# US 2020/0399699 A1 · Kits for diagnosing fragile x syndrome and uses thereof

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- Number: US 2020/0399699 A1
- Document ID: US20200399699A1
- Status: Published application
- Kind: A1
- Application number: 16/777,908
- Filed: 2020-01-31
- Published: 2020-12-24
- Earliest claimed: 2019-06-24
- Assignees: Origin Biotechnology Co., Ltd.
- Applicants: Origin Biotechnology Co., Ltd.
- Inventors: Yi-Yi KUO; Yu-Chiao HSIAO; I-Fan CHIU; Lai-Ha CHUNG; Shu-Ju LEE
- Web page: https://patentgrep.com/patent/US20200399699A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed herein is a method of performing polymerase chain reaction (PCR) to determine a repeating number of CGG sequence in fragile X mental retardation 1 (FMR1) gene. Also disclosed herein are a kit, and uses thereof in making a diagnosis of Fragile X syndrome (FXS) in a human subject based on the repeating number of the CGG sequence in a DNA sample isolated from the human subject. According to embodiments of the present disclosure, the kit comprises four primers, in which the first primer comprises a first polynucleotide sequence of SEQ ID NO: 1; the second primer comprises a second polynucleotide sequence of SEQ ID NO: 2; the third primer comprises a third polynucleotide sequence of SEQ ID NO: 3, and a non-human sequence disposed at and connected to the 5′-end of the third polynucleotide sequence; and the fourth primer comprises the non-human sequence.

## Claims

### Claim 1

A method of performing polymerase chain reaction (PCR) to determine a repeating number of the sequence of CGG in fragile X mental retardation 1 (FMR1) gene by amplifying a CGG repeat region of the FMR1 gene via a first to a fourth primers, wherein
the first primer comprises a first polynucleotide sequence, which is complementary to the upstream sequence of the CGG repeat region;
the second primer comprises a second polynucleotide sequence, which is complementary to the downstream sequence the CGG repeat region;
the third primer comprises a third polynucleotide sequence, and a non-human sequence disposed at and connected to the 5′-end of the third polynucleotide sequence, wherein the third polynucleotide sequence is complementary to the CGG repeat region; and
the fourth primer comprises the non-human sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein in the PCR, the concentration of the third primer is at least 1,000-fold lower than the concentration of each of the first, second, and fourth primers.

### Claim 3 (depends on 1)

The method of claim 1, wherein the third polynucleotide sequence comprises a repeating sequence of CGG, GGC, GCG, CCG, GCC, and/or CGC.

### Claim 4 (depends on 1)

The method of claim 1, wherein
the first polynucleotide sequence has the sequence of SEQ ID NO: 1;
the second polynucleotide sequence has the sequence of SEQ ID NO: 2;
the third polynucleotide sequence has the sequence of SEQ ID NO: 3; and
the non-human sequence has the polynucleotide sequence of SEQ ID NO: 4.

### Claim 5 (depends on 4)

The method of claim 4, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 6 (depends on 4)

The method of claim 4, wherein the first primer further comprises the non-human sequence disposed at the 5′-end of the first polynucleotide sequence, and the second primer further comprises the non-human sequence disposed at and connected to the 5′-end of the second polynucleotide sequence.

### Claim 7 (depends on 6)

The method of claim 6, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 12;
the second primer has the polynucleotide sequence of SEQ ID NO: 13;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 8 (depends on 1)

The method of claim 1, wherein
the first polynucleotide sequence has the sequence of SEQ ID NO: 1;
the second polynucleotide sequence has the sequence of SEQ ID NO: 2;
the third polynucleotide sequence has the sequence of SEQ ID NO: 3; and
the non-human sequence has the polynucleotide sequence of SEQ ID NO: 5.

### Claim 9 (depends on 8)

The method of claim 8, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.

### Claim 10 (depends on 8)

The method of claim 8, wherein the first primer further comprises the non-human sequence disposed at and connected to the 5′-end of the first polynucleotide sequence, and the second primer further comprises the non-human sequence disposed at and connected to the 5′-end of the second polynucleotide sequence.

### Claim 11 (depends on 10)

The method of claim 10, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 14;
the second primer has the polynucleotide sequence of SEQ ID NO: 15;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.

### Claim 12

A kit for making a diagnosis as to whether a human subject has Fragile X syndrome (FXS) or is a carrier of FXS, comprising,
a first primer comprising a first polynucleotide sequence of SEQ ID NO: 1;
a second primer comprising a second polynucleotide sequence of SEQ ID NO: 2;
a third primer comprising a third polynucleotide sequence of SEQ ID NO: 3, and a non-human sequence disposed at and connected to the 5′-end of the third polynucleotide sequence;
a fourth primer comprising the non-human sequence; and
polymerase chain reaction (PCR) reagents.

### Claim 13 (depends on 12)

The kit of claim 12, wherein the non-human sequence comprises the polynucleotide sequence of SEQ ID NO: 4.

### Claim 14 (depends on 13)

The kit of claim 13, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 15 (depends on 13)

The kit of claim 13, wherein the first primer further comprises the non-human sequence disposed at the 5′-end of the first polynucleotide sequence, and the second primer further comprises the non-human sequence disposed at and connected to the 5′-end of the second polynucleotide sequence.

### Claim 16 (depends on 15)

The kit of claim 15, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 12;
the second primer has the polynucleotide sequence of SEQ ID NO: 13;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 17 (depends on 12)

The kit of claim 12, wherein the non-human sequence comprises the polynucleotide sequence of SEQ ID NO: 5.

### Claim 18 (depends on 17)

The kit of claim 17, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.

### Claim 19 (depends on 17)

The kit of claim 17, wherein the first primer further comprises the non-human sequence disposed at and connected to the 5′-end of the first polynucleotide sequence, and the second primer further comprises the non-human sequence disposed at and connected to the 5′-end of the second polynucleotide sequence.

### Claim 20 (depends on 19)

The kit of claim 19, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 14;
the second primer has the polynucleotide sequence of SEQ ID NO: 15;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.

### Claim 21 (depends on 12)

The kit of claim 12, wherein the second primer is conjugated with a reporter molecule.

### Claim 22 (depends on 12)

The kit of claim 12, further comprising three DNA templates as positive controls, wherein the DNA templates respectively have 10-44, 45-54, and 55-200 repeats of the sequence of CGG within the polynucleotide sequences thereof.

### Claim 23 (depends on 12)

The kit of claim 12, further comprising a DNA template as a negative control, wherein the DNA template does not have the sequence of CGG within the polynucleotide sequence thereof.

### Claim 24 (depends on 12)

The kit of claim 12, wherein the PCR reagents comprise DNA polymerase, buffer, MgCl2, deoxypolynucleotide triphosphates (dNTPs), betaine, ethylene glycol, and glycerol.

### Claim 25 (depends on 12)

A method of making a diagnosis as to whether a human subject has Fragile X syndrome (FXS) or is a carrier of FXS by using the kit of claim 12, comprising,
(a) extracting a DNA sample from the human subject;
(b) mixing the DNA sample with the first to fourth primers in the PCR reagents;
(c) subjecting the mixture of the step (b) to PCR;
(d) determining a repeating number of the sequence of CGG in the product of the step (c); and
(e) making a diagnosis of FXS based on the repeating number determined by the step (d), wherein the repeating number of 201-375 indicates that the human subject has the FXS, and the repeating number of 55-200 indicates that the human subject is the carrier of the FXS.

### Claim 26 (depends on 25)

The method of claim 25, wherein in the mixture of the step (b), the concentration of the third primer is at least 1,000-fold lower than the concentration of each of the first, second, and fourth primers.

### Claim 27 (depends on 26)

The method of claim 26, wherein the concentration of the third primer is 0.1-0.5 nM, and the concentration of each of the first, second, and fourth primers is 0.5-1 μM.

### Claim 28 (depends on 25)

The method of claim 25, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 29 (depends on 25)

The method of claim 25, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 12;
the second primer has the polynucleotide sequence of SEQ ID NO: 13;
the third primer has the polynucleotide sequence of SEQ ID NO: 8; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 9.

### Claim 30 (depends on 25)

The method of claim 25, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 6;
the second primer has the polynucleotide sequence of SEQ ID NO: 7;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.

### Claim 31 (depends on 25)

The method of claim 25, wherein
the first primer has the polynucleotide sequence of SEQ ID NO: 14;
the second primer has the polynucleotide sequence of SEQ ID NO: 15;
the third primer has the polynucleotide sequence of SEQ ID NO: 10; and
the fourth primer has the polynucleotide sequence of SEQ ID NO: 11.