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Methods for preparing nucleic acid molecules for sequencing

A sequencing and nucleotide technology, applied in the field of rolling circle amplification, can solve problems such as high error rate

Pending Publication Date: 2020-11-13
UMC UTRECHT HLDG BV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these systems have even higher error rates than next-generation (second-generation) sequencers

Method used

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  • Methods for preparing nucleic acid molecules for sequencing
  • Methods for preparing nucleic acid molecules for sequencing
  • Methods for preparing nucleic acid molecules for sequencing

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Embodiment Construction

[0046] The means and methods described herein allow multiple determinations of the sequence of the same target DNA molecule. This can be used as a means of correcting errors. This differs from classical second-generation sequencing approaches that correct for errors by sequencing multiple independent molecules covering the same genomic locus. In this case, each read typically represents one sequencing event for one molecule. With the methods of the invention, a single (target) molecule is replicated repeatedly so that a single read represents multiple sequencing events for the same molecule.

[0047] The target nucleic acid is usually double-stranded DNA. Single-stranded DNA or RNA for which a defined sequence is desired can be readily converted to double-stranded DNA by methods known in the art. Such methods include, but are not limited to, cDNA synthesis, reverse transcriptase (RT) polymerase chain reaction (PCR), PCR, random primer extension, and the like. The target DN...

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Abstract

The invention relates to means and methods for preparing double stranded target DNA molecules for sequencing. In embodiments double stranded backbone DNA molecules comprising 5' and 3' ends are provided that are ligation compatible with 5' and 3' ends of said target DNA; form a first restriction enzyme recognition site when self-ligated; in a form that enables self-ligation. Methods may comprise providing, if not already present, said target DNA with 5' and 3' ends that are in a form that prevents self-ligation and that are ligation compatible with said backbone DNA 5' and 3' ends. Methods mayfurther comprise ligating said target DNA to said backbone DNA in the presence of a ligase and a first restriction enzyme that cuts said first restriction enzyme recognition site, thereby producing at least one DNA circle comprising a backbone DNA molecule and a target DNA molecule. Linear DNA may be removed at this time and subsequently a concatemer DNA molecule comprising an ordered array of copies of said at least one DNA circle through rolling circle amplification is produced that can be sequenced.

Description

technical field [0001] The present invention relates to means and methods for determining the sequence of nucleic acid molecules. In particular, the present invention relates to methods utilizing rolling circle amplification of nucleic acid molecules whose sequences are to be determined. Background technique [0002] Sequencing methods have evolved over time. The old Sanger sequencing method has been replaced by the now commonly used next-generation sequencing (NGS) method. Recently, these methods have been reviewed in the literature (Goodwin et al. 2016; NatureReviews|Genetics Volume 17:pp 333-351: doi:10.1038 / nrg.2016.49). The most commonly used NGS methods rely on the sequencing of short stretches of DNA. Sequencing technologies for short stretches of DNA are inherently error-prone. Errors are reduced by independently sequencing multiple copies of the same target sequence. However, for each individual sequence read, it is not possible to determine whether a change re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2521/501C12Q2525/131C12Q2525/151C12Q2525/191C12Q2525/307C12Q2531/125C12Q2535/122C12Q1/6869
Inventor 维加德·彼得·克洛斯特曼耶罗·德·里德阿莱西奥·马尔科齐
Owner UMC UTRECHT HLDG BV
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