Method for preparing polynucleotides for analysis

A technology of polynucleotides and target polynucleotides, applied in biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as readout step obstruction

Inactive Publication Date: 2008-03-12
LINGVITAE AS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] One difficulty with prior art methods is that the final readout step is often hampered by the need to distinguish between different amplified tags or cells

Method used

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  • Method for preparing polynucleotides for analysis
  • Method for preparing polynucleotides for analysis
  • Method for preparing polynucleotides for analysis

Examples

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Embodiment

[0086] To illustrate the "rollback" principle, a 114nt single-stranded molecule was used as the second polynucleotide substrate and the 38bp circular target template. The second polynucleotide was "anchored" by immobilizing the substrate molecule with biotin on paramagnetic beads coated with 1 [mu]M streptavidin.

[0087] The target molecule is hybridized to the second polynucleotide and phi29 DNA polymerase is added. The polymerase performs an extension reaction using the target as a template. The extended strand is complementary to the second polynucleotide, and according to the rollback theory, the extended strand will hybridize to the second polynucleotide to form a 114bp double-stranded molecule. Depending on the sequence of the target, the double-stranded molecule will contain recognition sites for certain restriction endonucleases (Figures 10, 11 and 12).

[0088] As shown in FIG. 10 , the target having the unit sequence 0100 forms a recognition site for BamHI at bit ...

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Abstract

A method for analysing a target polynucleotide having distinct units of nucleic acid sequence comprising: (i) forming a first polynucleotide which is a concatemer having multiple repeating target polynucleotide sequences; (ii) forming on the first polynucleotide a second polynucleotide hybridised to a portion of one or more of the target polynucleotides, such that the portion hybridised, or the portion not hybridised, corresponds to a sequence unit on the target, and determining the sequence unit on the target.

Description

technical field [0001] The present invention relates to methods of modifying polynucleotides so that the polynucleotides can be more easily analyzed. Background technique [0002] Advances in molecular research have been driven in part by improvements in techniques used to characterize molecules or their biological responses. In particular, the study of the nucleic acids DNA and RNA has benefited from technological developments in sequence analysis and the study of hybridization reactions. [0003] WO-A-00 / 39333 describes a method for sequencing polynucleotides by converting the sequence of a target polynucleotide into a second polynucleotide having a defined sequence and location information. It is reported that the sequence information of the target is "amplified" in the second polynucleotide, making it easier to distinguish individual bases in the target molecule. This can be achieved through the use of "amplified tags" with predetermined nucleic acid sequence units. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6813C12Q2525/151C12Q2531/125C12Q1/6806
Inventor P·莱克索
Owner LINGVITAE AS
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