Methods for detecting cystic fibrosis mutations using mitra tip extraction

Patent metadata

Field	Value
Publication number	US-11486005-B2
Application number	US-201715811550-A
Country	US
Kind code	B2
Filing date	Nov 13, 2017
Priority date	Nov 15, 2016
Publication date	Nov 1, 2022
Grant date	Nov 1, 2022

Abstract

The present disclosure provides methods for determining whether a patient exhibiting cystic fibrosis symptoms, or a patient at risk for cystic fibrosis, will benefit from treatment with one or more anti-cystic fibrosis therapeutic agents. These methods are based on detecting hereditary cystic fibrosis related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device. Kits for use in practicing the methods are also provided.

First claim

What is claimed is:

1. A method for detecting the presence or absence of mutations in a pool of nucleic acid samples comprising
(a) eluting at least two dried biological fluid samples, each having a volume of 20 μL or less and containing about 100 ng to 400 ng of genomic DNA, from at least two absorbent tips of microsampling devices, each of the at least absorbent tips comprising a separate dried biological fluid sample, by contacting the least two absorbent tips of the microsampling devices with a Proteinase K and a lysis buffer comprising guanidine hydrochloride, Tris⋅Cl, EDTA, and a nonionic surfactant;
(b) extracting from at least two nucleic acid samples of genomic DNA comprising a sample cystic fibrosis transmembrane regulator (CFTR) nucleic acid from the at least two dried biological fluid samples eluted from the at least two absorbent tips of microsampling devices;
(c) generating at least two libraries of amplicons corresponding to a plurality of target segments of the sample CFTR nucleic acid from each of the at least two nucleic acid samples, wherein each of the at least two libraries of amplicons comprises an index sequence;
(d) pooling the at least two libraries of amplicons; and
(e) detecting the presence or absence of each mutation from a panel of mutations in the at least two libraries of amplicons using high throughput massive parallel sequencing, wherein the panel of mutations comprises:

Conventional
HGVS cDNA
Conventional
HGVS cDNA

Name
Nomenclature
Name
Nomenclature

296+2T>A
c.164+2T>A
2143delT
c.2012delT

394delTT
c.262 263delTT
2183AA>G
c.2051

2052delAAinsG

405+1G>A
c.273+1G>A
2184delA
c.2052delA

406-1G>A
c.274-1G>A
2184insA
c.2052 2053insA

444delA
c.313delA
2307insA
c.2175 2176insA

457TAT>G
c.325
2347delG
c.2215delG

327delTATinsG

574delA
c.442delA
2585delT
c.2453delT

621+1G>T
c.489+1G>T
2622+1G>A
c.2490+1G>A

663delT
c.531delT
2711delT
c.2583delT

711+1G>T
c.579+1G>T
2789+5G>A
c.2657+5G>A

711+3A>G
c.579+3A>G
2869insG
c.2737 2738insG

711+5G>A
c.579+5G>A
3007delG
c.2875delG

712-1G>T
c.580-1G>T
3120+1G>A
c.2988+1G>A

852del22
c.720 741del22
3120G>A
c.2988G>A

935delA
c.803delA
3121-1G>A
c.2989-1G>A

936delTA
c.805 806delAT
3171delC
c.3039delC

1078delf
c.948delT
3199del6
c.3067

3072delATAGTG

1154insTC
c.1022
3272-26A>G
c.3140-26A>G

1023insTC

1161delC
c.1029delC
3659delC
c.3528delC

1213delT
c.1081delf
3667del4
c.3535

3538delACCA

1248+1G>A
c.1116+1G>A
3791delC
c.3659delC

1259insA
c.1127 1128insA
3821delT
c.3691delT

1288insTA
c.1153
3849+10kbC>T
c.3717+12191C>T

1154insAT

1341+1G>A
c.1209+1G>A
3876delA
c.3744delA

1461ins4
c.1329
3905insT
c.3773 3774insT

1330insAGAT

1525-1G>A
c.1393-1 G>A
4005+1 G>A
c.3873+1G>A

1548delG
c.1418delG
4016insT
c.3884 3885insT

1609delCA
c.1477
4209TGTT>AA
c.4077

1478delCA

4080delTGTTinsAA

1677delTA
c.1545
4382delA
c.4251delA

1546delTA

1717-1G>A
c.1585-1G>A
A455E
c.1364C>A

1717-8G>A
c.1585-8G>A
A559T
c.1675G>A

1811+
c.1679+
C524X
c.1572C>A

1.6kbA>G
1.6kbA>G

1812-1G>A
c.1680-1G>A
CFTRdele2,3
c.54-

5940 273+10250del

21kb

1898+1G>A
c.1766+1G>A
CFT

Assignees

• Quest Diagnostics Invest Llc

Inventors

• Sanders Heather
• Clarke Nigel J

Classifications

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title