KR20130027281A - Detection method of vsx1 single nucleotide polymorphism and detection kit for vsx1 snp - Google Patents

Abstract

The present invention relates to a method for detecting mutations in the VSX1 gene, which is the causative gene of the keratoconus using pyrosequencing, and a kit to which the method is applied.
According to the present invention, the mutation of the keratoconus causative gene can be detected accurately, quickly and simply, so that the keratoconus can be diagnosed or the risk for the keratoconus can be predicted in advance.

Description

Detection method of VSX1 single nucleotide polymorphism and Detection kit for VSX1 SNP}

The present invention relates to a method and kit for diagnosing a keratoconus, and more particularly, to a method for detecting a mutation of the VSX1 gene, which is a causative gene of a keratoconus using pyrosequencing, and a kit to which the method is applied.

The keratoconus is a non-inflammatory corneal dilator that usually occurs when the round cornea (the front of the eye) becomes thin and irregular (conical).

In the early stages, the keratoconus increases its slight visual blur and distortion and its sensitivity to glare and light.

This symptom usually occurs in late teens or late twenties, and can progress slowly after 10 to 20 years. Each eye can be affected separately.

As the keratoconus progresses, the cornea protrudes more and the vision may be more distorted. In a few cases, the cornea swells and causes sudden, significant loss of vision. Swelling occurs when a small defect in the prominent cone-like prominence of the cornea causes a development. Swelling can last for weeks or months as defects recover and are replaced by progressively injured tissue. If you experience this sudden swelling, your doctor may prescribe eye drops for temporary relief, but there is no medicine to prevent the disorder from progressing.

Genes such as VSX1 and AIPL1 have been reported as the causative genes of keratoconus. Heon et al. Reported for the first time a mutation in the VSX1 gene in patients with keratoconus and polymorphic corneal dystrophy (G160D, R166W, L159M, D144E, H244R, P247R) in a Canadian study. The mutation of the VSX1 gene in the keratoconus was studied (D144E, G160D, P247R, L179 and intron c.900 + 23A / G, c.900 + 84T / A), and Mintz--Hittner et al. It has been reported to be related to facial abnormalities, empty Turkey, corneal endothelial abnormalities, retinal and auditory bipolar cells. In addition, recent domestic reports have reported that T → A / T (IVS1-11 T> A) mutations in intron 1 of keratoconus patients are related to keratoconus.

As described above, research on the causative gene of the keratoconus has been made, but a method for efficiently diagnosing or predicting the keratoconus using such a gene is not known.

Therefore, the present inventors made intensive studies to develop a method for rapidly and accurately diagnosing and predicting a keratoconus. As a result, a specific region of the VSX1 gene sequence, which is the causative gene of the keratoconus, was used as a PCR primer or a sequencing primer. When using the VSX1 gene mutation detection method that performs pyrosequencing using the present invention, it was confirmed that it is possible to diagnose and predict the keratoconus quickly, easily and accurately, and thus, the present invention has been completed.

Accordingly, a main object of the present invention is to provide a method for quickly and accurately diagnosing or predicting a keratoconus.

Another object of the present invention to provide a kit that can diagnose or predict the keratoconus using the method as described above.

According to one aspect of the invention, the invention is

a) a forward primer consisting of 10 to 30 contiguous bases in the bases 2555 through 2670 of SEQ ID NO: 1 and 10 to 30 of the complementary base sequences of the bases 2674 through 2862 of SEQ ID NO: Performing polymerase chain reaction (PCR) on the sample DNA using a reverse primer composed of consecutive bases;

b) for the PCR product obtained in step a),

Consists of 10 to 18 consecutive bases in the 2654 th to 2671 th base sequences of SEQ ID NO: 1, or 10 to 18 consecutive bases in the complementary base sequences of the base sequences 2626 to 2690 th of SEQ ID NO: 1 It provides a method for detecting a mutation in the keratoconus causal gene comprising a; performing pyrosequencing using a sequencing primer consisting of.

In the method of the present invention, the forward primer is preferably composed of one base sequence selected from SEQ ID NO: 2 to 15, more preferably consisting of the base sequence of SEQ ID NO: 2.

In the method of the present invention, the reverse primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 16 to 34, more preferably consisting of the nucleotide sequence of SEQ ID NO.

In the method of the present invention, the sequencing primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 35 to 42, more preferably consisting of the nucleotide sequence of SEQ ID NO: 35.

The forward primer, the reverse primer and the primer for sequencing are preferably used in combination as shown in Table 1 below to efficiently detect mutations in the keratoconus causative gene. For example, a primer consisting of the nucleotide sequence of SEQ ID NO: 2 is used as a forward primer as a group 1 of Table 1, a primer consisting of the nucleotide sequence of SEQ ID NO: 16 is used as a reverse primer, and SEQ ID NO: A primer consisting of 35 base sequences can be used.

group Forward primer Reverse primer Sequencing Primer One SEQ ID NO: 2 SEQ ID NO: 16 SEQ ID NO: 35 2 SEQ ID NO: 2 SEQ ID NO: 17 SEQ ID NO: 35 3 SEQ ID NO: 2 SEQ ID NO: 17 SEQ ID NO: 36 4 SEQ ID NO: 2 SEQ ID NO: 18 SEQ ID NO: 35 5 SEQ ID NO: 2 SEQ ID NO: 19 SEQ ID NO: 35 6 SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 35 7 SEQ ID NO: 2 SEQ ID NO: 21 SEQ ID NO: 35 8 SEQ ID NO: 2 SEQ ID NO: 22 SEQ ID NO: 35 9 SEQ ID NO: 2 SEQ ID NO: 23 SEQ ID NO: 35 10 SEQ ID NO: 2 SEQ ID NO: 24 SEQ ID NO: 35 11 SEQ ID NO: 3 SEQ ID NO: 19 SEQ ID NO: 35 12 SEQ ID NO: 4 SEQ ID NO: 25 SEQ ID NO: 35 13 SEQ ID NO: 4 SEQ ID NO: 26 SEQ ID NO: 35 14 SEQ ID NO: 5 SEQ ID NO: 19 SEQ ID NO: 35 15 SEQ ID NO: 2 SEQ ID NO: 27 SEQ ID NO: 35 16 SEQ ID NO: 4 SEQ ID NO: 22 SEQ ID NO: 35 17 SEQ ID NO: 4 SEQ ID NO: 21 SEQ ID NO: 35 18 SEQ ID NO: 6 SEQ ID NO: 28 SEQ ID NO: 37 19 SEQ ID NO: 4 SEQ ID NO: 24 SEQ ID NO: 35 20 SEQ ID NO: 4 SEQ ID NO: 29 SEQ ID NO: 35 21 SEQ ID NO: 2 SEQ ID NO: 18 SEQ ID NO: 36 22 SEQ ID NO: 2 SEQ ID NO: 19 SEQ ID NO: 36 23 SEQ ID NO: 3 SEQ ID NO: 17 SEQ ID NO: 35 24 SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO: 35 25 SEQ ID NO: 3 SEQ ID NO: 17 SEQ ID NO: 36 26 SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO: 36 27 SEQ ID NO: 8 SEQ ID NO: 28 SEQ ID NO: 37 28 SEQ ID NO: 2 SEQ ID NO: 30 SEQ ID NO: 35 29 SEQ ID NO: 2 SEQ ID NO: 33 SEQ ID NO: 35 30 SEQ ID NO: 4 SEQ ID NO: 27 SEQ ID NO: 35 31 SEQ ID NO: 2 SEQ ID NO: 28 SEQ ID NO: 37 32 SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 37 33 SEQ ID NO: 6 SEQ ID NO: 26 SEQ ID NO: 37 34 SEQ ID NO: 2 SEQ ID NO: 17 SEQ ID NO: 38 35 SEQ ID NO: 2 SEQ ID NO: 32 SEQ ID NO: 35 36 SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 37 37 SEQ ID NO: 6 SEQ ID NO: 20 SEQ ID NO: 37 38 SEQ ID NO: 2 SEQ ID NO: 32 SEQ ID NO: 36 39 SEQ ID NO: 9 SEQ ID NO: 17 SEQ ID NO: 35 40 SEQ ID NO: 9 SEQ ID NO: 17 SEQ ID NO: 36 41 SEQ ID NO: 10 SEQ ID NO: 28 SEQ ID NO: 37 42 SEQ ID NO: 11 SEQ ID NO: 28 SEQ ID NO: 37 43 SEQ ID NO: 11 SEQ ID NO: 26 SEQ ID NO: 37 44 SEQ ID NO: 2 SEQ ID NO: 26 SEQ ID NO: 35 45 SEQ ID NO: 10 SEQ ID NO: 20 SEQ ID NO: 37 46 SEQ ID NO: 11 SEQ ID NO: 20 SEQ ID NO: 37 47 SEQ ID NO: 2 SEQ ID NO: 29 SEQ ID NO: 35 48 SEQ ID NO: 3 SEQ ID NO: 26 SEQ ID NO: 37 49 SEQ ID NO: 8 SEQ ID NO: 17 SEQ ID NO: 35 50 SEQ ID NO: 3 SEQ ID NO: 21 SEQ ID NO: 35 51 SEQ ID NO: 3 SEQ ID NO: 27 SEQ ID NO: 35 52 SEQ ID NO: 3 SEQ ID NO: 20 SEQ ID NO: 37 53 SEQ ID NO: 3 SEQ ID NO: 24 SEQ ID NO: 35 54 SEQ ID NO: 5 SEQ ID NO: 17 SEQ ID NO: 35 55 SEQ ID NO: 6 SEQ ID NO: 28 SEQ ID NO: 39 56 SEQ ID NO: 5 SEQ ID NO: 17 SEQ ID NO: 36 57 SEQ ID NO: 12 SEQ ID NO: 33 SEQ ID NO: 35 58 SEQ ID NO: 3 SEQ ID NO: 26 SEQ ID NO: 35 59 SEQ ID NO: 3 SEQ ID NO: 22 SEQ ID NO: 35 60 SEQ ID NO: 8 SEQ ID NO: 19 SEQ ID NO: 35 61 SEQ ID NO: 12 SEQ ID NO: 33 SEQ ID NO: 36 62 SEQ ID NO: 3 SEQ ID NO: 18 SEQ ID NO: 35 63 SEQ ID NO: 3 SEQ ID NO: 20 SEQ ID NO: 35 64 SEQ ID NO: 4 SEQ ID NO: 34 SEQ ID NO: 35 65 SEQ ID NO: 2 SEQ ID NO: 25 SEQ ID NO: 35 66 SEQ ID NO: 3 SEQ ID NO: 25 SEQ ID NO: 35 67 SEQ ID NO: 4 SEQ ID NO: 33 SEQ ID NO: 35 68 SEQ ID NO: 8 SEQ ID NO: 28 SEQ ID NO: 39 69 SEQ ID NO: 3 SEQ ID NO: 29 SEQ ID NO: 35 70 SEQ ID NO: 2 SEQ ID NO: 28 SEQ ID NO: 39 71 SEQ ID NO: 2 SEQ ID NO: 21 SEQ ID NO: 38 72 SEQ ID NO: 3 SEQ ID NO: 28 SEQ ID NO: 37 73 SEQ ID NO: 2 SEQ ID NO: 22 SEQ ID NO: 38 74 SEQ ID NO: 8 SEQ ID NO: 20 SEQ ID NO: 37 75 SEQ ID NO: 9 SEQ ID NO: 19 SEQ ID NO: 35 76 SEQ ID NO: 2 SEQ ID NO: 28 SEQ ID NO: 35 77 SEQ ID NO: 9 SEQ ID NO: 18 SEQ ID NO: 35 78 SEQ ID NO: 2 SEQ ID NO: 33 SEQ ID NO: 35 79 SEQ ID NO: 10 SEQ ID NO: 17 SEQ ID NO: 35 80 SEQ ID NO: 13 SEQ ID NO: 17 SEQ ID NO: 35 81 SEQ ID NO: 3 SEQ ID NO: 23 SEQ ID NO: 35 82 SEQ ID NO: 2 SEQ ID NO: 33 SEQ ID NO: 36 83 SEQ ID NO: 10 SEQ ID NO: 17 SEQ ID NO: 36 84 SEQ ID NO: 4 SEQ ID NO: 19 SEQ ID NO: 35 85 SEQ ID NO: 3 SEQ ID NO: 19 SEQ ID NO: 36 86 SEQ ID NO: 13 SEQ ID NO: 17 SEQ ID NO: 36 87 SEQ ID NO: 11 SEQ ID NO: 28 SEQ ID NO: 39 88 SEQ ID NO: 10 SEQ ID NO: 28 SEQ ID NO: 39 89 SEQ ID NO: 14 SEQ ID NO: 33 SEQ ID NO: 36 90 SEQ ID NO: 6 SEQ ID NO: 33 SEQ ID NO: 37 91 SEQ ID NO: 8 SEQ ID NO: 33 SEQ ID NO: 37 92 SEQ ID NO: 5 SEQ ID NO: 19 SEQ ID NO: 36 93 SEQ ID NO: 4 SEQ ID NO: 23 SEQ ID NO: 35 94 SEQ ID NO: 15 SEQ ID NO: 28 SEQ ID NO: 39 95 SEQ ID NO: 2 SEQ ID NO: 33 SEQ ID NO: 37 96 SEQ ID NO: 14 SEQ ID NO: 28 SEQ ID NO: 39 97 SEQ ID NO: 3 SEQ ID NO: 33 SEQ ID NO: 35 98 SEQ ID NO: 2 SEQ ID NO: 34 SEQ ID NO: 35 99 SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO: 40 100 SEQ ID NO: 6 SEQ ID NO: 28 SEQ ID NO: 41 101 SEQ ID NO: 6 SEQ ID NO: 28 SEQ ID NO: 42

In the method of the present invention, the polymerase chain reaction is subjected to an initial denaturation step at 90 to 98 ℃ for 5 to 20 minutes, 5 to 30 seconds denaturation at 90 to 98 ℃, 10 to 30 seconds annealing at 55 to 65 ℃ and It is preferable that the period which consists of 5-30 second extension in 70-75 degreeC consists of 20-50 cycles. More preferably, the initial denaturation step at 94 to 96 ℃ 5 to 20 minutes, 5 to 15 seconds denaturation at 94 to 96 ℃, 10 to 20 seconds annealing at 59 to 61 ℃ and 5 to 15 seconds at 71 to 73 ℃ The period consisting of the extension is preferably 30 to 50 cycles, most preferably undergoing an initial denaturation step at 95 ° C. for 11 minutes, 10 seconds at 95 ° C., 15 seconds annealing at 60 ° C., and 9 seconds at 72 ° C. It is good to consist of 40 cycles.

In the method of the present invention,

In performing a polymerase chain reaction (PCR) on the sample DNA in step a), a second forward primer and a sequence consisting of 10 to 30 consecutive bases in the base sequence of SEQ ID NO: 2487 to 2603 Further using a second reverse primer consisting of 10 to 30 consecutive bases from the complementary base sequence of the base sequence number 2617 to 2756 of No. 1,

In performing the pyro sequencing in step b), consisting of 10 to 23 consecutive bases in the base sequence of the 2584th to 2606th of SEQ ID NO: 1, or the base sequence from 2608th to 2627th of SEQ ID NO: It is preferable to further use the second sequencing primer consisting of 10 to 20 consecutive bases in the complementary base sequence of.

In the method of the present invention, the second forward primer is preferably composed of one base sequence selected from SEQ ID NO: 43 to 59, more preferably consisting of the base sequence of SEQ ID NO: 43.

In the method of the present invention, the second reverse primer is preferably composed of one base sequence selected from SEQ ID NO: 60 to 70, more preferably consisting of the base sequence of SEQ ID NO: 66.

In the method of the present invention, the second sequencing primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 71 to 78, more preferably consisting of the nucleotide sequence of SEQ ID NO: 74.

The second forward primer, the second reverse primer and the second sequencing primer are preferably used in combination as shown in Table 2 below to efficiently detect mutations in the keratoconus causative gene. For example, as in Group 1 of Table 2, a primer consisting of the nucleotide sequence of SEQ ID NO: 43 is used as the second forward primer, a primer consisting of the nucleotide sequence of SEQ ID NO: 66 is used as the second reverse primer, and the second sequence is used. As the primer for analysis, a primer consisting of the nucleotide sequence of SEQ ID NO: 74 may be used.

group Second forward primer Second reverse primer Second sequencing primer 102 SEQ ID NO: 43 SEQ ID NO: 66 SEQ ID NO: 74 103 SEQ ID NO: 44 SEQ ID NO: 60 SEQ ID NO: 71 104 SEQ ID NO: 45 SEQ ID NO: 60 SEQ ID NO: 71 105 SEQ ID NO: 46 SEQ ID NO: 60 SEQ ID NO: 71 106 SEQ ID NO: 45 SEQ ID NO: 61 SEQ ID NO: 71 107 SEQ ID NO: 46 SEQ ID NO: 61 SEQ ID NO: 71 108 SEQ ID NO: 45 SEQ ID NO: 62 SEQ ID NO: 71 109 SEQ ID NO: 45 SEQ ID NO: 63 SEQ ID NO: 71 110 SEQ ID NO: 46 SEQ ID NO: 63 SEQ ID NO: 71 111 SEQ ID NO: 45 SEQ ID NO: 64 SEQ ID NO: 72 112 SEQ ID NO: 45 SEQ ID NO: 64 SEQ ID NO: 73 113 SEQ ID NO: 45 SEQ ID NO: 64 SEQ ID NO: 74 114 SEQ ID NO: 45 SEQ ID NO: 65 SEQ ID NO: 71 115 SEQ ID NO: 45 SEQ ID NO: 66 SEQ ID NO: 73 116 SEQ ID NO: 45 SEQ ID NO: 66 SEQ ID NO: 74 117 SEQ ID NO: 47 SEQ ID NO: 66 SEQ ID NO: 74 118 SEQ ID NO: 47 SEQ ID NO: 66 SEQ ID NO: 73 119 SEQ ID NO: 46 SEQ ID NO: 66 SEQ ID NO: 74 120 SEQ ID NO: 46 SEQ ID NO: 66 SEQ ID NO: 73 121 SEQ ID NO: 48 SEQ ID NO: 66 SEQ ID NO: 74 122 SEQ ID NO: 48 SEQ ID NO: 66 SEQ ID NO: 73 123 SEQ ID NO: 49 SEQ ID NO: 66 SEQ ID NO: 73 124 SEQ ID NO: 49 SEQ ID NO: 66 SEQ ID NO: 74 125 SEQ ID NO: 50 SEQ ID NO: 61 SEQ ID NO: 71 126 SEQ ID NO: 51 SEQ ID NO: 64 SEQ ID NO: 74 127 SEQ ID NO: 45 SEQ ID NO: 67 SEQ ID NO: 74 128 SEQ ID NO: 45 SEQ ID NO: 67 SEQ ID NO: 72 129 SEQ ID NO: 45 SEQ ID NO: 67 SEQ ID NO: 73 130 SEQ ID NO: 52 SEQ ID NO: 66 SEQ ID NO: 74 131 SEQ ID NO: 52 SEQ ID NO: 66 SEQ ID NO: 73 132 SEQ ID NO: 51 SEQ ID NO: 64 SEQ ID NO: 72 133 SEQ ID NO: 51 SEQ ID NO: 64 SEQ ID NO: 73 134 SEQ ID NO: 46 SEQ ID NO: 62 SEQ ID NO: 71 135 SEQ ID NO: 51 SEQ ID NO: 66 SEQ ID NO: 74 136 SEQ ID NO: 51 SEQ ID NO: 66 SEQ ID NO: 73 137 SEQ ID NO: 49 SEQ ID NO: 64 SEQ ID NO: 74 138 SEQ ID NO: 49 SEQ ID NO: 64 SEQ ID NO: 73 139 SEQ ID NO: 49 SEQ ID NO: 64 SEQ ID NO: 72 140 SEQ ID NO: 48 SEQ ID NO: 64 SEQ ID NO: 72 141 SEQ ID NO: 48 SEQ ID NO: 64 SEQ ID NO: 73 142 SEQ ID NO: 48 SEQ ID NO: 64 SEQ ID NO: 74 143 SEQ ID NO: 50 SEQ ID NO: 60 SEQ ID NO: 71 144 SEQ ID NO: 51 SEQ ID NO: 67 SEQ ID NO: 74 145 SEQ ID NO: 53 SEQ ID NO: 60 SEQ ID NO: 71 146 SEQ ID NO: 44 SEQ ID NO: 61 SEQ ID NO: 71 147 SEQ ID NO: 51 SEQ ID NO: 67 SEQ ID NO: 72 148 SEQ ID NO: 51 SEQ ID NO: 67 SEQ ID NO: 73 149 SEQ ID NO: 54 SEQ ID NO: 66 SEQ ID NO: 73 150 SEQ ID NO: 54 SEQ ID NO: 66 SEQ ID NO: 74 151 SEQ ID NO: 55 SEQ ID NO: 60 SEQ ID NO: 71 152 SEQ ID NO: 44 SEQ ID NO: 60 SEQ ID NO: 75 153 SEQ ID NO: 54 SEQ ID NO: 64 SEQ ID NO: 73 154 SEQ ID NO: 54 SEQ ID NO: 64 SEQ ID NO: 74 155 SEQ ID NO: 54 SEQ ID NO: 64 SEQ ID NO: 72 156 SEQ ID NO: 45 SEQ ID NO: 60 SEQ ID NO: 75 157 SEQ ID NO: 56 SEQ ID NO: 66 SEQ ID NO: 73 158 SEQ ID NO: 56 SEQ ID NO: 66 SEQ ID NO: 74 159 SEQ ID NO: 46 SEQ ID NO: 61 SEQ ID NO: 75 160 SEQ ID NO: 45 SEQ ID NO: 61 SEQ ID NO: 75 161 SEQ ID NO: 53 SEQ ID NO: 66 SEQ ID NO: 73 162 SEQ ID NO: 45 SEQ ID NO: 62 SEQ ID NO: 75 163 SEQ ID NO: 45 SEQ ID NO: 63 SEQ ID NO: 75 164 SEQ ID NO: 45 SEQ ID NO: 68 SEQ ID NO: 71 165 SEQ ID NO: 46 SEQ ID NO: 63 SEQ ID NO: 75 166 SEQ ID NO: 57 SEQ ID NO: 60 SEQ ID NO: 71 167 SEQ ID NO: 47 SEQ ID NO: 60 SEQ ID NO: 71 168 SEQ ID NO: 51 SEQ ID NO: 60 SEQ ID NO: 71 169 SEQ ID NO: 51 SEQ ID NO: 61 SEQ ID NO: 71 170 SEQ ID NO: 47 SEQ ID NO: 61 SEQ ID NO: 71 171 SEQ ID NO: 57 SEQ ID NO: 61 SEQ ID NO: 71 172 SEQ ID NO: 57 SEQ ID NO: 63 SEQ ID NO: 71 173 SEQ ID NO: 51 SEQ ID NO: 63 SEQ ID NO: 71 174 SEQ ID NO: 45 SEQ ID NO: 65 SEQ ID NO: 75 175 SEQ ID NO: 51 SEQ ID NO: 69 SEQ ID NO: 71 176 SEQ ID NO: 47 SEQ ID NO: 63 SEQ ID NO: 71 177 SEQ ID NO: 57 SEQ ID NO: 62 SEQ ID NO: 71 178 SEQ ID NO: 51 SEQ ID NO: 62 SEQ ID NO: 71 179 SEQ ID NO: 58 SEQ ID NO: 66 SEQ ID NO: 74 180 SEQ ID NO: 58 SEQ ID NO: 66 SEQ ID NO: 73 181 SEQ ID NO: 57 SEQ ID NO: 66 SEQ ID NO: 74 182 SEQ ID NO: 57 SEQ ID NO: 66 SEQ ID NO: 73 183 SEQ ID NO: 45 SEQ ID NO: 70 SEQ ID NO: 74 184 SEQ ID NO: 45 SEQ ID NO: 70 SEQ ID NO: 72 185 SEQ ID NO: 45 SEQ ID NO: 70 SEQ ID NO: 73 186 SEQ ID NO: 50 SEQ ID NO: 61 SEQ ID NO: 75 187 SEQ ID NO: 53 SEQ ID NO: 61 SEQ ID NO: 71 188 SEQ ID NO: 51 SEQ ID NO: 65 SEQ ID NO: 71 189 SEQ ID NO: 46 SEQ ID NO: 62 SEQ ID NO: 75 190 SEQ ID NO: 51 SEQ ID NO: 70 SEQ ID NO: 74 191 SEQ ID NO: 53 SEQ ID NO: 65 SEQ ID NO: 71 192 SEQ ID NO: 51 SEQ ID NO: 70 SEQ ID NO: 72 193 SEQ ID NO: 51 SEQ ID NO: 70 SEQ ID NO: 73 194 SEQ ID NO: 57 SEQ ID NO: 69 SEQ ID NO: 71 195 SEQ ID NO: 55 SEQ ID NO: 61 SEQ ID NO: 71 196 SEQ ID NO: 59 SEQ ID NO: 66 SEQ ID NO: 73 197 SEQ ID NO: 44 SEQ ID NO: 66 SEQ ID NO: 74 198 SEQ ID NO: 50 SEQ ID NO: 60 SEQ ID NO: 75 199 SEQ ID NO: 59 SEQ ID NO: 61 SEQ ID NO: 75 200 SEQ ID NO: 45 SEQ ID NO: 64 SEQ ID NO: 76 201 SEQ ID NO: 46 SEQ ID NO: 60 SEQ ID NO: 77 202 SEQ ID NO: 57 SEQ ID NO: 60 SEQ ID NO: 78

According to another aspect of the invention, the invention

A forward primer consisting of 10 to 30 contiguous bases in the nucleotide sequences of 2555 th to 2670 th of SEQ ID NO: 1;

A reverse primer consisting of 10 to 30 contiguous bases in the complementary nucleotide sequences of the nucleotide sequences 2674 th to 2862 th of SEQ ID NO: 1; And

Consists of 10 to 18 consecutive bases in the 2654 th to 2671 th base sequences of SEQ ID NO: 1, or 10 to 18 consecutive bases in the complementary base sequences of the base sequences 2626 to 2690 th of SEQ ID NO: 1 It provides a kit for detecting a mutation of the keratoker causal gene by pyro sequencing comprising a sequencing primer consisting of.

In the kit of the present invention, the forward primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 2 to 15, more preferably consisting of the nucleotide sequence of SEQ ID NO: 2.

In the kit of the present invention, the reverse primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 16 to 34, more preferably consisting of the nucleotide sequence of SEQ ID NO.

In the kit of the present invention, the sequencing primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 35 to 42, more preferably consisting of the nucleotide sequence of SEQ ID NO: 35.

In the kit of the present invention, it is preferable to configure the forward primer, the reverse primer, and the sequencing primer as shown in Table 1 above, and may be configured in one or more groups.

In the kit of the present invention, the second forward primer consisting of 10 to 30 consecutive bases in the base sequence of the 2487 th to 2603 th SEQ ID NO: 1;

A second reverse primer consisting of 10 to 30 contiguous bases among the complementary nucleotide sequences of the nucleotide sequences 2617 to 2756 of SEQ ID NO: 1; And

Consists of 10 to 23 consecutive bases in the nucleotide sequences of SEQ ID NO: 1 2584 to 2606, or 10 to 20 consecutive bases in the complementary base sequences of the base sequences 2608 to 2627 of SEQ ID NO: It is preferable that the second sequencing primer consisting of; further comprises.

In the kit of the present invention, the second forward primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 43 to 59, more preferably consisting of the nucleotide sequence of SEQ ID NO: 43.

In the kit of the present invention, the second reverse primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 60 to 70, more preferably consisting of the nucleotide sequence of SEQ ID NO: 66.

In the kit of the present invention, the second sequencing primer is preferably composed of one nucleotide sequence selected from SEQ ID NO: 71 to 78, more preferably consisting of the nucleotide sequence of SEQ ID NO: 74.

In the kit of the present invention, the second forward primer, the second reverse primer, and the second sequencing primer may be bundled and configured as shown in Table 2, and may be configured in one or more groups.

In the present invention, it is preferable to apply a labeling material such as biotin to the forward primer or the reverse primer, so that the product produced after the polymerase chain reaction can be easily purified.

Mutations of the keratoconus causative gene to be detected in the present invention are G160V mutation and IVS1-11 mutation of the VSX1 gene.

The G160V mutant was substituted with thymine (T) in the VSX1 gene sequence, ie, guanine (G), which is the 2672th base in SEQ ID NO: 1, to which glycine (Glycine, Gly, G), the 160th amino acid of the VSX1 protein, was expressed. (valine, Val, V), and the IVS1-11 mutation indicates that the thymine (T), which is the 2607 base in SEQ ID NO: 1, is substituted with adenine (A).

The method of the present invention is to detect such a G160V or IVS1-11 mutation, it can be said that the design of an effective primer for identifying the nucleotide sequence of this mutation site through pyro sequencing.

In the present invention, a PCR is performed to amplify a mutation site in a first step, and pyro sequencing is performed on a PCR product in a second step.

In the first step, sample DNA is used as a template. It is preferable to use sample DNA as total DNA extracted from a sample of blood or the like for detecting a mutation of a keratoker causative gene or diagnosing or predicting a keratoconus. It is preferred that the resulting product, i.e., the amplified fragment, be purified from the PCR reaction solution.

In the second step, the PCR product is targeted and pyro sequencing is performed using sequencing primers. DNA amplified by PCR was made into single strands and combined with primers for sequencing, followed by a substrate with four enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrease. , Adenosine 5'-phosphosulfate (APS) and luciferin are added to react. When four dNTPs are sequentially added to the reaction solution, specific dNTPs are used in the reaction, and synthesis is performed along the single-stranded template DNA by DNA polymerase. If dNTPs that are complementary to the template come in, mixing occurs to release pyrophosphate (PPi), the amount of which is equal to the number of nucleotides to be mixed. ATP sulfurylase produces ATP using APS and PPi added to the reaction solution, which activates luciferase to emit light by converting luciferin into oxyluciferin. At this time, the amount of light emitted is proportional to the amount of ATP, which is in turn proportional to the amount of dNTP used for mixing. The emitted light is detected by the CCD camera and displayed as a program using software. Since the peak of the light signal is proportional to the mixed nucleotides, the type and number of the mixed nucleotides can be determined. Apyrase is an enzyme that breaks down nucleotides and breaks down unmixed dNTPs and ATPs. When one dNTP is decomposed, the next dNTP is added to the reaction solution. The addition of dNTPs is made one at a time. In addition, deoxyadenosine alpha-thio triphosphate (dATPαS) is used instead of dATP. The reason is that it is used for the reaction of the DNA polymerase but not for the activation of the luciferase. As the course of the experiment proceeds, the DNA chain complementary to the template DNA chain is synthesized, resulting in a signal peak of the pyrogram, and thus, each sequence can be analyzed.

As described above, according to the present invention, the mutation of the keratoconus causative gene can be detected accurately, quickly and simply, so that the keratoconus can be diagnosed or the risk for the keratoconus can be predicted in advance.

1 is a graph showing the results of analyzing the keratoconus causal gene VSX1 exon2 (G160V G> T) mutation according to the method of the present invention. VSX1 exon2 (G160V G> T) sequences are normal (not mutated). In the case of mutations, the peak height of G allele decreases in the gray area, and the peak of T allele appears.
Figure 2 is a graph showing the results of analyzing the keratoconus gene VSX1 intron1 (IVS1-11 T> A) mutation in accordance with the method of the present invention. VSX1 intron1 (IVS1-11 T> A) sequence is normal (not mutated). In the case of mutation, the peak height of A allele decreases in the gray area, and the peak of T allele appears.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Investigation of G160V Mutation of VSX1 Gene in Sample DNA

1-1. Sample DNA Extraction

Oral mucosal cells or hair roots of the subjects were collected and total DNA was extracted therefrom. DNA extraction was carried out using Qiagen's QIAamp DNA Mini kit, and the manufacturer's manual was followed.

1-2. Primer production

As shown in Table 3 below, the forward primer (G160V (G> T) _F), the reverse primer (G160V (G> T) _R), the sequencing primer (G160V (G> T) _Seq), the second forward primer (IVS1 -11 (T> A) _F), a second reverse primer (IVS1-11 (T> A) _R) and a second sequence primer (IVS1-11 (T> A) _Seq) were synthesized, respectively. And it was synthesized by attaching biotin to the second forward primer. The expected sizes of the PCR products are 98 bp and 200 bp, respectively.

Primer name SEQ ID NO: Sequence (5 '------- 3') PCR size G160V (G> T) _F 2 ATGAGGACAGCCAGTCTGAAGACA
98
G160V (G> T) _R 16 Biotin-GGACTGCCTGGCCCTATACCT G160V (G> T) _Seq 35 GCATCCCCCACCTTG IVS1-11 (T> A) _F 43 Biotin-TCCAGGAAATAGAGGGGATATG
200
IVS1-11 (T> A) _R 66 CATAAACCTTGGGCTGTGTCC IVS1-11 (T> A) _Seq 74 GCATCCCCCACCTTG

1-3. Polymerase chain reaction (PCR)

5 μl of the DNA solution extracted in 1-1 was used for PCR, and BioQuest's 2 × Hot Taq PCR Mix (cat No. QM14235) was used.

5 μl of DNA solution, 10 μl of 2X Hot Taq PCR Mix, and 3 μl of distilled water were mixed, and then 1 μl of the 1-2 forward primer (10 uM) and 1 μl of the reverse primer (10 uM) were added to each tube and / or the second forward direction. 1 μl of primer (10 uM) and 1 μl of second reverse primer (10 uM) were mixed to prepare a PCR mixture, and treated once at 11 minutes at 95 ° C., followed by a cycle of 95 ° C. 10 seconds, 60 ° C. 15 seconds, and 72 ° C. 9 seconds. After performing the PCR under the condition of repeating (cycle) 40 times, the PCR reaction solution was stored at 4 ° C. until use.

1-4. VSX1 gene analysis (IVS1-11, G160V SNP analysis by pyrosequencing)

20 μl of the biotinylated PCR product obtained in 1 to 3 was added with 3 μl of streptavidin sepharose (GE healthcare, Germany) and 37 μl of 2 × Binding buffer (# 979006, QIAGEN, Germany) solution. Sterile distilled water was added to make μl and mixed for 10 minutes at room temperature. 10 seconds in 70% ethanol solution, 10 seconds in Denaturation Solution (# 979007, QIAGEN, Germany) using PyroMarkTM Q24 User Manual (Pyrosequencing AB, Biotage, Uppsala, Sweden), in Wash Solution (# 979008, QIAGEN, Germany) Single strand DNA was obtained from the complex of biotinylated PCR product-streptavidin sepharose beads for 10 seconds. To each well, add 12 µl of Annealing buffer (# 979009, QIAGEN, Germany) and 1 µl of the primer for sequencing of 1-2 (10 uM) or 1 µl of the second sequencing primer (10 uM). After heating at room temperature and cooling slowly, pyro sequencing was performed using a PyroMark Gold Q24 reagent kit (enzyme and substrate mixture, dATP, dCTP, dGTP, dTTP, # 970802, QIAGEN, Germany). The analysis results using the pyro sequencing data and the PyroMarkTM Q24 software are shown in FIGS. 1 and 2.

Example 2. Kit Construction

A kit capable of performing about 24 reactions with the following configuration was produced.

① 250µl of BioQuest's 2x Hot-Taq master mixture

② below 25 μl of a mixture of G160V (G> T) _F primer and G160V (G> T) _R primer of Example 1-2 or IVS1-11 (T> A) _F primer and IVS1-11 (T> A 25 μl mixture of R primer

③ 30 μl of G160V (G> T) _Seq primer of Example 1-2 above or 30 μl of IVS1-11 (T> A) _F primer

Example 3. Kit Construction

The kit was manufactured by adding the following configuration to the configuration of Example 2.

④ streptavidin sepharose (GE healthcare, Germany)

⑤ 2 × Binding buffer (# 979006, QIAGEN, Germany)

⑥ Denaturation Solution (# 979007, QIAGEN, Germany)

⑦ Wash Solution (# 979008, QIAGEN, Germany)

⑧ Annealing buffer ((# 979009, QIAGEN, Germany)

⑨ PyroMark Gold Q24 reagent kit (enzyme and substrate mixture, dATP, dCTP, dGTP, dTTP, # 970802, QIAGEN, Germany)

<110> Specialty Lab Solution Co., Ltd. <120> Detection method of VSX1 single nucleotide polymorphism and          Detection kit for VSX1 SNP <130> P <160> 78 <170> Kopatentin 1.71 <210> 1 <211> 6669 <212> DNA <213> Homo sapiens <400> 1 tccagagagg ctcgcgcctt gcttgctaag gaaccatgac cggccgggac tcgctttccg 60 acgggcgcac tagcagcagg gcgctggtgc ctggcggttc ccctaggggc tcgcgccccc 120 ggggcttcgc catcacggac ctgctgggct tggaggccga gctgccggcg cccgctggcc 180 caggacaggg atctggctgc gagggtccgg cagtcgcgcc gtgcccgggc ccggggcttg 240 acggctccag cctggcgcgt ggggccctac cgctgggact cggcctcctc tgtggcttcg 300 gcacgcagcc gccggcggcc gctcgagcac cctgcctgct cctagcggac gtgccgttcc 360 tgccgcccag gggccccgag cccgctgccc cgctggctcc cagccgtccg ccgcctgcgc 420 tcggccgcca gaagcgcagc gacagcgtct ccacgtccgg taatcaggcc cgcgctttcc 480 gctcctgccc ctgtccccta ggctctgagc cgcgcagggg tcacagacca tcgccccacc 540 gcacccctgg ccccagcggc ccagatctag gcggggagcc cagagcgagg ccccgtcgcg 600 ggaggggcat tcgcggagct gcgccgcctg ccccttgctg catcctaaat ccctgcctct 660 catccggggt cctgttccct cagcgatgtg agaccgccac cacctggttc cgggtggaac 720 cttttgatga gaccctgggg tcttcaacaa ccatctccag agggccattt ttccccccag 780 cgccaggcgc cagtctccgc ctctggcagc tgcgtgggtc gggaaggccc gggcctgtgg 840 tggtcaccag cagcaccgaa caaggtacca ggtggaaacg cgggcggaaa tgatcgaatg 900 attgacgggt cctggaagag tacggggcct cgagccctgg aactgtccgc cccttggggt 960 gctgaaaagg gagtcccggg taccgcgctc ctctgccggc gcagcgtcct cccagtcccc 1020 tgtgcaggag aaagaagcaa ccttggggat tcattgtagt tacgcctaga gatgtgttcc 1080 tggcctaccc atttccccaa agaaatccag acgggtcgcg gggaaagaga aggaagcaat 1140 gaaattctat aaaggacaag ggctaaggaa acgagtggtt ttgggtgtgg gagagaaaag 1200 aaagaattac acaagaaagg atggtatgtg gtagaatcta gagctgagca tccaggcagc 1260 caaggcaaaa agagtagact gggtcttctg atactaaaag gaagcatgtc agatcaatag 1320 gcaggcaggt ttttgctcca ctctaaaagg actttatttc atgagctcta cgtaagataa 1380 aatgaaactc cctattagtg tcactaaaaa tgcaaaactg ttttcatggg atgggttctt 1440 tcttacaaaa ccaaaggcag ctaaatccaa ctctgccagc atttctgaaa gtctggagca 1500 gagctgtcca agaaaaatga aatgtgattt catgtttgac ggaaatttct cacagtcaca 1560 ttaaaagtga aatgaatcta aatactatat tttaacccaa caatgatagc aaatattatc 1620 attttacaca taatcaatac aaaaatattt tactttttag gagcactaag tcttctaact 1680 ccattaagtg atttatgttt acagcatgtc ttgatcttac tggccacatt tcatgtgctt 1740 aataacctta tgtgtctcat gtctcatcag gttggacagc ccaatacaga atgttttaat 1800 ccaagccagg tttctggtga actaattgat ggatgagtaa aactagtatt atttagttat 1860 ttttgccttt attcatttta aagatatttt gaggcaatta tgccttctag caatgaatct 1920 cacaatagtt tggccttcta accttctttt ccatctagac tttttgatgt attggatata 1980 aaaaggtaaa gcacagtgga gtggttctca aattttcaag ggcaactaca catcatctga 2040 gcacacttgt caagaatgag cctcctagat cccacctcca gagattctga tttggtgcat 2100 cagggaaggg ttgcagttct gacgagggtt ctgatgtcac aagccccaga cttcctttgg 2160 gaaatgttgg tgtagaggtt aagatgatga ggctgggggc ataggggtgg atgtgtttgg 2220 aagcagtgtg gggatagaaa gtgcaatcag tggctgagta gttttggctt cagataatct 2280 gggatttgaa ttctcaactt caccatttac tacctgtgtg attttgggca aattacttaa 2340 ccattcggtg cctcagtttc ctcatctgta aaaataggat gataataata gcagcagcca 2400 ttttggtgtt ttattattat attaagaata cttgagataa tgtatggaca gtgttaagca 2460 cagagcctgg gatctagtag gcactaaaaa tgctggctca tactgtaaac tacttaagta 2520 cccaagaggt tcataacttc aatcctcaca ttaaaatcca ggaaatagag gggatatgat 2580 cacccttaaa gtcctcttct tctttctgtg ccatcagatg aggacagcca gtctgaagac 2640 aggaatgacc taaaggcatc ccccaccttg ggcaagagga agaagcggcg gcacaggtat 2700 agggccaggc agtcccctct gcctgccttt cctcgggaca cagcccaagg tttatggcac 2760 ctgcatctga gaatttatgg cccggcatga tagcaggccc agtggtttcc tggttggcaa 2820 tgggaagggc acagaagcca tttgggaaga taattctgca gttcctagga ggctgggaaa 2880 atgctacaat cttgctagca tttttttggg agcattttaa agacaatttt gtggatctgt 2940 caaatggatt ttgtcagaat gagctgatgg aattcattaa gaaagagcta aagaactgtt 3000 ttcgtttgaa tcaaatttct ggacatcagc taatcattca gaggtggggt gttccattat 3060 catgaagtgg cttctagggt ctggacagca gaggaagcag gcacggtggt ccttaaggcc 3120 agggaggctg ctgtccccag gggacatgtg cccacctgtg tgttttgggg tccttgcagg 3180 acagttttca ctgctcacca gctggaagag ttggagaagg cattcagcga ggcccactac 3240 cctgatgtgt atgcccgaga aatgctggct gtgaaaactg agctccccga agaccggata 3300 caggtgtctg gggtcccttt tctccgctcc aaagatacca cagagaacgt gtcattccca 3360 cattcagtga gccaatcagc agtcccttct ctatagccaa cacgctccct ttgcatagaa 3420 actgagggtc ccttagctga aggcaccaca agagcttgcc cctatgaccc tcatgccttt 3480 tttcatttta ttattattta gtatcaaatc attctttaaa atcacatgat aatgtgtgct 3540 gcaactagaa actataaaga tatgtcaatg aaaaaaaaaa aaaagagact acctctccat 3600 ctcaaaccct gaaagaaatc aacacaaatg cctgaggaga cctttctcta ctttgtggag 3660 tatattatct gcccctgaat atgtatgtgc atatacatat ctacatgcat atatacacac 3720 actcacatgc atacatagaa ttttcctctt ggttttatca gaagatatgt cactaactct 3780 ttttttaaca gctaaaccgt atttcataat agtcctttat taacataatg tttttaatga 3840 gaaatatcac cagcaaccca atagcctaag ctcggaagga gccagtgccc cagtgagctc 3900 attccaatta atgagaaaaa gcaggaaaca ctgtagttga cctcttcaca caatgtggga 3960 aaagggagtt tgattcttta gtttttttca ttcctgactc tatggaaact tcagtggacg 4020 tggtcacaag tgtccctact gcgttgaatg cccgtgagtc cggaccgcac gtgggcgctg 4080 ctgggggctc ctgggcgctc tccctgaggg cggctggttg gccctcggga gctatttcct 4140 tcagaaatag catgggatca tgctcgggag agaagatccg agccctggga cgccgcctcc 4200 gctcctccag gcgcccctct tcactgtctc ttctgtgtgc cttctgcttc tcctgcctcc 4260 aaccaggtct ggtttcaaaa ccgcagggcc aaatggcgca agcgggagaa gcgctggggc 4320 ggcagcagcg tgatggccga gtacgggctg tacggggcca tggtgcgcca ctgcatcccg 4380 ctgccagact ccgtgctcaa ctccgccgag ggcggcctgc tgggctcctg cgcgccctgg 4440 ctcctgggta aggaagggcc cccgggatcg cacggctcga ggtgttgtag gaggtggggg 4500 aatcccaggg tgtccccacc gaggcagcca tttccaaagc aaagcaacgt cagtttactg 4560 agattcaggt ccagaaccgt tcttattatt tttaattaat aacaaagctt gcagcaactg 4620 gatggttgcc tcatttttca cagagactgt ctaggctgtg tggaagtctc accgggctat 4680 gcatgctaaa tatgtatttg ggatttaagg gtgaaatgac atcattgcat taacagcatc 4740 caagtgttga atgctgttag aatttttaga aaccatacaa tttagggcac ttggtaatag 4800 gaaacagggc agtgctgagc agcgacccct ggggacagtg agcccctcca tctgagtcag 4860 gagggctggc cccgggcttg gttctgccat gaacaacctg ccgagtggct tttctgtccc 4920 ttagattttt ggtgctcaaa gagtctccgc tgctgagacc cagcactctc aggatgctcc 4980 acccttgttg ccctgtttgc tgtcgccaga aaccaacagg cagggatcac agattgtggg 5040 aatgagtcac agatgagtac ctccgggcac ggggtaatta gttaggagga tcatggtggc 5100 attttaggtt tagatggagg aagttttttg ctcagcgaac agtttttgaa cagctaccaa 5160 gcagccctat cctgggcact ggaggttcca atcatagata tgtcctgact ttgctctcaa 5220 ggactgaaac actagaggga gcattgtagg cagtgaccca aactggctca tctgaaattg 5280 cacaaacagg tgggggaggg cacttactgg catctgcgag accgcagcct aggggaaagt 5340 gggcccaatg ccaatcactg tgtcatccta aacacctttg ctggatgtga cccggggtca 5400 gacacctgct ttaaacccct aaggtgggag ttacctacct gcctctcctg taggccccag 5460 agataggcac tgacaaggac aattctagtg ggatttagag aacaataaga aggaaattta 5520 cttcattgct gaatttaaat ttttattttt attttttcct ttttattttt ttttttacaa 5580 gggatgcata aaaaatccat ggggatgata aggaagccag gaagtgaaga taagttggca 5640 ggactctggg gctctgacca cttcaaagaa ggttctagcc agagtgagtc aggatcacag 5700 agaggctcag ataaagtgag ccctgagaat ggcttggaag atgtggctat tgacctctcc 5760 agctctgccc ggcaggagac caagaaagtg caccctgggg ctggtgctca aggaggctcc 5820 aactccacgg cactggaggg gccccagcca gggaaggtgg gagccacatg agacccacag 5880 gtcccactgt caaaggctga aaatgtgcat attcctcact ggcattttcc aaaagacaaa 5940 aattgtccaa gacatatact ctcagtttga ttttcttctg acaatgtcaa gataaaatgc 6000 aatgccactt gctttaagag gacagatgag tgacctaggg aaaattctat ttcattctag 6060 aatggaaacg gttccttaat ggcactttaa ccagttaact tggtgcaaaa cttttgattc 6120 tcttttgtag cttgagcagg gaggatccga agttcaaaaa tgatttcact gctgggcatc 6180 atttgagatg tgtggttcaa tgtactgtat agatgaatag gttaggtctg atgtcctcat 6240 tctcaattag gaaatataaa attgtttaat atccagaaga atctgatatc atcatagtga 6300 agactccata cagacacatg aatgaattat attgtcttta ccttgaactt ggccttggat 6360 atttcaattt tcttgttagg gagtaatttt gtgttttctt gagtgtcact tgataggcta 6420 tgcatattgg aattcaaaga aacccatact ctagtcgttt ctgcatttcc tttaaaaata 6480 gcatgggatc atatttaaac aattgttgga tagctggaaa cagtgttttt aggtcagacc 6540 aggcttttgg tctttcaaaa tttgtgattg aaagcacttt agtgacttgg attttgtaaa 6600 tctttccttt cctgcgtttc ctttgccatt tatttattct ttaaaaataa acattttgtg 6660 gtgtgttta 6669 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 2 atgaggacag ccagtctgaa gaca 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 3 agccagtctg aagacaggaa tga 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 4 aatccaggaa atagagggga tatg 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 5 ccaggaaata gaggggatat ga 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 6 gaggacagcc agtctgaaga ca 22 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 7 aaaggcatcc cccaccttg 19 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 8 aggacagcca gtctgaagac a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 9 taaaggcatc ccccaccttg 20 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 10 cagccagtct gaagacagga atga 24 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 11 gccagtctga agacaggaat ga 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 12 gtgccatcag atgaggacag c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 13 ctaaaggcat cccccacctt g 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 14 tgtgccatca gatgaggaca g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify G160V (G> T) region <400> 15 agatgaggac agccagtctg a 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 16 ggactgcctg gccctatacc t 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 17 gggactgcct ggccctata 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 18 gaggaaaggc aggcagagg 19 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 19 aggaaaggca ggcagagg 18 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 20 cactgggcct gctatcatgc 20 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 21 gccataaacc ttgggctgtg t 21 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 22 atcatgccgg gccataaatt 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 23 aaatggcttc tgtgcccttc c 21 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 24 ggccataaat tctcagatgc ag 22 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 25 aaaccactgg gcctgctat 19 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 26 ccataaacct tgggctgtgt 20 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 27 tgccaaccag gaaaccac 18 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 28 gctgtgtccc gaggaaagg 19 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 29 ggccataaat tctcagatgc a 21 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 30 ttctcagatg caggtgccat aaac 24 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 31 ctcagatgca ggtgccataa ac 22 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 32 gcctggccct atacctgtgc 20 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 33 ccgccgcttc ttcctctt 18 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify G160V (G> T) region <400> 34 actgcagaat tatcttccca aatg 24 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 35 gcatccccca ccttg 15 <210> 36 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 36 catcccccac cttgg 15 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 37 gccgcttctt cctct 15 <210> 38 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 38 ggcatccccc acctt 15 <210> 39 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 39 ccgcttcttc ctcttg 16 <210> 40 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 40 aggcatcccc cacct 15 <210> 41 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 41 ccgcttcttc ctctt 15 <210> 42 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of G160V (G> T) region <400> 42 cgccgcttct tcctc 15 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 43 tccaggaaat agaggggata tg 22 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 44 aaatgctggc tcatactgta aact 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 45 ggggatatga tcacccttaa agtc 24 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 46 ggggatatga tcacccttaa agt 23 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 47 ccaggaaata gaggggatat gat 23 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 48 tccaggaaat agaggggata tgat 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 49 agaggggata tgatcaccct taaa 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 50 aaatccagga aatagagggg atat 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 51 ccaggaaata gaggggatat gatc 24 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 52 agaggggata tgatcaccct taa 23 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 53 aaaatccagg aaatagaggg gata 24 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 54 gaggggatat gatcaccctt aaag 24 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 55 aaaatgctgg ctcatactgt aaac 24 <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 56 ggatatgatc acccttaaag tcct 24 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 57 ccaggaaata gaggggatat ga 22 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 58 tccaggaaat agaggggata tga 23 <210> 59 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to amplify IVS1-11 (T> A) region <400> 59 tcacccttaa agtcctcttc ttct 24 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 60 atgcctttag gtcattcctg tc 22 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 61 cttcagactg gctgtcctca t 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 62 ctgtcttcag actggctgtc c 21 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 63 ctgtcttcag actggctgtc ct 22 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 64 tgggggatgc ctttaggtca 20 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 65 ggatgccttt aggtcattcc tgtc 24 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 66 cataaacctt gggctgtgtc c 21 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 67 ggtgggggat gcctttag 18 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 68 tcttcagact ggctgtcctc atc 23 <210> 69 <211> 19 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 69 gggggatgcc tttaggtca 19 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> Reverse primer to amplify IVS1-11 (T> A) region <400> 70 caaggtgggg gatgccttta g 21 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 71 taaagtcctc ttcttctttc 20 <210> 72 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 72 cctcatctga tggca 15 <210> 73 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 73 cctcatctga tggcac 16 <210> 74 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 74 ctgtcctcat ctgatgg 17 <210> 75 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 75 aaagtcctct tcttcttt 18 <210> 76 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 76 gtcctcatct gatggc 16 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 77 ccttaaagtc ctcttcttct 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for the sequencing of IVS1-11 (T> A) region <400> 78 cttaaagtcc tcttcttctt 20

Claims (14)

a) a forward primer consisting of 10 to 30 consecutive bases in the bases 2555 to 2670 of SEQ ID NO: 1 and
Performing polymerase chain reaction (PCR) on the sample DNA using a reverse primer composed of 10 to 30 consecutive bases in the complementary base sequences of the base sequences from 2674 th to 2862 th of SEQ ID NO: 1;
b) for the PCR product obtained in step a),
Consists of 10 to 18 consecutive bases in the 2654 th to 2671 th base sequences of SEQ ID NO: 1, or 10 to 18 consecutive bases in the complementary base sequences of the base sequences 2626 to 2690 th of SEQ ID NO: 1 Performing pyrosequencing using a sequencing primer consisting of; method for detecting a mutation in the keratoconus causal gene comprising. The method of claim 1,
The forward primer consists of one nucleotide sequence selected from SEQ ID NOs: 2 to 15,
The reverse primer consists of one nucleotide sequence selected from SEQ ID NO: 16 to 34,
The sequencing primer is characterized in that consisting of one nucleotide sequence selected from SEQ ID NO: 35 to 42. The method of claim 1,
The forward primer consists of the nucleotide sequence of SEQ ID NO: 2,
The reverse primer consists of the nucleotide sequence of SEQ ID NO: 16,
The sequencing primer is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 35. According to claim 1, wherein the polymerase chain reaction
Denature at 90-98 ° C. for 5-20 minutes,
And a cycle consisting of 5 to 30 seconds denaturation at 90 to 98 ° C., 10 to 30 seconds annealing at 55 to 65 ° C., and 5 to 30 seconds extension at 70 to 75 ° C., consisting of 20 to 50 cycles. According to claim 1, wherein the polymerase chain reaction
Denature at 95 ° C. for 11 minutes,
Wherein a cycle consisting of 10 seconds denaturation at 95 ° C., 15 seconds annealing at 60 ° C. and 9 seconds extension at 72 ° C. consists of 40 cycles. The method of claim 1,
In performing a polymerase chain reaction (PCR) on the sample DNA in step a),
A second forward primer consisting of 10 to 30 contiguous bases among the base sequences of SEQ ID NO: 2487 to 2603; and
Further using a second reverse primer consisting of 10 to 30 consecutive bases in the complementary base sequence of the base sequence from SEQ ID NO: 2617 to 2756,
In performing the pyro sequencing in step b),
Consists of 10 to 23 consecutive bases in the nucleotide sequences of SEQ ID NO: 1 2584 to 2606, or 10 to 20 consecutive bases in the complementary base sequences of the base sequences 2608 to 2627 of SEQ ID NO: Method for using a second sequencing primer consisting of. The method according to claim 6,
The second forward primer consists of one base sequence selected from SEQ ID NOs: 43 to 59,
The second reverse primer consists of one base sequence selected from SEQ ID NO: 60 to 70,
The second sequencing primer is characterized in that consisting of one base sequence selected from SEQ ID NO: 71 to 78. The method according to claim 6,
The second forward primer consists of the nucleotide sequence of SEQ ID NO: 43,
The second reverse primer consists of the nucleotide sequence of SEQ ID NO: 66,
The second sequencing primer is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 74. A forward primer consisting of 10 to 30 contiguous bases in the nucleotide sequences of 2555 th to 2670 th of SEQ ID NO: 1;
A reverse primer consisting of 10 to 30 contiguous bases in the complementary nucleotide sequences of the nucleotide sequences 2674 th to 2862 th of SEQ ID NO: 1; And
Consists of 10 to 18 consecutive bases in the 2654 th to 2671 th base sequences of SEQ ID NO: 1, or 10 to 18 consecutive bases in the complementary base sequences of the base sequences 2626 to 2690 th of SEQ ID NO: 1 A sequencing primer consisting of; a kit for detecting mutations in the keratoconus causal gene by pyro sequencing comprising a. The method of claim 9,
The forward primer consists of one nucleotide sequence selected from SEQ ID NOs: 2 to 15,
The reverse primer consists of one nucleotide sequence selected from SEQ ID NO: 16 to 34,
The sequencing primer is a kit, characterized in that consisting of one nucleotide sequence selected from SEQ ID NO: 35 to 42. The method of claim 9,
The forward primer consists of the nucleotide sequence of SEQ ID NO: 2,
The reverse primer consists of the nucleotide sequence of SEQ ID NO: 16,
The sequencing primer is a kit, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 35. The method of claim 9,
A second forward primer consisting of 10 to 30 contiguous bases among the base sequences of SEQ ID NO: 2487 to 2603;
A second reverse primer consisting of 10 to 30 contiguous bases among the complementary nucleotide sequences of the nucleotide sequences 2617 to 2756 of SEQ ID NO: 1; And
Consists of 10 to 23 consecutive bases in the nucleotide sequences of SEQ ID NO: 1 2584 to 2606, or 10 to 20 consecutive bases in the complementary base sequences of the base sequences 2608 to 2627 of SEQ ID NO: The kit comprising a second sequencing primer consisting of. 13. The method of claim 12,
The second forward primer consists of one base sequence selected from SEQ ID NOs: 43 to 59,
The second reverse primer consists of one base sequence selected from SEQ ID NO: 60 to 70,
The second sequencing primer is a kit, characterized in that consisting of one nucleotide sequence selected from SEQ ID NO: 71 to 78. 13. The method of claim 12,
The second forward primer consists of the nucleotide sequence of SEQ ID NO: 43,
The second reverse primer consists of the nucleotide sequence of SEQ ID NO: 66,
The second sequencing primer is a kit, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 74.

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