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Methods, compositions, and kits for nucleic acid analysis

An oligonucleotide and subgroup technology, applied in combinatorial chemistry, nucleotide library, organic compound library, etc., can solve problems such as poor application of diagnostic sequencing and sequencing errors

Inactive Publication Date: 2016-02-17
TOMA BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, preamplification of nucleic acid libraries can introduce sequencing errors due to the inherent error rate of nucleic acid polymerases
The introduction of pre-amplification bias and errors caused by pre-amplification can have undesirable consequences for diagnostic sequencing applications where accurate detection of rare mutations from limited starting samples is often desired

Method used

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  • Methods, compositions, and kits for nucleic acid analysis
  • Methods, compositions, and kits for nucleic acid analysis
  • Methods, compositions, and kits for nucleic acid analysis

Examples

Experimental program
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Effect test

preparation example Construction

[0410] Preparation of target-enriched libraries

[0411] In another aspect, the present invention provides a method for preparing a target-enriched DNA library. The method can include hybridizing target-selective oligonucleotides to sequencing library members to produce hybridization products. The method may further comprise amplifying the hybridization product in a single round of amplification to generate an extended strand.

[0412] Methods of target enrichment can be as described in US Patent Application Publication No. 20120157322, which is incorporated herein by reference.

[0413] Hybridization and amplification can occur in the reaction mixture. The mixture may contain nucleotides (dNTPs), polymerases, and target-selective oligonucleotides. In some embodiments, the mixture comprises a plurality of target-selective oligonucleotides. The mixture may contain, for example, 1-10, 5-20, 10-50, 40-100, 80-200, 150-500, 300-1000, 800-2000, 1000-5000, 4000-10000, 8000-200...

Embodiment 1

[0536] Figure 24Methods for assessing cancer in a subject are shown. The subject has had a colonoscopy and was found to have a colon tumor. At time point 0, tumor biopsies and blood draws were taken from subjects and used to aid in the diagnosis of colon cancer in subjects. Tumor and normal cells from the first blood draw were sequenced. Sequencing revealed the presence of three mutations in the subject's tumor. The mutation is a point mutation in the APC, KRAS and TP53 genes. The stage of the subject's cancer was determined. The subject underwent the first treatment (surgery) to remove the tumor. During the first treatment, a second blood draw was performed. The subject's tumor is determined to have metastasized. The subject is administered a second therapy (chemotherapy) to control the cancer. Subsequent blood draws were performed to determine the mutational status of these three genes in cell-free DNA from blood.

Embodiment 2

[0537] Example 2: Validation assay for tumor-specific mutations in subjects with colon cancer

[0538] The NCI-H1573 (CRL-5877) cell line with the KRASG12A mutation (mu) was obtained as a frozen stock from the American Type Culture Collection (ATCC). Genomic DNA (gDNA) was prepared from cell line material using a commercially available kit (DNeasyBlood & Tissue Kit, QIAGEN) according to the manufacturer's suggested protocol. Estimates of DNA concentration were obtained spectrophotometrically by measuring OD260 (NanoDrop1000, ThermoFisher Scientific Inc.).

[0539] Genomic DNA from the NA18507 cell line was used as a surrogate for wild-type DNA (wt) and was obtained as a purified stock (Coriell). Combine two microliters of the mixture containing wt (30 ng) and mu (6 ng) DNA into a 20 μl ddPCR reaction mix from a 2x ddPCR supermix for probes at a final concentration of 0.2 uM of each Forward primer (wt: 5'-AGATTACGCGGCAATAAGGCTCGGTTGGCATTGGATACTACTTGCCTACGCCACC-3' (SEQ ID NO: ...

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Abstract

Aspects of the invention relate to methods and kits for assessing cancer. Some aspects of the invention relate to methods and kits for preparing a sample library for sequencing. Some aspects of the invention relate to methods and kits for allele detection. Some aspects of the invention relate to high efficiency ligation methods and kits. Some aspects of the invention relate to sensitive detection of amplicons.

Description

[0001] cross reference [0002] This application claims US Provisional Application No. 61 / 767,718, filed February 21, 2013, US Provisional Application No. 61 / 769,683, filed February 26, 2013, US Provisional Application No. 61 / 769,683, filed March 12, 2013. 61 / 777,702, U.S. Provisional Application No. 61 / 780,578, filed March 13, 2013, U.S. Provisional Application No. 61 / 824,894, filed May 17, 2013, and U.S. Provisional Application No. August 27, 2013 .61 / 870,634, incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing that has been electronically filed in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on February 19, 2014, is named 44288-703.601_SL.txt and is 1,580,104 bytes in size. Background technique [0005] Cancer presents a serious challenge to modern medicine. In 2007, cancer has been estimated to cause approximately 13% of all human deaths (7.9 million) world...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B40/04C40B40/06C40B40/08
CPCC12Q2600/106C12Q2600/156C12Q1/6806C12Q1/6886C12Q2521/501C12Q2525/204C12Q2535/122C40B40/04C40B40/06C40B40/08C12Q2600/158C12P19/34
Inventor 奥斯汀·索迈克尔·Y·卢塞罗
Owner TOMA BIOSCI
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