# Patent

## Title
Kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence

## Abstract

One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, including: at least one first primer consisting of a first segment and a second segment, wherein the first segment is a complementary strand of a first sequence and the second segment is a second sequence, and the 3′ end of the first segment connects to the 5′ end of the second segment; a second primer being a third sequence; at least one third primer consisting of a third segment and a fourth segment, wherein the third segment is a fourth sequence and the fourth segment is a complementary strand of a fifth sequence, and the 3′ end of the third segment connects to the 5′ end of the fourth segment; and a fourth primer being a complementary strand of a sixth sequence, wherein the specific region includes rs1799853, rs1057910, rs2108622, rs9923231 and rs9934438.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of Taiwan Patent Application No. 101128721, filed on Aug. 9, 2012, the entirety of which is incorporated by reference herein.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
A sequence listing submitted as a text file via EFS-Web is incorporated herein by reference. The text file containing the sequence listing is named “0965-A23948-US_Seq_Listing.txt”; its date of creation is Sep. 19, 2012; and its size is 120,699 bytes.
TECHNICAL FIELD
The technical field relates to a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence.
BACKGROUND
It has been known that polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910)), polymorphism of CYP4F2 (cytochrome P450, family 4, subfamily F, polypeptide 2) gene (rs2108622) and polymorphisms of VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (VKORC1-1639 (rs9923231) of or VKORC1 1173 (rs9934438)) significantly impact the maintenance dose for the drug warfarin (Clinical Chemistry 55:4, 804-812 (2009); Blood. 2008 Apr. 15; 111(8):4106-12. Epub 2008 Feb. 4.; Ann Clin Lab Sci. 2011 Summer; 41(3):229-35). Before prescribing the drug, prescreening patients for their genotypes will facilitate a faster individualized determination of the proper maintenance dose, minimizing the risk of adverse reactions and reoccurrence of thromboembolic episodes. However, if genotyping is performed to determine the loading therapy can be delayed by several hours to 1 day.
Loop-mediated isothermal amplification (LAMP), a special type of nucleic acid amplification, is a new technique developed by Eiken Genome of Japan in 2000. This method is able to complete an amplification of DNA or RNA in one hour by using four primers in an environment of 60-65° C. Since the loop-mediated isothermal amplification has the advantages of speed, simplicity of isothermal amplification, and assay-design flexibility, the loop-mediated isothermal amplification is very suitable for various types of nucleic acid testing, such as genotype identification.
FIGS. 1A and 1B illustrate the basic principles of the loop-mediated isothermal amplification, and the detailed principles and operation instructions of this method can be referred to in U.S. Pat. No. 7,175,985 B1 and the animation illustration provided on the website of Eiken Genome of Japan.
According to FIGS. 1A and 1B, it is understood that for the loop-mediated isothermal amplification, six regions are selected in a target gene 100, which are F1, F2, F3, B1c, B2c and B3c, respectively, and F1c, F2c, F3c, B1, B2 and B3 in the other strand shown in FIGS. 1A and 1B are complementary to the F1c, F2c, F3c, B1, B2 and B3 regions mentioned above, respectively. In this method, at least one of F1 and B1c regions which are selected has to contain a mutation or single-nucleotide polymorphism site which is targeted for detection, and the enzyme which is adopted is Bst DNA polymerase which is capable of performing amplification reaction in isothermality and opening the double-strand structure of a target or template DNA during amplification.
Furthermore, in this method, four primers are adopted, and they are forward inner primer (FIP) 101, forward outer primer 103, backward inner primer (BIP) 105 and backward outer primer 107.
The sequence of the forward inner primer (FIP) 101 consists of a first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of F1c region which is predicated) and a second segment (the sequence of the F2 region). For example, if the F1 region of the target gene is predicated as a sequence from a wild type, the sequence of the first segment is the complementary strand of the F1 region from the wild type, in contrast, if the F1 region of the target gene is predicated as a sequence from a mutant type, the sequence of the first segment is the complementary strand of the F1 region from the mutant type.
The sequence of the forward outer primer 103 is the sequence of the F3 region
Moreover, the sequence of the backward inner primer (BIP) 105 consists of a third segment (the sequence of the B1c region which is predicated, that is the complementary strand of the sequence of the B1 region which is predicated) and a fourth segment (the complementary strand of the sequence of the B2c region, that is the sequence of the B2 region). For example, if the B1c region of the target gene is predicated as sequence from a wild type, the sequence of the third segment is the sequence of the B1c region from the wild type, in contrast, if the B1c region of the target gene is predicated as a sequence from a mutant type, the sequence of the third segment is the sequence of the B1c region from the mutant type.
Furthermore, the sequence of the backward outer primer 107 is the complementary strand of the sequence of the B3c region (namely, the sequence of the B3 region)
While performing the loop-mediated isothermal amplification, the second segment (the sequence of the F2 region) of the forward inner primer (FIP) 101 will anneal to the F2c region of the other strand of the target gene mentioned above and proceed to a complementary strand synthesis reaction, and a first strand which has the sequences of the first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of the F1c region which is predicated), second segment (the sequence of the B2 region), F1, B1c, B2c and B3 regions is synthesized, and the forward outer primer 103 will push the first strand aside and thus a second strand which has the sequences of F3, F2, F1, B1c, B2c and B3 regions is synthesized.
Next, the fourth segment (the sequence of the B2 region) of the backward inner primer (BIP) 105 anneals to the B2c region of the foregoing first strand. And a third strand which has the sequence of the B1c region as predicated, the sequences of B2, B1, F1c, F2c, and the sequence of F1 which is predicated is synthesized by using the first strand as a template.
After that, the backward outer primer 107 will push the third strand aside and thus a fourth strand which has the sequences of the B3, B2, B1, F1c and F2c regions and the sequence of the F1 region which is predicated is synthesized. The predicated B1c region of the third strand and B1 region of the third strand will result in self-annealing, and similarly the predicated F1 region of the third strand and F1c region of the third strand will also result in self-annealing. Thus the third strand will become a strand with two ends each have a loop formed.
Then, the forward inner primer (FIP) and backward inner primer (BIP) continue the complementary strand synthesis reaction by using the complementary strand synthesis products of the third strand and/or the complementary strand thereof as the template in turn, and a double-strand product which has a plurality of loops is formed (please refer to FIG. 1B).
In addition, FIG. 2A shows results obtained from performing the loop-mediated isothermal amplification, the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated being the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively. When the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, since the synthesized third strand which is mentioned in the foregoing paragraph and which has the sequence of the B1c region which is predicated, the sequences of B2, B1, F1c, F2c, and the sequence of the F1 which is predicated, is able to become a strand whose two ends each have a loop formed, the complementary strand synthesis reaction can continue.
In contrast, FIG. 2B shows results obtained from performing the loop-mediated isothermal amplification The sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated is not the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively. When the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are not the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, the third strand can not self-anneal to form a strand whose two ends each have a loop formed. Thus complementary strand synthesis can not be continued.
Therefore, according to that mentioned above, by using appropriate primers for wild type and mutant type, respectively, a mutation and/or polymorphism of a specific region in a target nucleotide sequence can be detected.
However, for rs1799853 of the CYP2C9*2 gene, rs1057910 of the CYP2C9*3 gene, rs2108622 of the CYP4F2 gene, rs9923231 of VKORC1-1639 and rs9934438 of VKORC1 1173, primers designed by the software, PrimerExplorer V4, which is provided by Eiken Genome on its website to perform the loop-mediated isothermal amplification, no amplification results can be detected for previous mentioned five genes.
Therefore, a new kit is needed which can be used to quickly and accurately detect the single-nucleotide polymorphism of CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) of the CYP2C9 gene, rs2108622 of CYP4F2 gene, VKORC1-1639 (rs9923231) and VKORC1 1173 (rs9934438) of VKORC1 gene.
SUMMARY
One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer consisting of a first segment and a second segment; a second primer; at least one third primer consisting of a third segment and a fourth segment; and a fourth primer. In the first primer, the 3′ end of the first segment connects to the 5′ end of the second segment, wherein the first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 375 and position 406 of SEQ ID NO.: 3 and has to contain position 401 of SEQ ID NO.: 3, and wherein the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 320 and position 348 of SEQ ID NO.: 3. The second primer is a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 298 and position 328 of SEQ ID NO.: 3. In the third primer, the 3′ end of the third segment connects to the 5′ end of the fourth segment, wherein the third segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 396 and position 428 of SEQ ID NO.: 3 and has to contain position 401 of SEQ ID NO.: 3, and wherein the fourth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 451 and position 479 of SEQ ID NO.: 3. The fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 482 and position 514 of SEQ ID NO.: 3. The kit is used for detecting a mutation and/or polymorphism of a specific region in a target gene in a sample from a subject, wherein the target gene is CYP2C9 (SEQ ID NO.: 1), and the specific region is CYP2C9*2 (rs1799853), and the position of the occurrence of the mutation and/or polymorphism is located on position 8633 of SEQ ID NO: 1, and the kit is used in a nucleic acid amplification.
One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer consisting of a first segment and a second segment; a second primer; at least one third primer consisting of a third segment and a fourth segment; and a fourth primer. In the first primer, the 3′ end of the first segment connects to the 5′ end of the second segment, wherein the first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 376 and position 406 of SEQ ID NO.: 25 and has to contain position 401 of SEQ ID NO.: 25, and wherein the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 332 and position 359 of SEQ ID NO.: 25. The second primer is a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 301 and position 328 of SEQ ID NO.: 25. In the third primer, the 3′ end of the third segment connects to the 5′ end of the fourth segment, wherein the third segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 396 and position 425 of SEQ ID NO.: 25 and has to contain position 401 of SEQ ID NO.: 25, and wherein the fourth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 448 and position 478 of SEQ ID NO.: 25. The fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 470 and position 501 of SEQ ID NO.: 25. The kit is used for detecting a mutation and/or polymorphism of a specific region in a target gene in a sample from a subject, wherein the target gene is CYP2C9 (SEQ ID NO.: 1), and the specific region is CYP2C9*3 (rs1057910), and the position of the occurrence of the mutation and/or polymorphism is located on position 47639 of SEQ ID NO: 1, and the kit is used in a nucleic acid amplification.
One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer consisting of a first segment and a second segment; a second primer; at least one third primer consisting of a third segment and a fourth segment; and a fourth primer. In the first primer, the 3′ end of the first segment connects to the 5′ end of the second segment, wherein the first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 393 and position 423 of SEQ ID NO.: 48 and has to contain position 417 of SEQ ID NO.: 48, and wherein the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 352 and position 381 of SEQ ID NO.: 48. The second primer is a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 333 and position 358 of SEQ ID NO.: 48. In the third primer, the 3′ end of the third segment connects to the 5′ end of the fourth segment, wherein the third segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 411 and position 444 of SEQ ID NO.: 48 and has to contain position 417 of SEQ ID NO.: 48, and wherein the fourth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 460 and position 488 of SEQ ID NO.: 48. The fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 492 and position 519 of SEQ ID NO.: 48. The kit is used for detecting a mutation and/or polymorphism of a specific region in a target gene in a sample from a subject, wherein the target gene is CYP4F2 (SEQ ID NO.: 46), and the specific region is rs2108622, and the position of the occurrence of the mutation and/or polymorphism is located on position 23454 of SEQ ID NO: 46, and the kit is used in a nucleic acid amplification.
One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer consisting of a first segment and a second segment or consisting of a first segment, a second segment and a third segment; a second primer; at least one third primer consisting of a fourth segment and a fifth segment or consisting of a fourth segment, a fifth segment and a sixth segment; and a fourth primer. In the first primer, the 3′ end of the first segment connects to the 5′ end of the second segment, or the 3′ end of the first segment connects to the 5′ end of the third segment, 3′ end of the third segment connects to the 5′ end of the second segment and the third segment consists of about 2-10 thymines, wherein the first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 476 and position 505 of SEQ ID NO.: 71 and has to contain position 501 of SEQ ID NO.: 71, and wherein the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 451 and position 481 of SEQ ID NO.: 71. The second primer is a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 400 and position 442 of SEQ ID NO.: 71. In the third primer, the 3′ end of the fourth segment connects to the 5′ end of the fifth segment, or the 3′ end of the fourth segment connects to the 5′ end of the sixth segment, 3′ end of the sixth segment connects to the 5′ end of the fifth segment, and the sixth segment consists of about 2-10 thymines, wherein the fourth segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 496 and position 529 of SEQ ID NO.: 71 and has to contain position 501 of SEQ ID NO.: 71, and wherein the fifth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 523 and position 550 of SEQ ID NO.: 71. The fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 542 and position 572 of SEQ ID NO.: 71. The kit is used for detecting a mutation and/or polymorphism of a specific region in a target gene in a sample from a subject, wherein the target gene is VKORC1 (SEQ ID NO.: 69), and the specific region is VKORC1-1639 (rs9923231), and the position of the occurrence of the mutation and/or polymorphism is located on position 3586 of SEQ ID NO: 69, and the kit is used in a nucleic acid amplification.
One embodiment of the disclosure provides a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer consisting of a first segment and a second segment; a second primer; at least one third primer consisting of a third segment and a fourth segment; and a fourth primer. In the first primer, the 3′ end of the first segment connects to the 5′ end of the second segment, wherein the first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 373 and position 407 of SEQ ID NO.: 93 and has to contain position 401 of SEQ ID NO.: 93, and wherein the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 324 and position 353 of SEQ ID NO.: 93. The second primer is a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 288 and position 317 of SEQ ID NO.: 93. In the third primer, the 3′ end of the third segment connects to the 5′ end of the fourth segment, wherein the third segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 395 and position 427 of SEQ ID NO.: 93 and has to contain position 401 of SEQ ID NO.: 93, and wherein the fourth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 451 and position 479 of SEQ ID NO.: 93. The fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 487 and position 514 of SEQ ID NO.: 93. The kit is used for detecting a mutation and/or polymorphism of a specific region in a target gene in a sample from a subject, wherein the target gene is VKORC1 (SEQ ID NO.: 69), and the specific region is VKORC1 1173 (rs9934438), and the position of the occurrence of the mutation and/or polymorphism is located on position 6399 of SEQ ID NO: 69, and the kit is used in a nucleic acid amplification.
A detailed description is given in the following embodiments with reference to the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
The embodiments of disclosure can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:
FIGS. 1A and 1B illustrate the basic principle for the loop-mediated isothermal amplification;
FIG. 2A shows a result obtained from performing the loop-mediated isothermal amplification, the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated being the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively;
FIG. 2B shows a result obtained from performing the loop-mediated isothermal amplification, the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated not being the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively;
FIG. 3A shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Comparative Example 1 (negative image);
FIG. 3B shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Example 1 (negative image);
FIG. 3C shows a figure showing the result for sequencing a product obtained from performing a polymerase chain reaction by the primers of Example 1;
FIG. 4A shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Comparative Example 2 (negative image);
FIG. 4B shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Example 2 (negative image);
FIG. 4C shows a figure showing the result for sequencing a product obtained from performing a polymerase chain reaction by the primers of Example 2;
FIG. 5A shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Comparative Example 3 (negative image);
FIG. 5B shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Example 3 (negative image);
FIG. 5C shows a figure showing the result for sequencing a product obtained from performing a polymerase chain reaction by the primers of Example 3;
FIG. 6A shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Comparative Example 4 (negative image);
FIG. 6B shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Example 4 (negative image);
FIG. 6C shows a figure showing the result for sequencing a product obtained from performing a polymerase chain reaction by the primers of Example 4;
FIG. 7A shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Comparative Example 5 (negative image);
FIG. 7B shows an electrophoresis gel for a product obtained from performing loop-mediated isothermal amplification by the primers of Example 7 (negative image); and
FIG. 7C shows a figure showing the result for sequencing a product obtained from performing a polymerase chain reaction by the primers of Example 7.



DETAILED DESCRIPTION
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.
In order to overcome the problem wherein the results obtained by using the primers designed by the software package PrimerExplorer V4, provided by Eiken Genome on its website to perform the loop-mediated isothermal amplification, it cannot be known what are the single-nucleotide polymorphisms of the CYP2C9*2 (rs1799853) of the CYP2C9 gene, CYP2C9*3 (rs1057910) of the CYP2C9 gene, rs2108622 of the CYP4F2 gene, VKORC1-1639 (rs9923231) and VKORC1 1173 (rs992323) of the VKORC1 gene of a test sample, the disclosure provides a new kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence.
The kit of the disclosure mentioned above is suitable for a nucleic acid amplification, especially for a loop-mediated isothermal amplification (LAMP), wherein by using this kit, the detection results can be quickly and accurately obtained, and this kit is suitable for detecting a single-nucleotide polymorphism of CYP2C9*2 (rs1799853) of the CYP2C9 gene, CYP2C9*3 (rs1057910) of the CYP2C9 gene, rs2108622 of the CYP4F2 gene, VKORC1-1639 (rs9923231) or VKORC1 1173 (rs992323) of the VKORC1 gene.
With regard to the design for the primers in the kit of the disclosure, it is similar to that of the primer design software by Eiken Genome of Japan, as shown in FIGS. 1A and 1B. Design for the primers is performed by selecting six regions within a selected primer design region of the target gene 100, which are F1 (a first sequence), F2 (a second sequence), F3 (a third sequence), B1c (a fourth sequence), B2c (a fifth sequence) and B3c (a sixth sequence), and F1c, F2c, F3c, B1, B2 and B3 in the other strand are complementary to the F1c, F2c, F3c, B1, B2 and B3 regions mentioned above, respectively, wherein the selected primer design region comprises a specific region which may be a mutation and/or single-nucleotide polymorphism region requiring detection, and at least one of the selected F1 and B1c regions has to contain a mutation site or single-nucleotide polymorphism site requiring detection. Similarly, in the disclosure, at least four primers are designed according to the six selected regions mentioned above, which are forward inner primer (FIP) 101, forward outer primer 103, backward inner primer (BIP) 105 and backward outer primer 107. The sequence of the forward inner primer (FIP) 101 consists of a first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of the F1c region which is predicated) and a second segment (the sequence of the F2 region). For example, if the F1 region of the target gene is predicated as a sequence from a wild type, the sequence of the first segment is the complementary strand of the F1 region from the wild type. In contrast, if the F1 region of the target gene is predicated as sequence from a mutant type, the sequence of the first segment is the complementary strand of F1 region from the mutant type. The sequence of the forward outer primer 103 is the sequence of the F3 region. The sequence of the backward inner primer (BIP) 105 consists of a third segment (the sequence of the B1c region which is predicated, that is the complementary strand of the sequence of the B1 region which is predicated) and a forth segment (the complementary strand of the sequence of the B2c region, that is the sequence of the B2 region). For example, if the B1c region of the target gene is predicated as a sequence from a wild type, the sequence of the third segment is the sequence of the B1c region from the wild type. In contrast, if the B1c region of the target gene is predicated as a sequence from a mutant type, the sequence of the third segment is the sequence of B1c region from the mutant type. The sequence of the backward outer primer 107 is the complementary strand of the sequence of the B3c region (namely, the sequence of the B3 region). However, it should be noted that for the single-nucleotide polymorphisms of the CYP2C9*2 (rs1799853) of the CYP2C9 gene, CYP2C9*3 (rs1057910) of the CYP2C9 gene, rs2108622 of the CYP4F2 gene, VKORC1-1639 (rs9923231) and VKORC1 1173 (rs9934438) of the VKORC1 gene, etc., the design concepts for the primers in the kit of the present disclosure, F1, F2, F3, B1c, B2c and B3c regions, are different from those suggested by the primer design software of Eiken Genome of Japan. And by using the kit of the present disclosure, the types of the single-nucleotide polymorphisms mentioned above can be detected quickly and accurately.
In a loop-mediated isothermal amplification performed by the kit of the disclosure, the second segment (the sequence of the F2 region) of the forward inner primer (FIP) 101 will anneal to the F2c region of the other strand of the target gene 100 mentioned above and proceed to a complementary strand synthesis reaction, and a first strand which has the sequences of the first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of the F1c region which is predicated), the second segment (the sequence of the B2 region), F1, B1c, B2c and B3 regions is synthesized, and the forward outer primer 103 will push the first strand aside and thus a second strand which has the sequences of the F3, F2, F1, B1c, B2c and B3 regions is synthesized. Next, the fourth segment (the sequence of the B2 region) of the backward inner primer (BIP) 105 anneals to the B2c region of the foregoing first strand, and a third strand which has the sequence of B1c region which is predicated, the sequences of B2, B1, F1c, F2c, and the sequence of F1 which is predicated is synthesized by using the first strand as a template. After that, the backward outer primer 107 will push the third strand aside and thus a fourth strand which has the sequences of the B3, B2, B1, F1c and F2c regions and the sequence of the F1 region which is predicated is synthesized.
When the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, since the synthesized third strand mentioned above which has the sequence of the B1c region which is predicated, the sequences of B2, B1, F1c, F2c, and the sequence of F1 which is predicated, is able to become a strand whose two ends each have a loop formed, complementary strand synthesis reaction through forward inner primer (FIP) 101 and backward inner primer (BIP) 105 can continue (referring again to FIG. 2A). In contrast, when the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are not the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, since in the synthesized third strand mentioned above which has the sequence of the B1c region which is predicated, the sequences of B2, B1, F1c, F2c, and the sequence of F1 which is predicated, self-annealing can not be formed, neither between the B1c region which is predicated and the B1 region of the third strand, nor between the F1 region which is predicated and the F1c region of the third strand which cannot self-anneal, respectively, it can not make the third strand become a strand whose two ends each have a loop formed, and the complementary strand synthesis reaction through forward inner primer (FIP) 101 and backward inner primer (BIP) 105 can not be continued (referring again to FIG. 2B).
Therefore, according to the preceding, in one embodiment of the present disclosure, the present disclosure may provide a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, which is used for detecting a mutation and/or polymorphism of a specific region in a target gene of a sample from a subject. The target gene mentioned above may be CYP2C9 (SEQ ID NO.: 1), the specific region mentioned above is CYP2C9*2 (rs1799853) (SEQ ID NO.: 3), and the position of the occurrence of the preceding mutation and/or polymorphism (CYP2C9*2 (rs1799853) (C→T)) is located on position 8633 of SEQ ID NO: 1 (namely, position 401 of SEQ ID NO.: 3).
The kit of the present disclosure may comprise, but is not limited to, at least one first primer, a second primer, at least one third primer, and a fourth primer, and the selected primer design region used for designing these primers, which is mentioned in the foregoing paragraph, is SEQ ID NO.: 3.
The at least one first primer mentioned above consists of a first segment and a second segment, wherein the 3′ end of the first segment connects to the 5′ end of the second segment. The first segment may be a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 375 and position 406 of SEQ ID NO.: 3 and has to contain position 401 of SEQ ID NO.: 3. The second segment may be a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 320 and position 348 of SEQ ID NO.: 3.
The second primer mentioned above may be a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 298 and position 328 of SEQ ID NO.: 3.
The at least one third primer mentioned above may consist of a third segment and a fourth segment, wherein the 3′ end of the third segment connects to the 5′ end of the fourth segment. The third segment may be a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 396 and position 428 of SEQ ID NO.: 3 and has to contain position 401 of SEQ ID NO.: 3. The fourth segment may be a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 451 and position 479 of SEQ ID NO.: 3.
Furthermore, the fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 482 and position 514 of SEQ ID NO.: 3.
The kit of the disclosure is used in a nucleic acid amplification. The nucleic acid amplification may comprise, but is not limited to, a loop-mediated isothermal amplification (LAMP). In one embodiment, the kit of the present disclosure is applied in a loop-mediated isothermal amplification (LAMP), and the temperature of the loop-mediated isothermal amplification may be about 55-65° C., and in one embodiment, the temperature of the loop-mediated isothermal amplification is about 60-65° C.
When the sequence of the specific region contains a nucleotide sequence which is predicated, a product of the complementary strand synthesis is produced in the nucleic acid amplification. The product of the complementary strand synthesis corresponds to the third strand mentioned in the foregoing paragraphs, and the formation principle of the third strand may be referenced in the foregoing paragraphs. The product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the fourth sequence, the complementary strand of the fifth sequence, the complementary strand of the fourth sequence, the complementary strand of the first sequence, the complementary strand of the second sequence and the first sequence, wherein in the product of the complementary strand synthesis, the fourth sequence and the complementary strand of the fourth sequence result in self-annealing, and the first sequence and the complementary strand of the first sequence result in self-annealing, or the product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the complementary strand of the fourth sequence, the fifth sequence, the fourth sequence, the first sequence, the second sequence and the complementary strand of the first sequence, wherein in the product of the complementary strand synthesis, the complementary strand of the fourth sequence and the fourth sequence result in self-annealing, and the complementary strand of the first sequence and the first sequence result in self-annealing, and that makes the product of the complementary strand synthesis become a strand whose two ends each have a loop formed to continue the complementary strand synthesis.
In contrast, when the sequence of the specific region is not a nucleotide sequence which is predicated, the complementary strand synthesis in the nucleic acid amplification will be inhibited.
In one embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 16 and/or a primer whose sequence is SEQ ID NO: 17, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 21 and/or a primer whose sequence is SEQ ID NO: 22, and the fourth primer is a primer whose sequence is SEQ ID NO: 23. Moreover, in this embodiment, this kit is capable of detecting wild type and/or mutant type of CYP2C9*2 (rs1799853(C→T)).
In another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 16, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 21, and the fourth primer is a primer whose sequence is SEQ ID NO: 23. Moreover, in this embodiment, this kit is capable of detecting wild type mutant type of CYP2C9*2 (rs1799853 (C)).
In another further embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 17, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 22, and the fourth primer is a primer whose sequence is SEQ ID NO: 23. Moreover, in this embodiment, this kit is capable of detecting mutant type of CYP2C9*2 (rs1799853 (T)).
In addition, the foregoing kit of the present disclosure may further comprise a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity, and nucleotide substrates, but is not limited thereto. Examples of the DNA polymerase may comprise, but are not limited to Bst DNA polymerase. In one embodiment, the nucleotide substrates may be nucleotide substrates which are modified, for example a nucleotide substrate which is linked to an enzyme or a dye, but it is not limited thereto.
Furthermore, in a nucleic acid amplification, especially in a loop-mediated isothermal amplification, the ratio for the amount of usage of the first primer to the second primer is between about 1:1 and 1:10, and the ratio for the amount of usage of the third primer to the fourth primer is between about 1:1 and 1:10.
In one embodiment of the present disclosure, the present disclosure may provide a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, which is used for detecting a mutation and/or polymorphism of a specific region in a target gene of a sample from a subject. The target gene mentioned above may be CYP2C9 (SEQ ID NO.: 1). The specific region mentioned above is CYP2C9*3 (rs1057910) (SEQ ID NO.: 25). The position of the occurrence of the preceding mutation and/or polymorphism, CYP2C9*3 (rs1057910 (A→C)), is located on position 47639 of SEQ ID NO: 1 (namely, position 401 of SEQ ID NO.: 25).
The kit of the present disclosure may comprise, but is not limited to, at least one first primer, a second primer, at least one third primer and a fourth primer, and the selected primer design region used for designing these primers, which is mentioned in the foregoing paragraph, is SEQ ID NO.: 25.
The at least one first primer mentioned above consists of a first segment and a second segment, wherein the 3′ end of the first segment connects to the 5′ end of the second segment. The first segment may be a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 376 and position 406 of SEQ ID NO.: 25 and has to contain position 401 of SEQ ID NO.: 25. The second segment may be a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 332 and position 359 of SEQ ID NO.: 25.
The second primer mentioned above may be a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 301 and position 328 of SEQ ID NO.: 25.
The at least one third primer mentioned above may consist of a third segment and a fourth segment, wherein the 3′ end of the third segment connects to the 5′ end of the fourth segment. The third segment may be a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 396 and position 425 of SEQ ID NO.: 25 and has to contain position 401 of SEQ ID NO.: 25. The fourth segment may be a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 448 and position 478 of SEQ ID NO.: 25.
Furthermore, the fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 470 and position 501 of SEQ ID NO.: 25.
The kit of the disclosure is used in a nucleic acid amplification. The nucleic acid amplification may comprise, but is not limited to, a loop-mediated isothermal amplification (LAMP). In one embodiment, the kit of the present disclosure is applied in a loop-mediated isothermal amplification (LAMP), and the temperature of the loop-mediated isothermal amplification may be about 55-65° C., and in one embodiment, the temperature of the loop-mediated isothermal amplification is about 60-65° C.
When the sequence of the specific region contains a nucleotide sequence which is predicated, a product of the complementary strand synthesis is produced in the nucleic acid amplification. The product of the complementary strand synthesis corresponds to the third strand mentioned in the foregoing paragraphs, and the formation principle of the third strand may be referenced in the foregoing paragraphs. The product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the fourth sequence, the complementary strand of the fifth sequence, the complementary strand of the fourth sequence, the complementary strand of the first sequence, the complementary strand of the second sequence and the first sequence, wherein in the product of the complementary strand synthesis, the fourth sequence, and the complementary strand of the fourth sequence result in self-annealing, and the first sequence and the complementary strand of the first sequence result in self-annealing, or the product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the complementary strand of the fourth sequence, the fifth sequence, the fourth sequence, the first sequence, the second sequence and the complementary strand of the first sequence, wherein in the product of the complementary strand synthesis, the complementary strand of the fourth sequence and the fourth sequence result in self-annealing, and the complementary strand of the first sequence and the first sequence result in self-annealing, and that makes the product of the complementary strand synthesis become a strand whose two ends each have a loop formed to continue the complementary strand synthesis.
In contrast, when the sequence of the specific region is not a nucleotide sequence which is predicated, complementary strand synthesis in the nucleic acid amplification will be inhibited.
In one embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 38 and/or a primer whose sequence is SEQ ID NO: 39, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 40 and/or a primer whose sequence is SEQ ID NO: 43, and the fourth primer is a primer whose sequence is SEQ ID NO: 44. Moreover, in this embodiment, this kit is capable of detecting wild type and/or mutant type of CYP2C9*3 (rs1057910 (A→C)).
In another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 38, the second primer is a primer whose sequence is SEQ ID NO: 40, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 43, and the fourth primer is a primer whose sequence is SEQ ID NO: 45. Moreover, in this embodiment, this kit is capable of detecting wild type of CYP2C9*3 (rs1057910 (A)).
In further another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 39, the second primer is a primer whose sequence is SEQ ID NO: 40, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 44, and the fourth primer is a primer whose sequence is SEQ ID NO: 45. Moreover, in this embodiment, this kit is capable of detecting mutant type of CYP2C9*3 (rs1057910 (C)).
In addition, the foregoing kit of the present disclosure may further comprise a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity, and nucleotide substrates, but is not limited thereto. Examples of the DNA polymerase may comprise, but is not limited to, Bst DNA polymerase. In one embodiment, the nucleotide substrates may be nucleotide substrates which are modified, for example a nucleotide substrate which is linked to an enzyme or a dye, but it is not limited thereto.
Furthermore, in a nucleic acid amplification, especially in a loop-mediated isothermal amplification, the ratio for the amount of usage of the first primer to the second primer is between about 1:1 and 1:10, and the ratio for the amount of usage of the third primer to the fourth primer is between about 1:1 and 1:10.
In one embodiment of the present disclosure, the present disclosure may provide a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, which is used for detecting a mutation and/or polymorphism of a specific region in a target gene of a sample from a subject. The target gene mentioned above may be CYP4F2 (SEQ ID NO.: 46), the specific region mentioned above is rs2108622 (SEQ ID NO.: 48), and the position of the occurrence of the preceding mutation and/or polymorphism, rs2108622 (C→T), is located on position 23454 of SEQ ID NO: 46 (namely, position 417 of SEQ ID NO.: 48).
The kit of the present disclosure may comprise, but is not limited to, at least one first primer, a second primer, at least one third primer, and a fourth primer, and the selected primer design region used for designing these primers, which is mentioned in the foregoing paragraph, is SEQ ID NO.: 48.
The at least one first primer mentioned above consists of a first segment and a second segment, wherein the 3′ end of the first segment connects to the 5′ end of the second segment. The first segment may be a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 393 and position 423 of SEQ ID NO.: 48 and has to contain position 417 of SEQ ID NO.: 48. The second segment may be a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 352 and position 381 of SEQ ID NO.: 48.
The second primer mentioned above may be a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 333 and position 358 of SEQ ID NO.: 48.
The at least one third primer mentioned above may consist of a third segment and a fourth segment, wherein the 3′ end of the third segment connects to the 5′ end of the fourth segment. The third segment may be a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 411 and position 444 of SEQ ID NO.: 48 and has to contain position 417 of SEQ ID NO.: 48. The fourth segment may be a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 460 and position 488 of SEQ ID NO.: 48.
Furthermore, the fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 492 and position 519 of SEQ ID NO.: 48.
The kit of the disclosure is used in a nucleic acid amplification. The nucleic acid amplification may comprise, but is not limited to, a loop-mediated isothermal amplification (LAMP). In one embodiment, the kit of the present disclosure is applied in a loop-mediated isothermal amplification (LAMP), and the temperature of the loop-mediated isothermal amplification may be about 55-65° C., and in one embodiment, the temperature of the loop-mediated isothermal amplification is about 60-65° C.
When the sequence of the specific region contains a nucleotide sequence which is predicated, a product of the complementary strand synthesis is produced in the nucleic acid amplification. The product of the complementary strand synthesis corresponds to the third strand mentioned in the foregoing paragraphs, and the formation principle of the third strand may be referenced in the foregoing paragraphs. The product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the fourth sequence, the complementary strand of the fifth sequence, the complementary strand of the fourth sequence, the complementary strand of the first sequence, the complementary strand of the second sequence and the first sequence, wherein in the product of the complementary strand synthesis, the fourth sequence and the complementary strand of the fourth sequence result in self-annealing, and the first sequence and the complementary strand of the first sequence result in self-annealing, or the product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the complementary strand of the fourth sequence, the fifth sequence, the fourth sequence, the first sequence, the second sequence and the complementary strand of the first sequence, wherein in the product of the complementary strand synthesis, the complementary strand of the fourth sequence and the fourth sequence result in self-annealing, and the complementary strand of the first sequence and the first sequence result in self-annealing, and that makes the product of the complementary strand synthesis become a strand whose two ends each have a loop formed to continue the complementary strand synthesis
In contrast, when the sequence of the specific region is not a nucleotide sequence which is predicated, complementary strand synthesis in the nucleic acid amplification will be inhibited.
In one embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 61 and/or a primer whose sequence is SEQ ID NO: 62, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 66 and/or a primer whose sequence is SEQ ID NO: 67, and the fourth primer is a primer whose sequence is SEQ ID NO: 68. Moreover, in this embodiment, this kit is capable of detecting wild type and/or mutant type of CYP4F2 (rs2108622 (C→T)).
In another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 61, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 66, and the fourth primer is a primer whose sequence is SEQ ID NO: 68. Moreover, in this embodiment, this kit is capable of detecting wild type of CYP4F2 (rs2108622 (C)).
In another further embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 62, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 67, and the fourth primer is a primer whose sequence is SEQ ID NO: 68. Moreover, in this embodiment, this kit is capable of detecting mutant type of CYP4F2 (rs2108622 (T)).
In addition, the foregoing kit of the present disclosure may further comprise a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity, and nucleotide substrates, but is not limited thereto. Examples of the DNA polymerase may comprise, but are not limited to, Bst DNA polymerase. In one embodiment, the nucleotide substrates may be nucleotide substrates which are modified, for example a nucleotide substrate which is linked to an enzyme or a dye, but it is not limited thereto.
Furthermore, in a nucleic acid amplification, especially in a loop-mediated isothermal amplification, the ratio for the amount of usage of the first primer to the second primer is between about 1:1 and 1:10, and the ratio for the amount of usage of the third primer to the fourth primer is between about 1:1 and 1:10.
In one embodiment of the present disclosure, the present disclosure may provide a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, which is used for detecting a mutation and/or polymorphism of a specific region in a target gene of a sample from a subject. The target gene mentioned above may be VKORC1 (SEQ ID NO.: 69), the specific region mentioned above is VKORC1-1639 (rs9923231) (SEQ ID NO.: 71), and the position of the occurrence of the preceding mutation and/or polymorphism, VKORC1-1639 (rs9923231 (T→C)), is located on position 3586 of SEQ ID NO: 69 (namely, position 501 of SEQ ID NO.: 71).
The kit of the present disclosure may comprise, but is not limited to, at least one first primer, a second primer, at least one third primer, and a fourth primer, and the selected primer design region used for designing these primers, which is mentioned in the foregoing paragraph, is SEQ ID NO.: 71.
The at least one first primer consists of a first segment and a second segment, wherein the 3′ end of the first segment connects to the 5′ end of the second segment, or the at least one first primer consists of a first segment, a second segment, and a third segment, wherein the 3′ end of the first segment connects to the 5′ end of the third segment and 3′ end of the third segment connects to the 5′ end of the second segment, and the third segment consists of about 2-10 thymines. The first segment is a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 476 and position 505 of SEQ ID NO.: 71 and has to contain position 501 of SEQ ID NO.: 71, and the second segment is a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 451 and position 481 of SEQ ID NO.: 71.
The second primer mentioned above may be a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 400 and position 442 of SEQ ID NO.: 71.
The at least one third primer consists of a fourth segment and a fifth segment, wherein the 3′ end of the fourth segment connects to the 5′ end of the fifth segment, or the at least one third primer consists of a fourth segment, a fifth segment, and a sixth segment, wherein the 3′ end of the fourth segment connects to the 5′ end of the sixth segment and 3′ end of the sixth segment connects to the 5′ end of the fifth segment, and the sixth segment consisting of about 2-10 thymines. The fourth segment is a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 496 and position 529 of SEQ ID NO.: 71 and has to contain position 501 of SEQ ID NO.: 71, and the fifth segment is a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 523 and position 550 of SEQ ID NO.: 71.
Furthermore, the fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 542 and position 572 of SEQ ID NO.: 71.
The kit of the disclosure is used in a nucleic acid amplification. The nucleic acid amplification may comprise, but is not limited to, a loop-mediated isothermal amplification (LAMP). In one embodiment, the kit of the present disclosure is applied in a loop-mediated isothermal amplification (LAMP), and the temperature of the loop-mediated isothermal amplification may be about 55-65° C., and in one embodiment, the temperature of the loop-mediated isothermal amplification is about 60-65° C.
When the sequence of the specific region contains a nucleotide sequence which is predicated, a product of the complementary strand synthesis is produced in the nucleic acid amplification. The product of the complementary strand synthesis corresponds to the third strand mentioned in the foregoing paragraphs, and the formation principle of the third strand may be referenced in the foregoing paragraphs. The product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the fourth sequence, the complementary strand of the fifth sequence, the complementary strand of the fourth sequence, the complementary strand of the first sequence, the complementary strand of the second sequence and the first sequence, wherein in the product of the complementary strand synthesis, the fourth sequence and the complementary strand of the fourth sequence result in self-annealing, and the first sequence and the complementary strand of the first sequence result in self-annealing, or the product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the complementary strand of the fourth sequence, the fifth sequence, the fourth sequence, the first sequence, the second sequence and the complementary strand of the first sequence, wherein in the product of the complementary strand synthesis, the complementary strand of the fourth sequence and the fourth sequence result in self-annealing, and the complementary strand of the first sequence and the first sequence result in self-annealing, and that makes the product of the complementary strand synthesis become a strand whose two ends each have a loop formed to continue the complementary strand synthesis
In contrast, when the sequence of the specific region is not a nucleotide sequence which is predicated, complementary strand synthesis in the nucleic acid amplification will be inhibited.
In one embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 84 and/or a primer whose sequence is SEQ ID NO: 85, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 89 and/or a primer whose sequence is SEQ ID NO: 90, and the fourth primer is a primer whose sequence is SEQ ID NO: 91. Moreover, in this embodiment, this kit is capable of detecting wild type and/or mutant type of VKORC1-1639 (rs9923231 (T→C)).
In another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 84, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 89, and the fourth primer is a primer whose sequence is SEQ ID NO: 91. Moreover, in this embodiment, this kit is capable of detecting wild type of VKORC1-1639 (rs9923231 (T)).
In another further embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 85, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 90, and the fourth primer is a primer whose sequence is SEQ ID NO: 91. Moreover, in this embodiment, this kit is capable of detecting mutant type of VKORC1-1639 (rs9923231 (C)).
In addition, the foregoing kit of the present disclosure may further comprise a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity, and nucleotide substrates, but it is not limited thereto. Examples of the DNA polymerase may comprise, but are not limited to, Bst DNA polymerase. In one embodiment, the nucleotide substrates may be nucleotide substrates which are modified, for example a nucleotide substrate which is linked to an enzyme or a dye, but it is not limited thereto.
Furthermore, in a nucleic acid amplification, especially in a loop-mediated isothermal amplification, the ratio for the amount of usage of the first primer to the second primer is between about 1:1 and 1:10, and the ratio for the amount of usage of the third primer to the fourth primer is between about 1:1 and 1:10.
In one embodiment of the present disclosure, the present disclosure may provide a kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, which is used for detecting a mutation and/or polymorphism of a specific region in a target gene of a sample from a subject. The target gene mentioned above may be VKORC1 (SEQ ID NO.: 69), the specific region mentioned above is VKORC1 1173 (rs9934438) (SEQ ID NO.: 93), and the position of the occurrence of the preceding mutation and/or polymorphism, VKORC1 1173 (rs9934438 (A→G)), is located on position 6399 of SEQ ID NO: 69 (namely, position 401 of SEQ ID NO.: 93).
The kit of the present disclosure may comprise, but is not limited to, at least one first primer, a second primer, at least one third primer and a fourth primer, and the selected primer design region used for designing these primers, which is mentioned in the foregoing paragraph, is SEQ ID NO.: 93.
The at least one first primer mentioned above consists of a first segment and a second segment, wherein the 3′ end of the first segment connects to the 5′ end of the second segment. The first segment may be a complementary strand of a first sequence and has about 10-30 nucleotides, and the first sequence is located between position 373 and position 407 of SEQ ID NO.: 93 and has to contain position 401 of SEQ ID NO.: 93. The second segment may be a second sequence and has about 10-30 nucleotides, and the second sequence is located between position 324 and position 353 of SEQ ID NO.: 93.
The second primer mentioned above may be a third sequence and has about 10-30 nucleotides, and the third sequence is located between position 288 and position 317 of SEQ ID NO.: 93.
The at least one third primer mentioned above may consist of a third segment and a fourth segment, wherein the 3′ end of the third segment connects to the 5′ end of the fourth segment. The third segment may be a fourth sequence and has about 10-30 nucleotides, and the fourth sequence is located between position 395 and position 427 of SEQ ID NO.: 93 and has to contain position 401 of SEQ ID NO.: 93. The fourth segment may be a complementary strand of a fifth sequence and has about 10-30 nucleotides, and the fifth sequence is located between position 451 and position 479 of SEQ ID NO.: 93.
Furthermore, the fourth primer is a complementary strand of a sixth sequence and has about 10-30 nucleotides, and the sixth sequence is located between position 487 and position 514 of SEQ ID NO.: 93.
The kit of the disclosure is used in a nucleic acid amplification. The nucleic acid amplification may comprise, but is not limited to, a loop-mediated isothermal amplification (LAMP). In one embodiment, the kit of the present disclosure is applied in a loop-mediated isothermal amplification (LAMP), and the temperature of the loop-mediated isothermal amplification may be about 55-65° C., and in one embodiment, the temperature of the loop-mediated isothermal amplification is about 60-65° C.
When the sequence of the specific region contains a nucleotide sequence which is predicated, a product of the complementary strand synthesis is produced in the nucleic acid amplification. The product of the complementary strand synthesis corresponds to the third strand mentioned in the foregoing paragraphs, and the formation principle of the third strand may be referenced in the foregoing paragraphs. The product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the fourth sequence, the complementary strand of the fifth sequence, the complementary strand of the fourth sequence, the complementary strand of the first sequence, the complementary strand of the second sequence and the first sequence, wherein in the product of the complementary strand synthesis, the fourth sequence and the complementary strand of the fourth sequence result in self-annealing, and the first sequence and the complementary strand of the first sequence result in self-annealing, or the product of the complementary strand synthesis from the 5′ end to the 3′ end comprises the complementary strand of the fourth sequence, the fifth sequence, the fourth sequence, the first sequence, the second sequence and the complementary strand of the first sequence, wherein in the product of the complementary strand synthesis, the complementary strand of the fourth sequence and the fourth sequence result in self-annealing, and the complementary strand of the first sequence and the first sequence result in self-annealing, and that makes the product of the complementary strand synthesis become a strand whose two ends each have a loop formed to continue the complementary strand synthesis
In contrast, when the sequence of the specific region is not a nucleotide sequence which is predicated, the complementary strand synthesis in the nucleic acid amplification will be inhibited.
In one embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 106 and/or a primer whose sequence is SEQ ID NO: 107, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 111 and/or a primer whose sequence is SEQ ID NO: 112, and the fourth primer is a primer whose sequence is SEQ ID NO: 113. Moreover, in this embodiment, this kit is capable of detecting wild type and/or mutant type of VKORC1 1173 (rs9934438 (A→G)).
In another embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 106, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 111, and the fourth primer is a primer whose sequence is SEQ ID NO: 113. Moreover, in this embodiment, this kit is capable of detecting wild type of VKORC1 1173 (rs9934438 (A)).
In another further embodiment, in the kit of the present disclosure, the at least one first primer comprises a primer whose sequence is SEQ ID NO: 107, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 112, and the fourth primer is a primer whose sequence is SEQ ID NO: 113. Moreover, in this embodiment, this kit is capable of detecting mutant type of VKORC1 1173 (rs9934438 (G)).
In addition, the foregoing kit of the present disclosure may further comprise a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity, and nucleotide substrates, but it is not limited thereto. Examples of the DNA polymerase may comprise, but are not limited to, Bst DNA polymerase. In one embodiment, the nucleotide substrates may be nucleotide substrates which are modified, for example a nucleotide substrate which is linked to an enzyme or a dye, but it is not limited thereto.
Furthermore, in a nucleic acid amplification, especially in a loop-mediated isothermal amplification, the ratio for the amount of usage of the first primer to the second primer is between about 1:1 and 1:10, and the ratio for the amount of usage of the third primer to the fourth primer is between about 1:1 and 1:10.
EXAMPLES
A. Determination for Polymorphism of CYP2C9*2 (rs1799853) of the CYP2C9 Gene
Comparative Example 1
(1) Sample Obtainment
Genomic DNA sample was extracted from a human whole blood sample by a commercial nucleic acid purification kit (Product number: 101; Manufacturer: Chemagen). After that, the concentration of the genomic DNA sample was determined by a spectrophotometer (Product number: ND-1000, Manufacturer: NanoDrop).
(2) Primer Design
Through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, six regions were selected from a primer design region (SEQ ID NO.: 3) which was set for CYP2C9*2 (rs1799853) of the CYP2C9 gene and primers were designed according to the six regions. The locations of the six selected regions and the designed primers are shown in Table 1.







TABLE 1







Six selected regions and designed primers for CYP2C9*2 (rs1799853)


of the CYP2C9 gene through a website version of the design  


software package PrimerExplorer V4, provided 


by Eiken Genome, Japan.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
326
343
18
AAATGGAAGGAGATCCGG


(Forward outer



(SEQ ID NO.: 8)


primer)






 


F2 region
344
361
18
CGTTTCTCCCTCATGACG






(SEQ ID NO.: 5)


 


F1c region
384
403
20
ACGGTCCTCAATGCTCCTCT






(SEQ ID NO.: 4)


 


B1c region
404
423
20
GTTCAAGAGGAAGCCCGCTG






(SEQ ID NO.: 9)


 


B2 region
462
481
20
GGTCAGTGATATGGAGTAGG






(SEQ ID NO.: 10)


 


B3 region
487
509
23
GTCAGTAGAGAAGATAGTAGTCC


(Backward outer



(SEQ ID NO.: 13)


primer)






 


FIP-w


38
ACGGTCCTCAATGCTCCTCT-


(Forward inner



CGTTTCTCCCTCATGACG


primer for wild



(SEQ ID NO.: 6)


type)






 


FIP-m


38
ACAGTCCTCAATGCTCCTCT-


(Forward inner



CGTTTCTCCCTCATGACG


primer for mutant



(SEQ ID NO.: 7)


type)






 


BIP-w


43
CGTGTTCAAGAGGAAGCCCGCTG-


(Backward inner



GGTCAGTGATATGGAGTAGG


primer for wild



(SEQ ID NO.: 11)


type)






 


BIP-m


43
TGTGTTCAAGAGGAAGCCCGCTG-


(Backward inner



GGTCAGTGATATGGAGTAGG


primer for mutant



(SEQ ID NO.: 12)


type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions of the loop-mediated isothermal amplifications and the electrophoresis conditions are shown in Table 2. The results for electrophoresis are shown in FIG. 3A, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 2







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Comparative Example 1.









Constituents of loop-mediated isothermal
Reaction
Electrophoresis


amplification
conditions
conditions













Tris-HCl
20 mM
59° C.,
100 V,


(pH 8.8)

2 hours;
30 minutes in


Betaine
0.8M
80° C.,
2% agarose/1X


dNTPs
1.4 mM
5 minutes
TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.16 μM


Tween 20
0.1%


Bst polymerase
8U (8U/uL, 1 μL)


DNA
20 ng (10 ng/μL, 2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 3A, there is no amplified product present in the experimental group using primers for wild type or the experimental group using primers for mutant type. Therefore, according to the above mentioned, it is clear that the primers designed for CYP2C9*2 (rs1799853) of the CYP2C9 gene through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan cannot be used for detecting the polymorphism of CYP2C9*2 (rs1799853) of the CYP2C9 gene.
Example 1
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those performed in Comparative Example 1.
(2) Primer Design
In this example, six regions were selected from a primer design region (SEQ ID NO.: 3) which was set for CYP2C9*2 (rs1799853) of the CYP2C9 gene and primers were designed according to the six regions. The locations of the six selected regions and the designed primers are shown in Table 3.







TABLE 3







Six selected regions and designed primers for CYP2C9*2 (rs1799853)


of the CYP2C9 gene of Example 1.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
303
323
21
GAATTGTTTTCAGCAATGGAA


(Forward outer



(SEQ ID NO.: 18)


primer)






 


F2 region
325
343
19
GAAATGGAAGGAGATCCGG






(SEQ ID NO.: 15)


 


F1c region
380
400
21
GTCCTCAATGCTCCTCTTCCC






(SEQ ID NO.: 14)


 


B1c region
404
423
20
GTTCAAGAGGAAGCCCGCTG






(SEQ ID NO.: 19)


 


B2 region
456
474
19
GATATGGAGTAGGGTCACC






(SEQ ID NO.: 20)


 


B3 region
487
509
23
GTCAGTAGAGAAGATAGTAGTCC


(Backward outer 



(SEQ ID NO.: 23)


primer)






 


FIP-w


41
GGTCCTCAATGCTCCTCTTCCC-


(Forward inner



GAAATGGAAGGAGATCCGG


primer for wild



(SEQ ID NO.: 16)


type)






 


FIP-m


41
AGTCCTCAATGCTCCTCTTCCC-


(Forward inner



GAAATGGAAGGAGATCCGG


primer for mutant



(SEQ ID NO.: 17)


type)






 


BIP-w


42
CGTGTTCAAGAGGAAGCCCGCTG-


(Backward inner



GATATGGAGTAGGGTCACC


primer for wild



(SEQ ID NO.: 21)


type)






 


BIP-m


42
TGTGTTCAAGAGGAAGCCCGCTG-


(Backward inner



GATATGGAGTAGGGTCACC


primer for mutant



(SEQ ID NO.: 22)


type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions of the loop-mediated isothermal amplifications and the electrophoresis conditions are shown in Table 4. The results for electrophoresis are shown in FIG. 3B, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 4







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Example 1.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C.,
100 V,


(pH 8.8)

2 hours;
30 minutes in


Betaine
0.8M
80° C.,
2% agarose/1X


dNTPs
1.4 mM
5 minutes
TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.16 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL, 2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 3B, it is evident that amplified product is clearly presented in the experimental group using primers for wild type, whereas there is no amplified product presented in the experimental group using primers for mutant type. Therefore, according to the results, it is clear that the genotype of the sample is CC (wild type) for the single-nucleotide polymorphism of CYP2C9*2 (rs1799853) (C→T).
Sequencing
A polymerase chain reaction was performed on the foregoing genomic DNA sample with the forward outer primer (SEQ ID NO.: 18) and the backward outer primer (SEQ ID NO.: 23) to obtain an amplified product of F3 region to B3 region that contains the position of the single-nucleotide polymorphism, CYP2C9*2 (rs1799853). Then the product was sequenced (by using SEQ ID NO.: 18 as a primer) to determine the genotype of the single-nucleotide polymorphism, CYP2C9*2 (rs1799853). The result of the sequencing is shown in FIG. 3C, wherein the nucleotide marked by a box in the sequence shown at the top of the figure is the single-nucleotide polymorphism site of CYP2C9*2 (rs1799853). According to FIG. 3C, it is known that the genotype of the sample is CC for the single-nucleotide polymorphism of CYP2C9*2 (rs1799853) (C→T).
According to the results for Comparative Example 1 and Example 1, it is known that, as compared with primers of the comparative example, by using the primers of the kit of the present disclosure, the polymorphism of CYP2C9*2 (rs1799853) of the CYP2C9 gene can be quickly and accurately detected.
B. Determination for Polymorphism of CYP2C9*3 (rs1057910) of the CYP2C9 Gene
Comparative Example 2
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
Through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, six regions were selected from a primer design region (SEQ ID NO.: 25) which was set for CYP2C9*3 (rs1057910) of the CYP2C9 gene and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 5.







TABLE 5







Six selected regions and designed primers for CYP2C9*3 (rs1057910)


of the CYP2C9 gene through a website version of the design  


software package PrimerExplorer V4, provided 


by Eiken Genome, Japan.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence














F3 region
314
331
18
GTGATTGGCAGAAACCGG


(Forward outer



(SEQ ID NO.: 30)


primer)






 


F2 region
337
354
18
CTGCATGCAAGACAGGAG






(SEQ ID NO.: 27)


 


F1c region
381
400
20
GTATCTCTGGACCTCGTGCA






(SEQ ID NO.: 26)


 


B1c region
401
420
20
ATTGACCTTCTCCCCACCAG






(SEQ ID NO.: 31)


 


B2 region
438
462
25
TAGTTTCTGAATTTAATGTCACAGG






(SEQ ID NO.: 32)


 


B3 region
463
482
20
AACTTACCTTGGGAATGAGA


(Backward outer



(SEQ ID NO.: 35)


primer)






 


FIP-w


39
T-GTATCTCTGGACCTCGTGCA-


(Forward inner



CTGCATGCAAGACAGGAG


primer for wild



(SEQ ID NO.: 28)


type)






 


FIP-m


39
G-GTATCTCTGGACCTCGTGCA-


(Forward inner



CTGCATGCAAGACAGGAG


primer for



(SEQ ID NO.: 29)


mutant type)






 


BIP-w


45
A-TTGACCTTCTCCCCACCAG-


(Backward inner



TAGTTTCTGAATTTAATGTCACAGG


primer for wild



(SEQ ID NO.: 33)


type)






 


BIP-m


45
C-TTGACCTTCTCCCCACCAG-


(Backward inner



TAGTTTCTGAATTTAATGTCACAGG


primer for



(SEQ ID NO.: 34)


mutant type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, and then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 6. The results for electrophoresis are shown in FIG. 4A, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 6







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Comparative Example 2.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.16 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 4A, there is no amplified product present in the experimental group using primers for wild type or the experimental group using primers for mutant type. Therefore, according to the above mentioned, it is clear that the primers designed for CYP2C9*3 (rs1057910) of the CYP2C9 gene through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, cannot be used for detecting the polymorphism of CYP2C9*3 (rs1057910) of the CYP2C9 gene.
Example 2
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
In this example, six regions were selected from a primer design region (SEQ ID NO.: 25) which was set for CYP2C9*3 (rs1057910) of the CYP2C9 gene and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 7.







TABLE 7







Six selected regions and designed primers for CYP2C9*3 (rs1057910)


of the CYP2C9 gene of Example 2.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
306
323
18
TTGAACGTGTGATTGGCA


(Forward outer



(SEQ ID NO.: 40)


primer)






 


F2 region
337
354
18
CTGCATGCAAGACAGGAG






(SEQ ID NO.: 37)


 


F1c region
381
400
20
GTATCTCTGGACCTCGTGCA






(SEQ ID NO.: 36)


 


B1c region
401
420
20
ATTGACCTTCTCCCCACCAG






(SEQ ID NO.: 41)


 


B2 region
453
473
21
TGGGAATGAGATAGTTTCTGA






(SEQ ID NO.: 42)


 


B3 region
475
496
22
GTGTAGGAGAAACAAACTTACC


(Backward outer



(SEQ ID NO.: 45)


primer)






 


FIP-w


39
T-GTATCTCTGGACCTCGTGCA-


(Forward inner



CTGCATGCAAGACAGGAG


primer for wild



(SEQ ID NO.: 38)


type)






 


FIP-m


39
G-GTATCTCTGGACCTCGTGCA-


(Forward inner



CTGCATGCAAGACAGGAG


primer for mutant



(SEQ ID NO.: 39)


type)






 


BIP-w


41
A-TTGACCTTCTCCCCACCAG-


(Backward inner



TGGGAATGAGATAGTTTCTGA


primer for wild



(SEQ ID NO.: 43)


type)






 


BIP-m


41
C-TTGACCTTCTCCCCACCAG-


(Backward inner



TGGGAATGAGATAGTTTCTGA


primer for mutant



(SEQ ID NO.: 44)


type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 8. The results for electrophoresis are shown in FIG. 4B, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 8







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Example 2.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.16 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 4B, it can be seen that amplified product is clearly presented in the experimental group using primers for wild type whereas there is no amplified product presented in the experimental group using primers for mutant type. Therefore, according to the results, it is clear that the genotype of the sample is AA (wild type) for the single-nucleotide polymorphism of CYP2C9*3 (rs1057910) (A→C).
Sequencing
A polymerase chain reaction was performed on the foregoing genomic DNA sample with the forward outer primer (SEQ ID NO.: 40) and the backward outer primer (SEQ ID NO.: 45) to obtain an amplified product of F3 region to B3 region that contains the position of the single-nucleotide polymorphism, CYP2C9*3 (rs1057910). Then the products were sequenced (by using SEQ ID NO.: 40 as a primer) to determine the genotype of the single-nucleotide polymorphism, CYP2C9*3 (rs1057910). The result of the sequencing is shown in FIG. 4C, wherein the nucleotide marked by a box in the sequence shown at the top of the figure is the single-nucleotide polymorphism site of CYP2C9*3 (rs1057910). According to FIG. 4C, it is known that the genotype of the sample is AA for the single-nucleotide polymorphism of CYP2C9*3 (rs1057910) (A→C).
According to the results for Comparative Example 2 and Example 2, it is known that, as compared with primers of the comparative example, by using the primers of the kit of the present disclosure, the polymorphism of CYP2C9*3 (rs1057910) of the CYP2C9 gene can be quickly and accurately detected.
C. Determination for Polymorphism of rs2108622 of the CYP4F2 Gene
Comparative Example 3
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
Through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, six regions were selected from a primer design region (SEQ ID NO.: 48) which was set for rs2108622 of the CYP4F2 gene, and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 9.







TABLE 9







Six selected regions and designed primers for rs2108622 of the


CYP4F2 gene through a website version of the design software package 


PrimerExplorer V4, provided by Eiken Genome of Japan.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
307
326
20
TGAGGGAGGTGATGTTGGAT


(Forward outer



(SEQ ID NO.: 53)


primer)






 


F2 region
347
364
18
CCCCTCCTCTAGGAGCCT






(SEQ ID NO.: 50)


 


F1c region
398
416
19
TGTGGCCGGACCCTGAGGT






(SEQ ID NO.: 49)


 


B1c region
419
439
21
GCTGGGTTGTGATGGGTTCCG






(SEQ ID NO.: 54)


 


B2 region
481
498
18
GGCCTTCTCCTGACTGCT






(SEQ ID NO.: 55)


 


B3 region
501
518
18
AGGGGCCCCTCAGTGAAG


(Backward outer



(SEQ ID NO.: 58)


primer)






 


FIP-w


39
TG-TGTGGCCGGACCCTGAGGT-


(Forward inner



CCCCTCCTCTAGGAGCCT


primer for wild



(SEQ ID NO.: 51)


type)






 


FIP-m


39
TA-TGTGGCCGGACCCTGAGGT-


(Forward inner



CCCCTCCTCTAGGAGCCT


primer for mutant



(SEQ ID NO.: 52)


type)






 


BIP-w


42
ACA-


(Backward inner



GCTGGGTTGTGATGGGTTCCG-


primer for wild



GGCCTTCTCCTGACTGCT


type)



(SEQ ID NO.: 56)


 


BIP-m


42
ATA-


(Backward inner



GCTGGGTTGTGATGGGTTCCG-


primer for mutant



GGCCTTCTCCTGACTGCT


type)



(SEQ ID NO.: 57)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 10. The results for electrophoresis are shown in FIG. 5A, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 10







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Comparative Example 3.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.2 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 5A, there is no amplified product present in the experimental group using primers for wild type or the experimental group using primers for mutant type. Therefore, according to the above mentioned, it is clear that the primers designed for rs2108622 of the CYP4F2 gene through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, cannot be used for detecting the polymorphism of rs2108622 of the CYP4F2 gene.
Example 3
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
In this example, six regions were selected from a primer design region (SEQ ID NO.: 48) which was set for rs2108622 of the CYP4F2 gene and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 11.







TABLE 11







Six selected regions and designed primers for rs2108622 of the


CYP4F2 gene of Example 3.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
338
353
16
AAACCCTGCCCCCTCC


(Forward outer



(SEQ ID NO.: 63)


primer)






 


F2 region
357
376
20
AGGAGCCTTGGAATGGACAA






(SEQ ID NO.: 60)


 


F1c region
398
416
19
TGTGGCCGGACCCTGAGGT






(SEQ ID NO.: 59)


 


B1c region
419
439
21
GCTGGGTTGTGATGGGTTCCG






(SEQ ID NO.: 64)


 


B2 region
465
483
19
GCTCCCTTCTCTCCCACAG






(SEQ ID NO.: 65)


 


B3 region
497
514
18
GCCCCTCAGTGAAGGAGG


(Backward outer



(SEQ ID NO.: 68)


primer)






 


FIP-w


41
TG-TGTGGCCGGACCCTGAGGT-


(Forward inner



AGGAGCCTTGGAATGGACAA


primer for wild



(SEQ ID NO.: 61)


type)






 


FIP-m


41
TA-TGTGGCCGGACCCTGAGGT-


(Forward inner



AGGAGCCTTGGAATGGACAA


primer for 



(SEQ ID NO.: 62)


mutant type)






 


BIP-w


43
ACA-


(Backward inner



GCTGGGTTGTGATGGGTTCCG-


primer for wild



GCTCCCTTCTCTCCCACAG


type)



(SEQ ID NO.: 66)


 


BIP-m


43
ATA-


(Backward inner



GCTGGGTTGTGATGGGTTCCG-


primer for 



GCTCCCTTCTCTCCCACAG


mutant type)



(SEQ ID NO.: 67)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 12. The results for electrophoresis are shown in FIG. 5B, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 12







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Example 3.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
63° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.2 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 5B, amplified product is clearly presented in the experimental group using primers for wild type whereas there is no amplified product present in the experimental group using primers for mutant type. Therefore, according to the results, it is clear that the genotype of the sample is CC (wild type) for the single nucleotide polymorphism of CYP4F2 gene (rs2108622) (C→T).
Sequencing
A polymerase chain reaction was performed on the foregoing genomic DNA sample with the forward outer primer (SEQ ID NO.: 63) and the backward outer primer (SEQ ID NO.: 68) to obtain an amplified product of F3 region to B3 region that contains the position of the single-nucleotide polymorphism, rs2108622 of the CYP4F2 gene. Then the products were sequenced (by using SEQ ID NO.: 63 as a primer) to determine the genotype of the single-nucleotide polymorphism, rs2108622 of the CYP4F2 gene. The result for the sequencing is shown in FIG. 5C, wherein the nucleotide marked by a box in the sequence shown at the top of the figure is the single-nucleotide polymorphism site of rs2108622 of the CYP4F2 gene. According to FIG. 4C, it is known that the genotype of the sample is CC for the single-nucleotide polymorphism of the CYP4F2 gene (rs2108622) (C→T).
According to the results for Comparative Example 3 and Example 3, it is known that, as compared with primers of the comparative example, by using the primers of the kit of the present disclosure, the polymorphism of rs2108622 of the CYP4F2 gene can be quickly and accurately detected.
D. Determination for Polymorphism of VKORC1-1639 (rs9923231) of VKORC1
Comparative Example 4
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
Through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, six regions were selected from a primer design region (SEQ ID NO.: 71) which was set for VKORC1-1639 (rs9923231) of the VKORC1 gene and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 13.







TABLE 13







Six selected regions and designed primers for VKORC1-1639


(rs9923231) of the VKORC1 gene through a website version of the design 


software package PrimerExplorer V4, provided by Eiken Genome, Japan.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
415
433
19
AGGCTTGTCTTAAACTCCT


(Forward outer



(SEQ ID NO.: 76)


primer)






 


F2 region
437
454
18
CTCAAGTGATCCACCCAC






(SEQ ID NO.: 73)


 


F1c region
477
496
20
CGGTGGCTCACGCCTATAAT






(SEQ ID NO.: 72)


 


B1c region
498
518
21
ACCAGGCCAATGGTTGTTTTT






(SEQ ID NO.: 77)


 


B2 region
553
571
19
GGTAGGTGCAACAGTAAGG






(SEQ ID NO.: 78)


 


B3 region
572
590
19
GGAAGAGAGTTCCCAGAAG


(Backward outer



(SEQ ID NO.: 81)


primer)






 


FIP-w


43
AGGTG-


(Forward inner



CGGTGGCTCACGCCTATAAT-


primer for wild



CTCAAGTGATCCACCCAC


type)



(SEQ ID NO.: 74)


 


FIP-m


43
GGGTG-


(Forward inner



CGGTGGCTCACGCCTATAAT-


primer for mutant



CTCAAGTGATCCACCCAC


type)



(SEQ ID NO.: 75)


 


BIP-w


40
ACC-T-GGCCAATGGTTGTTTTT-


(Backward inner



GGTAGGTGCAACAGTAAGG


primer for wild



(SEQ ID NO.: 79)


type)






 


BIP-m


40
ACC-C-GGCCAATGGTTGTTTTT-


(Backward inner



GGTAGGTGCAACAGTAAGG


primer for mutant



(SEQ ID NO.: 80)


type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 14. The results for electrophoresis are shown in FIG. 6A, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 14







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Comparative Example 4.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.8 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 6A, there is no amplified product present in the experimental group using primers for wild type or the experimental group using primers for mutant type. Therefore, according to the above mentioned, it is clear that the primers designed for VKORC1-1639 (rs9923231) of the VKORC1 gene through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, cannot be used for detecting the polymorphism of VKORC1-1639 (rs9923231) of the VKORC1 gene.
Example 4
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
In this example, six regions were selected from a primer design region (SEQ ID NO.: 71) which was set for VKORC1-1639 (rs9923231) of the VKORC1 gene and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 15.







TABLE 15







Six selected regions and designed primers for VKORC1-1639


(rs9923231) of the VKORC1 gene of Example 4.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
415
437
23
AGGCTTGTCTTAAACTCCTGACC


(Forward outer



(SEQ ID NO.: 86)


primer)






 


F2 region
456
476
21
TCGGCCTCCCAAAATGCTAGG






(SEQ ID NO.: 83)


 


F1c region
481
501
21
TGGTGCGGTGGCTCACGCCTA






(SEQ ID NO.: 82)


 


B1c region
501
524
24
AGGCCAATGGTTGTTTTTCAGGTC






(SEQ ID NO.: 87)


 


B2 region
528
545
18
CTGGGAAGTCAAGCAAGA






(SEQ ID NO.: 88)


 


B3 region
547
567
21
GGTGCAACAGTAAGGGATCCC


(Backward outer



(SEQ ID NO.: 91)


primer)






 


FIP-w


38
AGGTGCGGTGGCTCACGCCTA-


(Forward inner



TTTT-TCGGCCTCCCAAA


primer for wild



(SEQ ID NO.: 84)


type)






 


FIP-m


41
GGGTGCGGTGGCTCACGCCTA-


(Forward inner



TTTT-TCGGCCTCCCAAAATG


primer for 



(SEQ ID NO.: 85)


mutant type)






 


BIP-w


46
TGGCCAATGGTTGTTTTTCAGGTC-


(Backward inner



TTTT-CTGGGAAGTCAAGCAAGA


primer for wild



(SEQ ID NO.: 89)


type)






 


BIP-m


46
CGGCCAATGGTTGTTTTTCAGGTC-


(Backward inner



TTTT-CTGGGAAGTCAAGCAAGA


primer for 



(SEQ ID NO.: 90)


mutant type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 16. The results for electrophoresis are shown in FIG. 6B, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 16







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Example 4.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
0.8 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 6B, amplified product is clearly presented in the experimental group using primers for wild type whereas there is no amplified product presented in the experimental group using primers for mutant type. Therefore, according to the results, it is clear that the genotype of the sample is TT (wild type) for the single-nucleotide polymorphism of VKORC1-1639 (rs9923231) (T→C).
Sequencing
A polymerase chain reaction was performed on the foregoing genomic DNA sample with the forward outer primer (SEQ ID NO.: 86) and the backward outer primer (SEQ ID NO.: 91) to obtain an amplified product of F3 region to B3 region that contains the position of the single-nucleotide polymorphism, VKORC1-1639 (rs9923231). Then the products were sequenced (by using SEQ ID NO.: 86 as a primer) to determine the genotype of the single-nucleotide polymorphism, VKORC1-1639 (rs9923231). The result for the sequencing is shown in FIG. 6C, wherein the nucleotide marked by a box in the sequence shown at the top of the figure is the single-nucleotide polymorphism site of VKORC1-1639 (rs9923231). According to FIG. 6C, it is known that the genotype of the sample is TT for the single-nucleotide polymorphism of VKORC1-1639 (rs9923231) (T→C).
According to the results for Comparative Example 4 and Example 4, it is known that, as compared with primers of the comparative example, by using the primers of the kit of the present disclosure, the polymorphism of VKORC1-1639 (rs9923231) of the VKORC1 gene can be quickly and accurately detected.
E. Determination for Polymorphism of VKORC1 1173 (rs9934438) of VKORC1
Comparative Example 5
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
Through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, six regions were selected from a primer design region (SEQ ID NO.: 93) which was set for VKORC1 1173 (rs9934438) of the VKORC1 gene, and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 17.







TABLE 17







Six selected regions and designed primers for VKORC1 1173


(rs9934438) of the VKORC1 gene through a website version of the design 


software package PrimerExplorer V4, provided by Eiken Genome, Japan.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
305
322
18
TGTCAACCCAGTGCCTTG


(Forward outer



(SEQ ID NO.: 98)


primer)






 


F2 region
329
348
20
GCCCGAGAAAGGTGATTTCC






(SEQ ID NO.: 95)


 


F1c region
378
398
21
TGGACTAGGATGGGAGGTCGG






(SEQ ID NO.: 94)


 


B1c region
402
422
21
GTCGATGATCTCCTGGCACCG






(SEQ ID NO.: 99)


 


B2 region
454
472
19
AGGGGAGGATAGGGTCAGT






(SEQ ID NO.: 100)


 


B3 region
492
509
18
CCTACGGAGTAGCCACGT


(Backward outer 



(SEQ ID NO.: 103)


primer)






 


FIP-w


45
CTCT-


(Forward inner



TGGACTAGGATGGGAGGTCGG-


primer for wild



GCCCGAGAAAGGTGATTTCC


type)



(SEQ ID NO.: 96)


 


FIP-m


45
CCCT-


(Forward inner



TGGACTAGGATGGGAGGTCGG-


primer for mutant



GCCCGAGAAAGGTGATTTCC


type)



(SEQ ID NO.: 97)


 


BIP-w


42
GA-


(Backward inner



GTCGATGATCTCCTGGCACCG-


primer for wild



AGGGGAGGATAGGGTCAGT


type)



(SEQ ID NO.: 101)


 


BIP-m


42
GG-


(Backward inner



GTCGATGATCTCCTGGCACCG-


primer for mutant



AGGGGAGGATAGGGTCAGT


type)



(SEQ ID NO.: 102)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 18. The results for electrophoresis are shown in FIG. 7A, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 18







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Comparative Example 5.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
61° C., 2 hours;
100 V,


(pH 8.8)

80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
1.6 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 7A, there is no amplified product present in the experimental group using primers for wild type or the experimental group using primers for mutant type. Therefore, according to the above mentioned, it is clear that the primers designed for VKORC1 1173 (rs9934438) of the VKORC1 gene through a website version of the design software package PrimerExplorer V4, provided by Eiken Genome of Japan, cannot be used for detecting the polymorphism of VKORC1 1173 (rs9934438) of the VKORC1 gene.
Example 5
(1) Sample Obtainment
The sample source which was used was identical to that of Comparative Example 1. DNA extraction steps were the same as those used in Comparative Example 1.
(2) Primer Design
In this example, six regions were selected from a primer design region (SEQ ID NO.: 93) which was set for VKORC1 1173 (rs9934438) of the VKORC1 gene, and primers were designed according to the six regions. The locations of the selected six regions and the designed primers are shown in Table 19.







TABLE 19







Six selected regions and designed primers for VKORC1 1173


(rs9934438) of the VKORC1 gene of Example 5.











Selected regions






and designed
5′
3′




primers
position
position
Length
Sequence





F3 region
293
312
20
CCAGGTTAGGACTGTCAACC


(Forward outer



(SEQ ID NO.: 108)


primer)






 


F2 region
329
348
20
GCCCGAGAAAGGTGATTTCC






(SEQ ID NO.: 105)


 


F1c region
378
398
21
TGGACTAGGATGGGAGGTCGG






(SEQ ID NO.: 104)


 


B1c region
402
422
21
GTCGATGATCTCCTGGCACCG






(SEQ ID NO.: 109)


 


B2 region
456
474
19
AGAGGGGAGGATAGGGTCA






(SEQ ID NO.: 110)


 


B3 region
492
509
18
CCTACGGAGTAGCCACGT


(Backward outer



(SEQ ID NO.: 113)


primer)






 


FIP-w


45
CTCT-


(Forward inner



TGGACTAGGATGGGAGGTCGG-


primer for wild



GCCCGAGAAAGGTGATTTCC


type)



(SEQ ID NO.: 106)


 


FIP-m


45
CCCT-


(Forward inner



TGGACTAGGATGGGAGGTCGG-


primer for 



GCCCGAGAAAGGTGATTTCC


mutant type)



(SEQ ID NO.: 107)


 


BIP-w


42
GA-GTCGATGATCTCCTGGCACCG-


(Backward inner



AGAGGGGAGGATAGGGTCA


primer for wild



(SEQ ID NO.: 111)


type)






 


BIP-m


42
GG-GTCGATGATCTCCTGGCACCG-


(Backward inner



AGAGGGGAGGATAGGGTCA


primer for 



(SEQ ID NO.: 112)


mutant type)









(3) Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplifications were performed on the sample through primers for wild type and primers for mutant type, respectively. Then the products were analyzed through electrophoresis. The operating conditions for the loop-mediated isothermal amplifications and electrophoresis conditions are shown in Table 20. The results for electrophoresis are shown in FIG. 7B, wherein the lanes indicated by W, Wn, M and Mn respectively show the results for an experimental group using primers for wild type, a negative control group using primers for wild type, an experimental group using primers for mutant type and a negative control group using primers for mutant type.







TABLE 20







Operating conditions of the loop-mediated isothermal amplifications


and electrophoresis conditions for Example 5.









Constituents of loop-mediated
Reaction
Electrophoresis


isothermal amplification
conditions
conditions













Tris-HCl
20 mM
55° C., 2 hours;
100 V,



(pH 8.8)
80° C.,
30 minutes in


Betaine
0.8M
5 minutes
2% agarose/1X


dNTPs
1.4 mM

TAE buffer


KCl
10 mM


MgSO4
8 mM


(NH4)2SO4
10 mM


Outer primer
0.2 μM


Inner primer
1.6 μM


Tween 20
0.1%


Bst polymerase
8U (8 U/μL, 1 μL)


DNA
20 ng (10 ng/μL,



2 μL)


Total volume
12.5 μL









According to the results shown in FIG. 7B, amplified product is clearly presented in the experimental group using primers for wild type whereas there is no amplified product presented in the experimental group using primers for mutant type. Therefore, according to the results, it is clear that the genotype of the sample is AA (wild type) for the single-nucleotide polymorphism of VKORC1 1173 (rs9934438) of VKORC1 (A→G).
Sequencing
A polymerase chain reaction was performed on the foregoing genomic DNA sample with the forward outer primer (SEQ ID NO.: 108) and the backward outer primer (SEQ ID NO.: 113) to obtain an amplified product of F3 region to B3 region that contains the position of the single-nucleotide polymorphism, VKORC1 1173 (rs9934438). Then the products were sequenced (by using SEQ ID NO.: 108 as a primer) to determine the genotype of the single-nucleotide polymorphism, VKORC1 1173 (rs9934438). The result for the sequencing is shown in FIG. 7C, wherein the nucleotide marked by a box in the sequence shown at the top of the figure is the single-nucleotide polymorphism site of VKORC1 1173 (rs9934438). According to FIG. 7C, it is known that the genotype of the sample is AA for the single-nucleotide polymorphism of VKORC1 1173 (rs9934438) of VKORC1 (A→G).
According to the results for Comparative Example 5 and Example 5, it is known that, as compared with primers of the comparative example, by using the primers of the kit of the present disclosure, the polymorphism of VKORC1 1173 (rs9934438) of the VKORC1 gene can be quickly and accurately detected.
It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents.

## Claims

1. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer, a second primer, at least one third primer; and a fourth primer, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 16 and/or a primer whose sequence is SEQ ID NO: 17, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 21 and/or a primer whose sequence is SEQ ID NO: 22, and the fourth primer is a primer whose sequence is SEQ ID NO: 23.

2. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 1, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 16, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 21, and the fourth primer is a primer whose sequence is SEQ ID NO: 23.

3. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 1, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 17, the second primer is a primer whose sequence is SEQ ID NO: 18, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 22, and the fourth primer is a primer whose sequence is SEQ ID NO: 23.

4. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 1, further comprising:
a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity; and
nucleotide substrates.

5. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer, a second primer, at least one third primer; and a fourth primer, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 38 and/or a primer whose sequence is SEQ ID NO: 39, the second primer is a primer whose sequence is SEQ ID NO: 40, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 43 and/or a primer whose sequence is SEQ ID NO: 44, and the fourth primer is a primer whose sequence is SEQ ID NO: 45.

6. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 5, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 38, the second primer is a primer whose sequence is SEQ ID NO: 40, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 43, and the fourth primer is a primer whose sequence is SEQ ID NO: 45.

7. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 5, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 39, the second primer is a primer whose sequence is SEQ ID NO: 40, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 44, and the fourth primer is a primer whose sequence is SEQ ID NO: 45.

8. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 5, further comprising:
a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity; and
nucleotide substrates.

9. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer, a second primer, at least one third primer; and a fourth primer, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 61 and/or a primer whose sequence is SEQ ID NO: 62, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 66 and/or a primer whose sequence is SEQ ID NO: 67, and the fourth primer is a primer whose sequence is SEQ ID NO: 68.

10. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 9, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 61, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 66, and the fourth primer is a primer whose sequence is SEQ ID NO: 68.

11. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 9, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 62, the second primer is a primer whose sequence is SEQ ID NO: 63, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 67, and the fourth primer is a primer whose sequence is SEQ ID NO: 68.

12. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 9, further comprising:
a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity; and
nucleotide substrates.

13. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer, a second primer, at least one third primer; and a fourth primer, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 84 and/or a primer whose sequence is SEQ ID NO: 85, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 89 and/or a primer whose sequence is SEQ ID NO: 90, and the fourth primer is a primer whose sequence is SEQ ID NO: 91.

14. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 13, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 84, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 89, and the fourth primer is a primer whose sequence is SEQ ID NO: 91.

15. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 13, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 85, the second primer is a primer whose sequence is SEQ ID NO: 86, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 90, and the fourth primer is a primer whose sequence is SEQ ID NO: 91.

16. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 13, further comprising:
a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity; and
nucleotide substrates.

17. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence, comprising: at least one first primer, a second primer, at least one third primer; and a fourth primer, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 106 and/or a primer whose sequence is SEQ ID NO: 107, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 111 and/or a primer whose sequence is SEQ ID NO: 112, and the fourth primer is a primer whose sequence is SEQ ID NO: 113.

18. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 17, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 106, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 111, and the fourth primer is a primer whose sequence is SEQ ID NO: 113.

19. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 17, wherein the at least one first primer comprises a primer whose sequence is SEQ ID NO: 107, the second primer is a primer whose sequence is SEQ ID NO: 108, the at least one third primer comprises a primer whose sequence is SEQ ID NO: 112, and the fourth primer is a primer whose sequence is SEQ ID NO: 113.

20. The kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence as claimed in claim 17, further comprising:
a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement activity; and
nucleotide substrates.