactive Publication Date: 2009-07-29
APPL BIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although electrophoretic separation is not required, pyrosequencing has a number of disadvantages that still limit its widespread application (Franca et al., Quarterly Reviews of Biophysics, 35(2):169-200, 2002)
Sequencing by hybridization has also been proposed as an alternative (US Patent 5,202,231; WO 99 / 60170; WO 00 / 56937; Drmanac et al., Advances in Biochemical Engineering / Biotechnology, 11:16-101, 2002), but also has many disadvantages, including in distinguishing highly Errors may occur when similar sequences

Method used

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  • Reagents, methods, and libraries for gel-free bead-based sequencing
  • Reagents, methods, and libraries for gel-free bead-based sequencing
  • Reagents, methods, and libraries for gel-free bead-based sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0260] Example 1 provides an exemplary protocol.

[0261] Sequencing can be performed in the 5'→3' direction with an extension probe containing a 3'-O-P-S-5' junction. Figure 5A shows the use of 5'-O-P-O-X-O-P-S-NNNNN B * -One cycle of hybridization, ligation and cleavage performed by the extension probe in the 3' form, where N represents any nucleotide, and N B Represents the part that the ligase cannot extend (such as N B is a nucleotide lacking a 3' hydroxyl group or having a blocking moiety attached), * Represents a detectable moiety, and X represents a nucleotide whose species corresponds to the detectable moiety. Alternatively, a bulk blocking moiety can be attached to the 3' terminal nucleotide to prevent multiple ligation. For example, attaching a bulky group to, for example, the 2' or 3' position of the sugar moiety of the nucleotide will prevent ligation. Fluorescent labels can be used as suitable macrogroups.

[0262] A template comprising a binding region 40...

Embodiment 2

[0446] Example 2: Efficient Cleavage and Ligation of Phosphorothioated Oligonucleotides Containing Nucleotides with Reduced Degeneracy

[0447] However, another consideration for probe length is the fidelity of the extended oligo and its impact on subsequent ligation efficiency. The fidelity of T4 DNA ligase has been shown to decrease rapidly after the 5th base after the junction (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). If a mismatch is introduced on the 5' side of the newly ligated junction, ligation efficiency can be reduced by depletion, however, without a phase shift or increase in background signal (a major hurdle encountered in polymerase-based sequencing by synthetic approaches). ).

[0448] Preferably, the probe set should be able to hybridize to any DNA sequence in order to resequence uncharacterized DNA. However, the complexity of labeled probe sets increases exponentially with the length and number of 4-fold degenerate bases. Furthermor...

Embodiment 3

[0453] Example 3: Fidelity of Probe Ligation

[0454] Bacterial NAD-dependent ligases such as Taq DNA ligase have been reported to have high sequence fidelity at the ligation, with essentially no nick-closing activity for mismatches on the 3' side but some tolerance for mismatches on the 5' side (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). On the other hand, T4 DNA ligase has been reported to be slightly less stringent, allowing mismatches at the 3'- and 5'-sides of the junction. Therefore, it was of interest to evaluate the fidelity of probe ligation with T4 DNA ligase compared to Taq DNA ligase in our system.

[0455] Using standard ABI sequencing techniques, we developed two methods to assess the sequence fidelity of ligated oligonucleotides. The first approach was designed to clone and sequence the ligation products. In this method, ligated extension products are ligated to adapter sequences, cloned and transformed into bacteria. Individual colon...

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Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and / or agents appropriate to remove a damaged base from a nucleic acid. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to microparticles, which are immobilized in or on a semi-solid support or attached to a substrate. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage.

Description

[0001] governmental support [0002] This invention was made with government support (grant number R01-HG-003570 awarded by NIH). The government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application claims the benefit of and priority to co-pending US Provisional Application 60 / 793,702, filed April 19, 2006, which is hereby incorporated by reference in its entirety. This application claims to be related to provisional applications USSN 60 / 649,294, filed February 1, 2005; USSN 60 / 656,599, filed February 25, 2005; USSN 60 / 673,749, filed April 21, 2005, July 15, 2005 Priority and benefit of USSN 60 / 699,541, filed September 30, 2005 and USSN 11 / 345,979, both filed September 30, 2005, all of which are incorporated herein by reference. Background of the invention [0005] Nucleic acid sequencing technology is important in a variety of fields from basic research to clinical diagnosis. Results obtained from such techniques...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6874C12Q1/6837C12Q1/6869C12Q2565/501C12Q2533/107C12Q2531/137C12Q2537/1373C12Q2535/00
Inventor K·麦柯南A·布兰查德G·科斯塔
Owner APPL BIOSYSTEMS INC
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