# US 2021/0340626 A1 · Method and Kit for Identifying Gene Mutations

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- Number: US 2021/0340626 A1
- Document ID: US20210340626A1
- Status: Published application
- Kind: A1
- Application number: 17/374,478
- Filed: 2021-07-13
- Published: 2021-11-04
- Earliest claimed: 2015-11-04
- Assignees: University of Pretoria
- Applicants: University of Pretoria
- Inventors: Robin John GREEN; Refiloe MASEKELA; Cheryl STEWART; Michael Sean PEPPER
- Web page: https://patentgrep.com/patent/US20210340626A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- G16B 30/10: Sequence alignment; Homology search
- 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

This invention relates to a method of identifying mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a kit for performing the method, and furthermore to isolated nucleotide sequences being complementary to one or more mutations of the CFTR gene. According to a first aspect of the invention there is provided a method of identifying mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, including the steps of providing one or more nucleic acid sequences, fully complementary to one or more segments of the CFTR gene, wherein the one or more nucleic acid sequences correspond to the mutation to be identified; providing a biological sample of an individual to be tested for CF; isolating nucleic acids from the biological sample; and testing the biological sample for the presence of one or more of the nucleic acid sequences using a suitable detection method.

## Claims

### Claim 1

A method of identifying mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene of an individual, including the steps of:
providing one or more nucleic acid sequences fully complementary to one or more segments of the CFTR gene, wherein the said one or more nucleic acid sequences corresponds to the mutation to be identified and is selected from the group consisting of SEQ ID NOs: 1-3;
providing a biological sample of the individual to be tested for CF;
isolating nucleic acids from the biological sample; and
testing the biological sample for the presence of one or more of the nucleic acid sequences using a suitable detection method.

### Claim 2 (depends on 1)

The method of identifying mutations in the CFTR gene of an individual according to claim 1, wherein the detection method utilizes one or more methods selected from the group consisting of an amplification refractory mutation system (ARMS), next generation sequencing (NGS), quantitative polymerase chain reaction (qPCR) and microarrays.

### Claim 3 (depends on 2)

The method of identifying mutations in the CFTR gene of an individual according to claim 2, wherein the ARMS detection method comprises one or more tetra-ARMS primers selected from the group consisting of SEQ ID NOs: 4-15.

### Claim 4 (depends on 2)

The method of identifying mutations in the CFTR gene of an individual according to claim 2, wherein the NGS detection method comprises one or more probes selected from the group consisting of SEQ ID NOs: 16-21.

### Claim 5 (depends on 2)

The method of identifying mutations in the CFTR gene of an individual according to claim 2, wherein the qPCR detection method comprises the use of one or more primers and probes selected from the group consisting of SEQ ID NOs: 22-27, as well as the corresponding hybprobes selected from the group consisting of SEQ ID NOs: 28-33.

### Claim 6 (depends on 2)

The method of identifying mutations in the CFTR gene of an individual according to claim 2, wherein the microarray detection method comprises the use of one or more probes selected from the group consisting of SEQ ID NOs: 34-36.

### Claim 7

A method for diagnosing cystic fibrosis (CF) in an individual comprising the steps of:
obtaining a biological sample from the individual;
isolating nucleic acids from the sample; and
testing the biological sample for the presence of one or more nucleic acid sequences fully complementary to one or more segments of the CFTR gene, wherein said one or more nucleic acid sequences corresponds to the mutation to be identified and is selected from the group consisting of SEQ ID NOs: 1-3.

### Claim 8

A method for determining if an individual or the individual's offspring will have a predisposition to CF, the method comprising the steps of:
obtaining a biological sample from the individual;
isolating nucleic acids from the sample;
testing the biological sample for the presence of one or more nucleic acid sequences fully complementary to one or more segments of the CFTR gene, wherein said one or more nucleic acid sequences corresponds to the mutation to be identified and is selected from the group consisting of SEQ ID NOs: 1-3; and
determining from the test results if the individual or the individual's offspring has a predisposition for CF; and
advising the individual accordingly.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the individual is of African origin.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the biological sample contains DNA.

### Claim 11

One or more nucleic acid sequences fully complementary to one or more segments of the CFTR gene, wherein said one or more nucleic acid sequences correspond to one or more mutations in the CFTR gene selected from the group consisting of SEQ ID NOs: 1-3.

### Claim 12

A kit for identifying mutations in the CFTR gene of an individual, wherein the kit is provided with primers and probes required to detect the presence of one or more nucleic acids fully complementary to one or more segments of the CFTR gene, with the said one or more nucleic acid sequences corresponding to one or more mutations in the CFTR gene selected from the group consisting of SEQ ID NOs: 1-3.

### Claim 13 (depends on 7)

The method according to claim 7, wherein the individual is of African origin.

### Claim 14 (depends on 8)

The method according to claim 8, wherein the individual is of African origin.

### Claim 15 (depends on 7)

The method according to claim 7, wherein the biological sample contains DNA.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the biological sample is any sample obtained from the individual's blood, serum, plasma, urine, skin, or hair.

### Claim 17 (depends on 8)

The method according to claim 8, wherein the biological sample contains DNA.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the biological sample is obtained from the individual's blood, serum, plasma, urine, skin, or hair.

### Claim 19 (depends on 10)

The method according to claim 10, wherein the biological sample is obtained from the individual's blood, serum, plasma, urine, skin, or hair.