US 2016/0098516 A1Published application

Methods and systems for detection of a genetic mutation

OwnerIl-Jin KIM, David JABLONS +2inventors

InventorsIl-Jin KIM, David JABLONS, Pedro Juan Mendez ROMERO, Jun-Hee YOON

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Abstract

Methods and systems for the detection of genetic mutations from a tissue sample (e.g., a preserved tissue sample) are provided. The method includes the steps of a) extracting a nucleic acid from a tissue or biological sample; b) preparing a targeted nucleic acid amplicon library from the extracted nucleic acid; c) sequencing the targeted nucleic acid amplicon library to produce tissue sample target nucleic acid sequence data; and d) analyzing the sample target nucleic acid sequence data to determine whether it contains a mutation (e.g., a mutation associated with a risk for a particular disease). The methods described herein advantageously can be performed in less than 36 hours.

Claims10

Dependent claims

A method for extracting nucleic acid from a preserved tissue sample, the method comprising the steps of:

(a) incubating the preserved tissue sample with a tissue digestion solution to form a tissue digestion mixture, wherein the tissue digestion solution is selected from the group consisting of:
(i) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, KH2PO4 at a concentration of 0.1 mM to 5 mM, and Tween 20;
(ii) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, KH2PO4 at a concentration of 0.1 mM to 5 mM, and Triton-X100;
(iii) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, and KH2PO4 at a concentration of 0.1 mM to 5 mM;
(iv) a tissue digestion solution comprising TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, and KCl at a concentration of 0.2 mM to 200 mM;
(v) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM;
(vi) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM and Triton-X100;
(vii) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM and Tween 20;
(viii) a tissue digestion solution comprising TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, KCl at a concentration of 0.2 mM to 200 mM, and Triton-X100;
(ix) a tissue digestion solution comprising a TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, KCl at a concentration of 0.2 mM to 200 mM, and Tween 20;
and
(x) a tissue digestion solution comprising a TAPS sodium salt at a concentration of 0.5 mM to 25 mM, KCl at a concentration of 0.2 mM to 200 mM, β-Mercaptoethanol at a concentration of 0.1 mM to 1 mM, and Triton-X100,
(b) heating the tissue digestion mixture at 80 to 110° C. for 1-30 minutes;
(c) adding a protease solution comprising a proteinase to the tissue digestion mixture to form a protein degradation mixture and incubating the protein degradation mixture at 50 to 70° C. for 1-30 minutes;
and
(d) incubating the protein degradation mixture at 80 to 110° C. for 1-30 minutes;
thereby extracting nucleic acid from the preserved tissue sample.

14 more elements

A method for determining whether or not a nucleic acid from a preserved tissue sample has a mutation, the method comprising the steps of:

(a) incubating the preserved tissue sample with a tissue digestion solution to form a tissue digestion mixture, wherein the tissue digestion solution is selected from the group consisting of:
(i) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, KH2PO4 at a concentration of 0.1 mM to 5 mM, and Tween 20;
(ii) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, KH2PO4 at a concentration of 0.1 mM to 5 mM, and Triton-X100;
(iii) a tissue digestion solution comprising NaCl at a concentration of 10 mM to 140 mM, Na2HPO4 at a concentration of 0.5 mM to 10 mM, and KH2PO4 at a concentration of 0.1 mM to 5 mM;
(iv) a tissue digestion solution comprising TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, and KCl at a concentration of 0.2 mM to 200 mM;
(v) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM;
(vi) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM and Triton-X100;
(vii) a tissue digestion solution comprising HEPES buffer at a concentration of 1 mM to 100 mM and Tween 20;
(viii) a tissue digestion solution comprising TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, KCl at a concentration of 0.2 mM to 200 mM, and Triton-X100;
(ix) a tissue digestion solution comprising a TAPS sodium salt at a concentration of 0.5 mM to 25 mM, DTT at a concentration of 0.05 mM to 5 mM, KCl at a concentration of 0.2 mM to 200 mM, and Tween 20;
and
(x) a tissue digestion solution comprising a TAPS sodium salt at a concentration of 0.5 mM to 25 mM, KCl at a concentration of 0.2 mM to 200 mM, β-Mercaptoethanol at a concentration of 0.1 mM to 1 mM, and Triton-X100,
(b) heating the tissue digestion mixture at 80 to 110° C. for 1-30 minutes;
(c) adding a proteinase solution comprising a proteinase to the tissue digestion mixture to form a protein degradation mixture and incubating the protein degradation mixture at 50 to 70° C. for 1-30 minutes;
(d) incubating the protein degradation mixture at 80 to 110° C. for 1-30 minutes;
thereby extracting nucleic acid from the preserved tissue sample;
(e) amplifying nucleic acid extracted from the tissue sample, the step of amplication using 5′ phosphorylated oligonucleotides that target a nucleic acid of interest;
(f) directly ligating an oligonuclotide comprising an adaptor nucleic acid and a bar code nucleic acid to each of the amplified target nucleic acids, thereby making a targeted nucleic acid amplicon library comprising tissue sample target nucleic acid;
(g) sequencing the library;
(h) obtaining a tissue sample target nucleic acid sequence data and database target nucleic acid sequence data, wherein the database target nucleic acid sequence data is located in a mutation database;
(i) comparing the tissue sample target nucleic acid sequence data with the database target nucleic acid sequence data to determine if the sample target nucleic acid sequence data contains a registered mutation from the mutation database;
(j) determining the reliability of the mutation that is registered in the mutation database by determining the mutant allele frequency of the mutation that is registered in the mutation database;
and
(k) generating a result as to whether the tissue sample target nucleic acid sequence data contains a mutation, thereby detecting the mutation.

21 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/867,934Filed 28 Sep 2015
Priority: 26 Sep 2014
Published: 7 Apr 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

C12N 15/1003
Processes for the isolation, preparation or purification of DNA or RNA: extracting or separating nucleic acids from biological samples, e.g. pure…
C12N 15/1065
Isolating an individual clone by screening libraries: preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis,…
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6869
Involving nucleic acids: methods for sequencing