Test liquid with double probes of bridge type probe and lock type probe and detection method of test liquid

Abstract

The invention relates to the technical field of biology, in particular to test liquid with a bridging probe and a locking probe and a detection method of the test liquid. The invention aims to solve the problems that the existing gene detection efficiency is low, the detection cost is high, and the condition that n nucleotide sequences exist in a DNA sequence and are not quantitatively repeated cannot be accurately measured. The invention relates to a test solution with double probes of a bridging probe and a locking probe and a detection method of the test solution. The method comprises the following steps: sequentially connecting and combining bridging probes end to end and covering a gene mutation DNA region to form a bridging type cluster, connecting the bridging type probe cluster and a lock-type probe end to end to form annular DNA, combining a universal primer with a middle region of the lock-type probe, carrying out rolling circle type DNA amplification at room temperature, adding a conventional SYBR green fluorescent dye and detecting an SYBR signal, and finally, recording data through a fluorometer, judging whether the patient has mutation or not according to the existence of fluorescence, or judging whether a plurality of repeated mutations exist or not according to the intensity of the fluorescence. According to the test, the result can be judged within 30-45 minutes, and the efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a test solution with a dual-probe of a bridging probe and a padlock probe and a detection method thereof. Background Art

[0002] With the development of gene sequencing technology, the demand for DNA detection technology has also been continuously increasing. Currently, the common detection technologies in the market are polymerase chain reaction (PCR) and rolling circle amplification reaction (RCA), but both have great disadvantages. First of all, PCR operation is cumbersome. In the reaction process, specific DNA fragments are synthesized by in vitro enzymatic synthesis, which consists of a cycle of high-temperature denaturation, low-temperature annealing, and appropriate-temperature extension and then this cycle is recycled. However, in the experiment, non-specific amplification is likely to occur (for example, primer dimers, that is, the primers generate hairpin structures or other secondary structures during annealing, or the primers non-specifically bind to certain positions of the template, which will also produce non-specific amplification and affect the final result). RCA is an amplification method with much higher efficiency than PCR, but it itself is only rolling circle replication and cannot be used as a detection method. Although the RCA detection method with a padlock probe can be used for detection, it cannot detect the case of indefinite repetition of n nucleotide sequences in a DNA sequence. Therefore, we have designed a test solution with a bridging probe and a padlock probe, which not only solves the problem of low efficiency of PCR but also makes up for the deficiencies of RCA detection. Summary of the Invention

[0003] In order to detect special DNA sequences faster and more efficiently, the present invention provides a test solution with a bridging probe and a padlock probe, including:

[0004] Padlock probe DNA and bridging probe DNA, and the padlock probe DNA includes:

[0005] Padlock probe DNA target DNA recognition part: at the 3' and 5' ends of the padlock probe, used to pair with the upstream and downstream DNAs of the target mutant DNA sample region;

[0006] Padlock probe DNA universal primer recognition part: binding to the universal linear primer to facilitate amplification;

[0007] Padlock probe DNA randomly generated part: used to increase the basic length of the whole primer and stabilize primer pairing.

[0008] Bridging probe DNA: used to specifically match the region of mutant DNA.

[0009] Further, the test solution further includes: T4 DNA ligase, ligation buffer (10×), Phi29 buffer (10×), ddH2O, paired target DNA primers, Phi29 DNA polymerase, ATP, dNTPs, green fluorescent dye SYBR.

[0010] Further, there is one or more of the bridging probe DNA, and the linear primer is a primer with paired nucleotide repeats.

[0011] A detection method based on a test solution having a bridging probe and a padlock probe includes the following steps:

[0012] Sequentially add phosphorylated padlock probe DNA, bridging probe DNA, target DNA sample, T4 DNA ligase, ligation buffer (10×), Phi29 buffer (10×), ddH2O, paired target DNA primers, Phi29 DNA polymerase, ATP, dNTPs, green fluorescent dye SYBR, and let stand in a 30-degree incubator for 10 - 15 minutes while detecting SYBR signals and recording data with a fluorometer;

[0013] Further, the operating steps based on a 100-μL system are as follows:

[0014] Add 2 μL of padlock probe DNA, 10 μL of bridging probe DNA, 10 μL of ligation buffer (10×), 10 μL of Phi29 buffer (10×), 5 μL of 0.1 mM ATP, 1 μg - 5 μg of target DNA sample, 2 μL of T4 DNA ligase, 2 μL of Phi29 DNA polymerase, 2 μL of 100 mM dNTPs, 2 μL of 10 mM SYBR, 5 μL of 100 mM paired target DNA primers, and make up to 100 μL with ddH2O, and let stand in a 30-degree incubator for 10 - 15 minutes.

[0015] Principle: Use T4 polynucleotide kinase (T4 PNK) to transfer a phosphate group on ATP to the 5'-hydroxyl group of the DNA molecule, making the primer DNA with hydroxyl groups at both ends become one end with a hydroxyl group and the other end with a phosphate group; use T4 ligase to connect the two ends of the primer combined with the bacterial single-stranded DNA part, making the primer form a loop as an amplification template; free nucleotides specifically bind to and extend with the circularized primer under the action of Phi29 DNA polymerase, greatly amplifying the signal and making the result visual in subsequent operations.

[0016] For the reaction steps of implementing the fluorescent dye are as follows:

[0017] (1) Obtain the target gene to be detected or the generated DNA sequence

[0018] 2. Perform the same steps as for the bridged padlock probe.

[0019] 3. Add 0.2 μL of the green fluorescent dye to every 20 μL of the reaction.

[0020] 4. Detect the SYBR signal and record the data using a fluorometer.

[0021] Furthermore, the padlock probe DNA sequence is: The paired target DNA sample sequence has 50 - 100 CAG repeats, and the sequences upstream and downstream of the paired target DNA sample repeat region are specific DNA sequences.

[0022] Furthermore, when the target DNA sample is the DMPK gene, its sequence is: -(CTG)n, where when n ≥ 50, the paired target DNA primer sequence is -(GAC)n-, where n is one of the numbers 1, 2, 3, 4, 5, 6, 7, 8.

[0023] Furthermore, when the target DNA sample has a dynamic mutation, its sequence is the gene exon (CAG)n, where when n ≥ 20, the paired target DNA primer sequence is -(GTC)n-, where n is one of the numbers 1, 2, 3, 4, 5, 6, 7, 8.

[0024] Further, when the targeted DNA sample is a single nucleotide polymorphism or gene mutation, the paired targeted DNA primer sequence (3'->5') is one of the following: -(CTGCACGCGCCGG)-, -(GTCTTCACGGACC)-, -(AAAGGGTCCTTGG)-, -(GGGGACTTAGGTC)-, -(AGGACTACGGGTG)-, -(CCCGGATGCCGAC)-, -(GCGGGGCCTCGGT)-, -(TCAAAGCACTACG)-, -(ACTAAAAGATGTA)-, -(AATACTCCTAAAG)-, -(TGAAAAGACTGAC)-, -(AAAAAAATGACCA)-, -(TCCACCGACTTCT)-, -(TCCACCAACTTCT)-, -(GTATTATAGTTCC)-, -(TACTAGCGCACCT)-, -(TCCTGTTCACGGC)-, -(GCATAAAACACAG)-, -(TGAGACGGGTGCA)-, -(TGCTGGGCTCCGG)-, -(TGCTGGGATCCGG)-, -(TGCTGGGTTCCGG)-, -(TGCTGGGGTCCGG)-, -(TCATCATGCAGCT)-, -(TTGGGCGAGCCAA)-, -(TTGGGCGTGCCAA)-, -(TTGGGCGCGCCAA)-, -(TTGGGCGGGCCAA)-, -(TTGGGAGAGCCAA)-, -(TTGGGAGGGCCAA)-, -(AACTGCAAGGTGC)-, -(AACTGCAGGGTGC)-, -(CTACAGAGAAATC)-, -(CTACAGAAAAATC).

[0025] The advantages of this patent lie in its high sensitivity and the property of isothermal amplification. The padlock probe can sensitively detect the matching DNA sequences. The ligation reaction and rolling circle amplification reaction can continuously perform DNA amplification and extension at the same temperature without changing the temperature. Both T4 ligase and phi29 DNA polymerase can work at room temperature, meaning that the reaction can be carried out at room temperature. The reaction results can be quantified by electrophoresis to determine the number of target DNA templates. By adding the targeted DNA sample, the situation of an indefinite number of repeats of n nucleotide sequences in a DNA sequence can be detected. The probe and primer can stably pair with the DNA sequence. Since the required steps can be carried out simultaneously, with the template set, the test can determine the result within 10 - 15 minutes. Since the working environment of the required enzymes is around 30 degrees Celsius, the whole process can be carried out at 30 degrees Celsius or even at room temperature without adjusting the temperature. Thus, it can better meet the needs of more industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 FIG. for the ligation reaction and rolling circle amplification reaction of the padlock probe in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0027] Figure 2 FIG. for the circular rolling circle reaction with a probe length of 162 bases in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0028] Figure 3 FIG. for the circular rolling circle reaction with a probe length of 322 bases in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0029] Figure 4 FIG. for the rolling circle diagram of real-time quantitative detection with a fluorescent dye in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0030] Figure 5 FIG. for the test result diagram of Propionibacterium acnes DNA identification in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0031] Figure 6 FIG. for the detection diagram of non-sequenced DNA with a bridging probe in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention;

[0032] Figure 7 FIG. for the DNA experimental test diagram of patients with myotonic dystrophy in the experiment of the test solution with double probes of bridging probe and padlock probe and its detection method of the present invention; Detailed implementation mode

[0033] Next, the present invention will be described in detail through embodiments and accompanying drawings:

[0034] The present invention provides a test solution with a bridging probe and a padlock probe and its detection method. The detection object is a specific nucleotide sequence. This version is a new type of amplification method, which is characterized in that the ligation reaction and the rolling circle amplification reaction are carried out simultaneously, greatly saving time costs.

[0035] As Figure 1 shown: The padlock probe ligation and rolling circle amplification reactions are divided into two steps. The first step is the ligation reaction, which is the content of Figure a. Under the guidance of the bacterial DNA template, the phosphorylated padlock probe DNA is complementary paired with the bacterial DNA. The long and flexible single-stranded padlock probe will bend, reducing the distance between the two ends of the padlock probe DNA to a state where ligation can occur. T4 DNA ligase will carry out the ligation reaction on the padlock probe DNA at an efficient temperature of 30 degrees Celsius, connecting the two originally separated ends, making the padlock probe DNA into circular DNA. The second step is the rolling circle amplification reaction, which is the content of Figure b. First, the paired target DNA primer will be complementary paired with the circularized DNA. At a suitable temperature of 30 degrees, phi29 DNA polymerase can amplify and extend the primer length. The amplification method can be single-point or multi-point amplification. The basic single-point amplification is shown in the figure below Figure b. The effect of multi-point amplification is shown in the right part of Figure b. Finally, the circularized DNA will continuously coil and eventually exceed the length of the original padlock probe DNA. It should be noted that the suitable working temperatures for both the ligation reaction and the rolling circle amplification reaction are around 30 degrees. This means that the first and second steps can be carried out simultaneously in the same reaction solution.

[0036] The third step is the identification method of the amplified DNA. The basic version of the circularized rolling circle amplification reaction can be judged whether the amplification is successful by electrophoresis. For the amplification result of an unsuccessful reaction, the electrophoresis band will be shown at a low position, that is, the original length of the padlock probe. The successful amplification result will be shown at a high position, that is, at a position greater than the original length of the padlock probe. The length of the probe can be changed according to requirements, and the amplification results of probes with different original lengths will be at different positions.

[0037] For the amplification result of the bridging probe DNA, theoretically, the length of the probe can be designed arbitrarily. As Figure 2In this experiment, we selected the probe length to be 162 bases. In the experiment with 162 bases, the rolling circle reaction could be successfully carried out and detected. By comparing with the marker in lane 1 of the electrophoresis result graph, the black band at the bottom of lane 2 corresponded to the size of 162 base pairs. This was the size of the original probe without reaction. The black band slightly above lane 2 corresponded to a size greater than 162 base pairs, which was the result of successful amplification. Secondly, this experimental result could also prove that the rolling circle amplification reaction could be used for quantitative analysis of the target DNA content in the sample. For lanes 3 to 14, the target DNA content in the sample decreased continuously, and the results of successful reactions also gradually decreased. According to the quantitative method of amplification results established by the negative and positive control groups, the rolling circle amplification could determine the target DNA content in the sample. The probe with a size of 162 bases was practical. The size of the base pairs to be synthesized was only 162 bases, but it also met the conditions for stable amplification.

[0038] As Figure 3 shown, in another experiment, we used a probe with a size of 322 bases. The results of lanes 1 and 3 were the positive control groups. Only the specific probe with a size of 322 was added to the reaction samples. The result of the reaction was that all the probes were successfully amplified and migrated to a position greater than 322 in size. This proved that the probe with a size of 322 could be successfully amplified and was more stable. Secondly, an equal amount of specific probe and a non-specific probe were added to the reaction sample in lane 2. An equal amount of specific probe and four non-specific probes were added to the reaction sample in lane 4. The results showed that even under the interference of non-specific probes, the specific probe could still successfully react.

[0039] As Figure 4 shown, in addition to presenting the reaction results by electrophoresis, fluorescent dyes could also be used for real-time quantitative reaction results. We call this method Worm qL-RCA.

[0040] Worm qL-RCA is applied to RCA based on the principle of qPCR. Initially, bacteria cannot be detected because the fluorescent dye does not bind to double-stranded DNA. During DNA amplification, double-stranded DNA is formed, and at the same time, the green fluorescent dye can bind to the hydrogen bonds between double-stranded DNA and emit fluorescence. Then, the fluorescence intensity above the background signal level is measured and used to quantify the number of newly generated double-stranded DNA. Under rolling circle amplification, the more DNA is amplified and the longer the base pair length, the more double-stranded DNA the green fluorescent dye can bind to. Subsequently, we can quantify rolling circle amplification at any time based on the fluorescence intensity. When the fluorescence intensity exceeds a threshold, we can conclude that the reaction is successful. Through qL-RCA, the detection of various genes can be achieved, including the detection of DNA related to bacteria and human diseases.

[0041] In the experimental demonstration, we take the detection of the generated Propionibacterium acnes as an example. As Figure 5 shown, the cycle threshold (CT) of Propionibacterium acnes DNA amplification is 17×20 seconds (340 seconds), which is the standard value for comparing the initial content of Propionibacterium acnes DNA in the test sample. In addition, the CT value is inversely proportional to the DNA sample amount, that is, the lower the CT value, the higher the DNA sample amount. At the same time, we set the standard that a CT value lower than 30×20 seconds (600 seconds) indicates a high content of the target bacteria, and higher than 30×20 seconds (600 seconds) indicates a low content of the target bacteria. According to these standards, the CT value of sample 2 (shown as the yellow dot) is 23×20 seconds (460 seconds), followed by 26×20 seconds (520 seconds) of sample 3 (shown as the light blue dot). The high target content of the detected samples proves the accuracy of the experiment. In comparison with these two sets of data, the CT value of sample 1 (shown as the gray dot) is 30×20 seconds (600 seconds), higher than the set standard and sufficient to be diagnosed as a high concentration. Finally, no CT value is shown on the random DNA (shown as the orange dot), indicating the success of the experiment. In short, real-time qL-RCA has successfully opened up a new world for us to identify test results, making up for the limitation of the long time-consuming gel electrophoresis.

[0042] For the detection of indefinite sequences, the above method cannot effectively detect. Then a higher version of the bridging probe is needed to solve it.

[0043] For some diseases, the DNA sequences of patients may contain a relatively large and variable number of trinucleotide repeats. Myotonic Dystrophy type 1 (DM1) is one such disease. Myotonic Dystrophy (DM) is a dominant hereditary multi-system disease. The main symptoms of this disease are muscle weakness, myotonia, and damage to other multiple organs, such as the nervous system, cardiovascular system, endocrine system, etc. The main cause of DM1 is the abnormal amplification of the CTG trinucleotide sequence in the non-coding region of the DMPK gene on chromosome 19q13.2. In DM1 patients, the number of CTG amplifications can exceed 100 times, while in the normal population, the number of CTG amplifications is only 5 - 40 times. Due to the slow progression of the disease, the current methods for diagnosing Myotonic Dystrophy (type 1) are relatively inefficient. It takes about 5 years from the first appearance of symptoms to a confirmed diagnosis. The diagnosis of DM generally requires the use of electromyography, biopsy, clinical symptoms, and genetic testing. For genetic testing, it is very difficult to achieve specific primer pairing and stable amplification with ordinary PCR and triple-prime PCR targeting trinucleotide repeats. These detection methods also lack speed. Additionally, because padlock probes need to pair with the patient's DNA for detection, the variable number of trinucleotide repeats makes the pairing and detection of L-RCA very difficult. We designed a bridging probe for the detection of this disease.

[0044] As Figure 6 shown: In Figure (a): The padlock probe first pairs with the left and right sides of the trinucleotide repeat in the patient's DNA. At the same time, a primer designed to pair with four trinucleotide repeats (12 nucleotides) pairs with the sequence containing the trinucleotide repeat in the middle. Subsequently, as shown in Figure (b), T4 DNA ligase can link the padlock probe and the small primer of the variably paired trinucleotide sequence into a loop. If the trinucleotide repeat in the patient's DNA is not a multiple of four and cannot be perfectly paired by a certain 12-nucleotide primer, T4 DNA ligase actually also has the ability to correct and trim, enabling the primer of the trinucleotide repeat to be perfectly paired with the DNA sequence. Finally, in the same way as L-RCA, the bridging probe can achieve amplification and detection. Additionally, SYBR Green can also be added to the detection method of the bridging probe to achieve Worm qL-RCA. Since more trinucleotide repeats result in a longer total DNA sequence, the number of trinucleotide repeats in the detected DNA can be determined by comparing it with synthetically prepared DNA (30 CTG repeats, 50 CTG repeats, 100 CTG repeats) in electrophoresis. Secondly, due to the slower rate of ligation, amplification, and fluorescence emission for sequences with more trinucleotide repeats and longer lengths, the number of trinucleotide repeats in the detected DNA can also be used as a control in Worm qL-RCA to determine whether the disease is confirmed.

[0045] These are two primers used for detecting myotonic dystrophy. The first is a padlock probe sequence containing 300 nucleotides. To ensure the effectiveness of the probe, during the artificial synthesis of this probe, a double-stranded DNA is first synthesized, with a hydroxyl group at the 5' end of one DNA strand and the 5' end of the other strand phosphorylated. Lambda exonuclease can catalyze the cleavage of the DNA strand with a phosphorylated 5' end starting from the phosphorylated 5' end. BS Worm DNA-300 can then form a single-stranded probe. The second is a primer sequence containing 12 nucleotides targeting trinucleotide repeats. In fact, it is feasible for the length of this primer to be a multiple of three. The 12-nucleotide length is the most stable primer designed by us.

[0046] As Figure 7 shown, in the experiment of testing the detection of myotonic dystrophy, the Worm qL-RCA detection method was successful. In the figure, the trend marked in blue is the artificial synthetic DNA containing 100 trinucleotide repeats used to simulate patient DNA detected by Worm qL-RCA, the orange is the artificial synthetic DNA containing 50 trinucleotide repeats detected by Worm qL-RCA, and the gray is the random DNA detected by Worm qL-RCA. The fluorescence values of the trends detecting the simulated patient DNA are all greater than the set threshold. In contrast, the final fluorescence value of the randomly generated DNA did not exceed the threshold, so the detection feasibility of the padlock probe can be seen. The trend of the simulated patient DNA containing 50 trinucleotide repeats can also be seen to have a faster amplification and fluorescence emission rate than the trend of the simulated patient DNA containing 100 trinucleotide repeats. This phenomenon proves the previously set hypothesis that fewer trinucleotide repeats will lead to faster amplification and fluorescence emission..

[0047] For general DNA detection, the DNA sequence to be detected is a definite nucleotide sequence. This sequence can be detected by basic PCR or RCA. For this situation, a padlock probe without a primer for paired target DNA can solve the problem.

[0048] For the situation where there are three or n nucleotide sequence indefinite repeats in a DNA sequence, such as a sequence with an indefinite number of ctg repeats, a padlock probe containing a primer for paired target DNA can solve the problem.

[0049] The above are only the preferred embodiments of the present invention and are not used to limit the present invention; for those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A test solution with dual probes of a bridging probe and a padlock probe, characterized in that, The test solution includes: padlock probe DNA and bridging probe DNA. The padlock probe DNA includes: The target DNA recognition part of the padlock probe DNA: at the 3' and 5' ends of the padlock probe, which is used to pair with the upstream and downstream DNAs of the targeted mutant DNA region; The universal primer recognition part of the padlock probe DNA: binds to the universal linear primer for easy recognition; The randomly generated part of the padlock probe DNA: used to increase the basic length of the whole primer and stabilize primer pairing; The bridging probe DNA: used to specifically match the region of the mutant DNA and serve as a DNA module to achieve docking with the padlock probe to form circular DNA.

2. The test solution with a double-probe of a bridging probe and a locking probe according to claim 1, characterized in that, The test solution also includes: T4 DNA ligase, ligation buffer (10×), Phi29 buffer (10×), ddH2O, paired target universal DNA primer, Phi29 DNA polymerase, ATP, dNTPs, green fluorescent dye SYBR.

3. The test solution with double probes of bridging probe and padlock probe according to claim 1, wherein the bridging probe DNA has one or more, and the universal linear primer is a primer with paired nucleotide repeats.

4. A detection method for a test solution based on a dual-probe with a bridging probe and a padlock probe, characterized in that, It includes the following detection steps: Sequentially add phosphorylated padlock probe DNA, bridging probe DNA, target DNA sample, T4 DNA ligase, ligation buffer (10×), Phi29 buffer (10×), ddH2O, paired universal DNA primer, Phi29 DNA polymerase, ATP, dNTPs, green fluorescent dye SYBR, and incubate in a 30-degree constant temperature incubator for 30 - 45 minutes while detecting the SYBR signal and recording data with a fluorometer.

5. The detection method based on the test solution with a dual-probe of a bridging probe DNA and a padlock probe according to claim 4, wherein, The detection step based on a 100 μL system is: add 2 μL of padlock probe DNA, 10 μL of bridging probe DNA, 10 μL of ligation buffer (10×), 10 μL of Phi29 buffer (10×), 5 μL of 0.1 mM ATP, 1 μg - 5 μg of target DNA sample, 2 μL of T4 DNA ligase, 2 μL of Phi29 DNA polymerase, 2 μL of 100 mM dNTPs, 2 μL of 10 mM SYBR, 5 μL of 100 mM paired universal DNA primer, and make up to 100 μL with ddH2O, then incubate in a 30-degree constant temperature incubator for 10 - 15 minutes.

6. The detection method of the test solution based on the double probes of the bridging probe and the locking probe according to claim 5, characterized in that, The lock-type probe DNA sequence is as follows: The binding region sequence containing the paired universal DNA primer, and the paired target DNA sample containing 50-100 CAG repeat regions upstream and downstream sequences are specific DNA sequences.

7. The detection method of the test solution based on the double probes of the bridging probe and the locking probe according to claim 6, characterized in that, When the target DNA sample is the DMPK gene with the sequence -(CTG)n, where n ≥ 50 times, the sequence of the paired target DNA primer is -(GAC)n-, where n is one of the numbers 1, 2, 3, 4, 5, 6, 7, 8, and the union forms a bridging probe library.

8. The detection method of the test solution based on the double probes of the bridge probe and the lock probe according to claim 6, characterized in that, When the target DNA sample is a dynamic mutation with the sequence (CAG)n in the gene exon, where n ≥ 20 times, the sequence of the paired target DNA primer is -(GTC)n-, where n is one of the numbers 1, 2, 3, 4, 5, 6, 7, 8, and the union forms a bridging probe library.

9. The detection method of the test solution based on the double probes of the bridging probe and the locking probe according to claim 6, wherein, When the targeted DNA sample is a single nucleotide polymorphism or gene mutation, the paired targeted DNA primer sequence (3'->5') is one of the following: -(CTGCACGCGCCGG)-, -(GTCTTCACGGACC)-, -(AAAGGGTCCTTGG)-, -(GGGGACTTAGGTC)-, -(AGGACTACGGGTG)-, -(CCCGGATGCCGAC)-, -(GCGGGGCCTCGGT)-, -(TCAAAGCACTACG)-, -(ACTAAAAGATGTA)-, -(AATACTCCTAAAG)-, -(TGAAAAGACTGAC)-, -(AAAAAAATGACCA)-, -(TCCACCGACTTCT)-, -(TCCACCAACTTCT)-, -(GTATTATAGTTCC)-, -(TACTAGCGCACCT)-, -(TCCTGTTCACGGC)-, -(GCATAAAACACAG)-, -(TGAGACGGGTGCA)-, -(TGCTGGGCTCCGG)-, -(TGCTGGGATCCGG)-, -(TGCTGGGTTCCGG)-, -(TGCTGGGGTCCGG)-, -(TCATCATGCAGCT)-, -(TTGGGCGAGCCAA)-, -(TTGGGCGTGCCAA)-, -(TTGGGCGCGCCAA)-, -(TTGGGCGGGCCAA)-, -(TTGGGAGAGCCAA)-, -(TTGGGAGGGCCAA)-, -(AACTGCAAGGTGC)-, -(AACTGCAGGGTGC)-, -(CTACAGAGAAATC)-, -(CTACAGAAAAATC).

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