# US 10,370,704 B2 · Mutation identification method

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- Number: US 10,370,704 B2
- Document ID: US10370704B2
- Status: Granted
- Kind: B2
- Application number: 15/381,346
- Filed: 2016-12-16
- Published: 2017-11-09
- Granted: 2019-08-06
- Earliest claimed: 2016-05-09
- Assignees: Health In Code, S.L.
- Applicants: Health In Code, S.L.
- Inventors: Carmen María Gayoso Babío; Óskar Martínez De Ilárduya Ruiz De Larramendi; Iván Aarón Lesende Rodríguez
- Web page: https://patentgrep.com/patent/US10370704B2

## CPC

- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to a method for identifying the position of a genetic mutation and to the use of said method for simplifying the screening of said genetic mutation.

## Claims

### Claim 1

A method for identifying the position of a genetic mutation, comprising:
a) determining an ascertained nucleic acid (ANA) region comprising the genetic mutation;
b) performing polymerase chain reaction (PCR) with primers to generate a PCR product, wherein the PCR template is DNA extracted from a biological sample obtained from a subject, and the primers are:
b1) a degenerate primer, and
b2) a specific primer:
hybridizing specifically within the ANA region, at a distance of 0.2-100 Kb from any end of the ANA region, if the genetic mutation comprises a gain of genetic material, a translocation or a gene fusion, and extending by PCR out of the ANA region, or
hybridizing specifically outside the ANA region, at a distance of 0.2-100 Kb from any end of the ANA region, if the mutation comprises a loss of genetic material, and extending by PCR into the ANA region,
and wherein in the PCR, the hybridizing comprises two consecutive hybridizations, a first hybridization at a hybridization temperature equal to the Tm of the specific primer ±10° C., and a second hybridization at a hybridization temperature equal to the Tm of the degenerate primer ±5° C.;
c) sequencing the PCR product obtained in step b) to obtain a sequence; and
d) aligning the sequence obtained in step c) with a reference genome for identifying the position of the genetic mutation.

### Claim 2 (depends on 1)

The method of claim 1, wherein step a) is performed based on DNA or RNA extracted from a biological sample obtained from the subject.

### Claim 3 (depends on 1)

The method of claim 1, wherein step a) is performed on DNA by multiplex ligation-dependent probe amplification (MLPA), comparative genomic hybridization (CGH) array, single nucleotide polymorphism (SNP) array, next generation sequencing (NGS), Southern blot, karyotyping, fluorescence in situ hybridization (FISH), RT-PCR or Δ-PCR.

### Claim 4 (depends on 1)

The method of claim 1, wherein in the PCR template is gDNA, cDNA or ciDNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the degenerate primer is a 5′ end phosphorylated degenerate primer.

### Claim 6 (depends on 1)

The method of claim 1, wherein the specific primer comprises at its 5′ end a sequence complementary to a sequence of its 3′ end.

### Claim 7 (depends on 1)

The method of claim 1, wherein the degenerate primer has between 5 and 16 nucleotides.

### Claim 8 (depends on 1)

The method of claim 1, wherein the hybridization temperature of the second hybridization is between 20-40° C.

### Claim 9 (depends on 1)

The method of claim 1, wherein the second hybridization is performed for 1 to 10 seconds.

### Claim 10 (depends on 1)

The method of claim 1, wherein the specific primer hybridizes at 0.5-80 Kb from the 3′ or 5′ end of the ANA region.

### Claim 11 (depends on 1)

The method of claim 1, wherein the degenerate primer has the sequence shown in SEQ ID NO 1.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sequencing of step c) is performed by next generation sequencing (NGS).

### Claim 13 (depends on 1)

The method of claim 1, wherein after step d), in which the position of the mutation is identified, further comprising step e) comprising reconfirming the position of the mutation by Sanger sequencing.

### Claim 14 (depends on 1)

The method of claim 1, wherein the genetic mutation is selected from the group consisting of insertion, deletion, duplication, transgene, translocation and gene fusion.

### Claim 15 (depends on 1)

A method for screening a genetic mutation, comprising:
i) identifying the position of a genetic mutation using the method of claim 1;
ii) performing PCR to generate another PCR product, wherein the PCR template is DNA extracted from a sample obtained from a subject, and the primers are a forward primer and a reverse primer hybridizing specifically upstream and downstream, respectively, of the exact position of the genetic mutation;
iii) analyzing the size of the PCR product amplified in ii), wherein a PCR product having the expected size according to the forward and reverse primers of step ii) is indicative of the presence of the genetic mutation, and the absence of said PCR product is indicative of the absence of the genetic mutation.

### Claim 16 (depends on 15)

The method of claim 15, further comprising after step iii) a step iv) comprising sequencing the PCR product obtained in step ii).

### Claim 17 (depends on 1)

A method for screening a genetic mutation, comprising:
i) identifying the position of a genetic mutation using the method of claim 1;
ii) performing PCR to generate another PCR product, wherein the PCR template is DNA extracted from a sample obtained from a subject; and the primers are a forward primer and a reverse primer hybridizing specifically upstream and downstream, respectively, of the exact position of the genetic mutation; and
iii) sequencing the PCR product obtained in step ii), wherein the result of the sequencing shows the presence or absence of the genetic mutation.

### Claim 18 (depends on 15)

The method of claim 15, wherein the subject is diagnosed as having a genetic mutation-associated pathology when the PCR product of step iii) has the expected size according to the forward and reverse primers used in step ii).

### Claim 19 (depends on 17)

The method of claim 17, wherein the subject is diagnosed as having a genetic mutation-associated pathology when the sequencing of step iii) shows the presence of the genetic mutation.

### Claim 20 (depends on 1)

The method of claim 1, wherein the biological sample is selected from the group consisting of fresh or fixed tissue obtained from biopsy or autopsy, blood, plasma, serum, urine, feces, saliva, ejaculate, bone marrow, cerebrospinal fluid, ascitic fluid, synovial fluid, amniotic fluid, buccal or buccal-pharyngeal discharge, pleural fluid, peritoneal fluid, pericardial fluid, intra-articular fluid, and mixtures thereof.