Probe, primer and kit for detecting Y chromosome copy number variation
By designing probes and primers covering 118 target sites of the Y chromosome, combined with reference gene data correction, the problems of high cost and low throughput of Y chromosome CNV variant detection in the prior art are solved, and efficient, accurate and low-cost Y chromosome copy number variant detection is achieved.
Patent Information
- Application Number
- CN202510150983.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-11
- Publication Date
- 2025-04-25
AI Technical Summary
In the prior art, CNV variant detection for Y chromosomes has problems such as high cost, low throughput, high sample DNA requirements, and false positives are prone to detection results.
A probe and primer for Y chromosome copy number variation detection was designed to cover 118 target sites for 61 genes on the Y chromosome, and data correction was carried out in combination with reference genes. The ligation reaction and PCR amplification were used to perform ligation reactions and PCR amplification. The amplified fragments were separated by capillary electrophoresis, and the copy number was calculated.
Copy number variation detection of multiple sites on the Y chromosome is realized, with high resolution and accurate measurement of sites within 6 copies. It has high detection efficiency, low cost, high accuracy of detection results, and CV is less than 10%.
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Figure CN119876380A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of molecular biology technology, and specifically relates to a probe, primers, and a kit for detecting Y chromosome copy number variation. Background Art
[0002] Recent studies have shown that genetic defects are an important cause of male infertility. The Y chromosome plays an important role in this. The acrocentromere of the Y chromosome is divided into two parts, the long arm and the short arm. For example, the long arm 1 region 1 band contains an azoospermia factor. The deletion of this gene fragment is called YCMs, which is the main cause of male spermatogenesis disorders. Studies have confirmed that 10%-15% of azoospermia and 5%-10% of severe oligospermia patients will have YCMs, and this defect will be inherited to offspring, so the detection of Y chromosome CNV variation is of great significance.
[0003] At present, the main methods for detecting CNV variation of Y chromosome are: multiplex ligation probe amplification (MLPA), real-time fluorescence quantitative PCR (RT-qPCR), microarray comparative gene hybridization (array-CGH), affymetrix CNV chip technology and second-generation gene sequencing technology. Among them, arrat-CGH, affymetrix CNV chip technology and second-generation sequencing are mainly used for copy number variation detection of the whole genome, which is accurate and efficient, but the cost is relatively high; although RT-qPCR technology is simple and easy to operate, its throughput is low and it is difficult to detect multiple fragments at the same time; MLPA is a relatively mature method for copy number detection of specific gene fragments so far, which can be achieved through the first-generation sequencing platform. However, this method also has certain limitations, such as high requirements for sample DNA, low detection throughput, and easy false positive results. Summary of the invention
[0004] The purpose of this application is to provide a probe, primers and kit for detecting Y chromosome copy number variation, so as to solve the various problems existing in the prior art for CNV variation detection of Y chromosome, and to provide a fast, comprehensive, accurate and low-cost Y chromosome copy number variation detection technology.
[0005] In order to achieve the above-mentioned object, the first aspect of the present application provides a probe and primer for detecting Y chromosome copy number variation, wherein the probe comprises:
[0006] 118 probe sets covering a total of 118 target sites on 61 genes on chromosome Y, the positions of the 118 target sites are shown in Table 1 of the specification from No. 1 to No. 118;
[0007] at least one of the probe sets covering at least one target site located on a reference gene;
[0008] Wherein, each of the probe sets comprises a 5' end probe and a 3' end probe which are at least partially complementary to the 5' end and the 3' end of the target site, respectively;
[0009] The primers include a forward primer and a reverse primer for the probe set.
[0010] In one or more embodiments, each of the 5' end probes comprises a first universal primer binding site and a first linker sequence arranged in sequence, and each of the 3' end probes comprises a second linker sequence and a second universal primer binding site arranged in sequence, wherein the first linker sequence and the second linker sequence are at least partially complementary to the 5' end and the 3' end of the target site, respectively;
[0011] The primers include:
[0012] a first universal primer set targeting the first universal primer binding site, comprising at least one first universal primer, each of the first universal primers being at least partially complementary to the first universal primer binding site of at least one of the 5' end probes;
[0013] The second universal primer set targeting the second universal primer binding site includes at least one second universal primer, and each of the second universal primers is at least partially complementary to the second universal primer binding site of at least one of the 3' end probes.
[0014] In one or more embodiments, the first connection sequences of the 118 probe sets for the 118 target sites on the Y chromosome are shown as SEQ ID NOs. 1 to 118, and the second connection sequences are shown as SEQ ID NOs. 119 to 236.
[0015] In one or more embodiments, the probe further comprises:
[0016] Five probe sets covering five target sites on chromosome X, wherein the positions of the five target sites on chromosome X are shown in Table 1 of the specification as NO.119 to NO.123, the first connection sequences of the five probe sets for chromosome X are shown in SEQ ID NOs.237 to 241, and the second connection sequences are shown in SEQ ID NOs.242 to 246.
[0017] In one or more embodiments, the probe includes 32 probe groups covering 32 target sites located on a reference gene, the positions of the 32 target sites of the reference gene are shown in NO.124 to NO.155 in Table 1 of the specification, the first connection sequence of the 32 probe groups for the reference gene is shown in SEQ ID NO.247 to 278, and the second connection sequence is shown in SEQ ID NO.279 to 310.
[0018] In one or more embodiments, the first universal primer set includes four first universal primers, the sequences of which are shown in SEQ ID NOs. 311 to 314, the 5' ends of the four first universal primers are respectively labeled with different fluorescent groups, and the second universal primer set includes two second universal primers, the sequences of which are shown in SEQ ID NOs. 315 to 316;
[0019] The first universal primer binding site of the 5' end probe of each probe set is selected from one of the sequences shown in SEQ ID NOs. 317-320, and the second universal primer binding site of the 3' end probe of each probe set is selected from one of the sequences shown in SEQ ID NOs. 321-322.
[0020] In order to achieve the above-mentioned object, the second aspect of the present application provides a kit for detecting Y chromosome copy number variation, comprising:
[0021] A first ligation premix solution includes 4×GC Solution and a probe mixture solution, wherein the probe mixture solution includes the probe described in any one of the above embodiments;
[0022] The second ligation premix includes 10× Taq buffer and Taq ligase;
[0023] Ligation reaction termination solution, including EDTA;
[0024] A PCR premix comprises 10×PCR buffer, dNTP, MgCl2, a primer mixture and Taq DNA polymerase, wherein the primer mixture comprises the primers described in any one of the above embodiments.
[0025] In order to achieve the above object, the second aspect of the present application provides a method for using the kit described in any one of the above embodiments, comprising:
[0026] The first ligation premix solution and the sample DNA are mixed, and then the second ligation premix solution is added to perform a ligation reaction so that the probe is connected to the target site;
[0027] Adding the ligation reaction termination solution;
[0028] Add PCR premix solution and perform PCR amplification;
[0029] The amplified PCR product is denatured and then subjected to capillary electrophoresis to separate the amplified fragments, obtain the information of each amplified fragment and calculate the copy number.
[0030] In one or more embodiments, the probe comprises a plurality of probe groups, each of the probe groups comprises a plurality of probe groups, the plurality of probe groups comprises at least one detection probe group for chromosome Y and at least one reference probe group for a reference gene, and the plurality of probe groups of each of the probe groups correspond to the same forward primer and reverse primer;
[0031] The step of obtaining information of each amplified fragment and calculating the copy number comprises:
[0032] The R value of the detection probe group a is obtained by dividing the fluorescence peak height value of the amplified fragment corresponding to a detection probe group a in the probe group by the fluorescence peak height value of the amplified fragment corresponding to a reference probe group b in the same probe group;
[0033] The R value of the detection probe group a in the test sample is divided by the R value of the detection probe group a in n normal male reference samples, and then multiplied by the copy number of the detection probe group a in the normal reference sample to obtain n corrected copy values of the detection probe group a for the reference probe group b, wherein the test sample is a sample of the Y chromosome copy number to be detected, and the normal male reference sample is a male sample with a known normal Y chromosome copy number;
[0034] Based on the n corrected copy number values, the relative copy number of the detection probe group a relative to the reference probe group b is calculated;
[0035] Repeat the above steps to respectively calculate the relative copy numbers of the detection probe group a for all reference probe groups in the same probe group, and obtain i relative copy numbers of the detection probe group a;
[0036] Based on the i relative copy numbers, the copy number of the amplified fragment corresponding to the detection probe group a is obtained.
[0037] In one or more embodiments, the step of calculating the relative copy number of the detection probe set a relative to the reference probe set b based on the n correction copy number values comprises:
[0038] When n is greater than 1, determining whether the coefficient of variation of the n corrected copy numbers is less than or equal to a first threshold value, and if so, taking the average value of the n corrected copy numbers as the relative copy number of the detection probe group a relative to the reference probe group b;
[0039] If the coefficient of variation of the n corrected copy values is greater than a first threshold, the corrected copy values farthest from the median of the n corrected copy values are sequentially eliminated until the coefficient of variation of the remaining m corrected copy values is less than a second threshold, wherein m is greater than or equal to 2n / 3, and the average value of the m corrected copy values is taken as the relative copy number of the detection probe group a relative to the reference probe group b;
[0040] If there are no remaining m correction copy number values whose coefficient of variation is less than the second threshold, taking the median of the n correction copy number values as the relative copy number of the detection probe group a to the reference probe group b;
[0041] The step of obtaining the copy number of the amplified fragment corresponding to the detection probe group a based on the i relative copy numbers comprises:
[0042] Determine whether the coefficient of variation of the i relative copy numbers is less than or equal to a third threshold value, and if so, take the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe group a;
[0043] If the coefficient of variation of the i relative copy numbers is greater than the third threshold, the relative copy numbers farthest from the median of the i relative copy numbers are sequentially eliminated until the coefficient of variation of the remaining j relative copy numbers is less than the fourth threshold, wherein j is greater than or equal to 2n / 3, and the average value of the j relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a;
[0044] If there are no remaining j relative copy numbers whose coefficient of variation is less than the fourth threshold, the median of the i relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a.
[0045] Different from the prior art, the beneficial effects of this application are:
[0046] The primers and probes of the present application can cover a total of 118 target sites in 61 genes on the Y chromosome, and can detect copy number variation at multiple sites on the Y chromosome at one time. They can detect both large-fragment copy number variation and tiny duplication deletions, with high resolution, and can accurately quantify sites within 6 copies.
[0047] The method of using the kit of the present application is simple, and only includes a ligation reaction and a PCR reaction. The entire process does not exceed 24 hours in total, and the detection efficiency is high;
[0048] The kit of the present application is based on the principle of high-specific connection. The detection region connection product is amplified using universal primers, and the amplification efficiency is basically the same. There are multiple reference gene sites for data correction calculation, and the average detection CV is <10%, which can ensure the accuracy of the test results.
[0049] This application completes the detection based on the first-generation sequencing platform, which is cheaper than the second-generation platform. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0051] Figure 1 It is a schematic diagram of the detection principle of this application;
[0052] Figure 2 is a chromatogram of each fragment after PCR amplification of each sample in Example 3 of the present application;
[0053] Figure 3 It is a data diagram of the copy number of each target site after PCR amplification of the test sample 1 in Example 3 of the present application;
[0054] Figure 4 This is a data diagram of the copy number of each target site after PCR amplification of the test sample 2 in Example 3 of the present application;
[0055] Figure 5 This is a data diagram of the copy number of each target site after PCR amplification of the test sample 3 in Example 3 of the present application. DETAILED DESCRIPTION
[0056] In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.
[0057] In response to the problems of high cost and low throughput in the detection of Y chromosome copy number variation in the prior art, the applicant has developed a primer probe and kit for Y chromosome copy number variation based on the multiple nucleic acid analysis method previously proposed by the applicant. The primer probe and kit proposed by the applicant can achieve rapid, high-throughput, high-accuracy, low-cost, and high-resolution detection of copy number variation at multiple sites on the Y chromosome, and is highly popular.
[0058] Specifically, the principle of the detection method of this application is first introduced, please refer to Figure 1 , Figure 1 It is a schematic diagram of the detection principle of this application.
[0059] like Figure 1 As shown, the copy number changes of nucleic acid sequences containing 2 or more nucleotides in a sample can be simultaneously detected in a single test.
[0060] A probe set is designed for each target site, and each probe set may include a 5' end probe and a 3' end probe, wherein the 5' end probe includes a first universal primer binding site X, a first filling sequence A1 and a first specific sequence L1 arranged in sequence; the 3' end probe includes a second specific sequence L2, a second filling sequence A2 and a second universal primer binding site Y arranged in sequence.
[0061] The first specific sequence L1 and the second specific sequence L2 can be complementary to the 5' end and 3' end of the target site, respectively.
[0062] The first filling sequence A1 and the second filling sequence A2 are used to increase the length difference of subsequent amplification products. By changing the length of the first filling sequence A1 and the second filling sequence A2, it is helpful to improve the multiplicity of analysis.
[0063] Each group of probes is connected to the corresponding target site respectively, and there may be no gap between the 5' end of the 5' end probe and the 3' end of the 3' end probe of each probe group, thereby obtaining a connection product.
[0064] In some embodiments, a gap may be provided between the 5' end of the 5' end probe and the 3' end of the 3' end probe of each probe group, and the gap may be closed by extending one of the probes to obtain a ligation product, which can achieve the effect of this embodiment.
[0065] Based on the forward primer complementary to the first universal primer binding site X and the reverse primer complementary to the second universal primer binding site Y, the ligation product can be amplified.
[0066] By performing electrophoretic separation on the amplified fragments, the information of each amplified fragment can be obtained, thereby calculating the copy number.
[0067] In order to facilitate copy number calculation and further increase the multiplicity of the detection method, in one embodiment, different fluorescent sequences may be labeled on the primer sequence, and a third filling sequence of different lengths may be connected to the primer sequence.
[0068] Furthermore, for all probe sets, the first universal primer binding site X of at least some of the probe sets may be the same, so that at least some of the ligation products can be amplified based on the same universal primer.
[0069] Based on the above design, the length and fluorescence of the amplified fragment are analyzed to obtain the information of the amplified fragment and compare it with the standard value to calculate the copy number.
[0070] In some embodiments, a probe group covering a reference gene can also be introduced to compare the fluorescence peak of the amplified fragment of the target gene with the fluorescence peak of the amplified fragment of the reference gene, thereby standardizing the fluorescence peak of the amplified fragment and determining the change in copy number.
[0071] Specifically, the probe group covering the reference gene can correspond to the same forward primer and reverse primer as the partial probe group covering the target site, so that correction can be performed based on the fluorescence peak of the reference gene, and finally the copy number of each target site amplified fragment can be calculated based on the copy number of the normal reference sample.
[0072] In one embodiment, all probes can be divided into multiple probe groups, each probe group can correspond to the same forward primer and reverse primer, and each probe group can include multiple probe groups, wherein the multiple probe groups can include at least one detection probe group for the target gene, and at least one reference probe group for the reference gene.
[0073] In the subsequent calculation process, the fluorescence peak values of the amplified fragments corresponding to all the detection probe groups in each probe group may be standardized based on the fluorescence peak values of the amplified fragments corresponding to the reference probe group in the probe group.
[0074] In particular, in the subsequent data processing process, a normal reference sample with a known normal copy number can be introduced, and the data of the test sample after standardization can be compared with the data of the normal reference sample to correct the data of the test sample to ensure data accuracy.
[0075] Based on the primer and probe combination designed above, a kit for detecting copy number variation at each site of the Y chromosome can be constructed.
[0076] In one embodiment, the kit may include a first ligation premix solution, a second ligation premix solution, a ligation reaction termination solution, and a PCR premix solution.
[0077] The first connection premix includes 4×GC Solution and probe mixture, and the probe mixture includes the probes designed in the above embodiments;
[0078] The second ligation master mix includes 10× Taq buffer and Taq ligase.
[0079] Ligation reaction stop solution, including EDTA.
[0080] The PCR premix includes 10×PCR buffer, dNTP, MgCl2, primer mixture and Taq DNA polymerase, and the primer mixture includes the primers designed in the above embodiments.
[0081] Specifically, the method of using the kit may include:
[0082] 1. Mix the first ligation premix and sample DNA, and then add the second ligation premix. At this time, the two probes of each probe group will hybridize with the 5' end and 3' end of the target site respectively, and connect under the action of ligase to obtain a ligation product;
[0083] 2. Add ligation reaction termination solution to the ligation product to terminate the ligation reaction;
[0084] 3. Mix the terminated ligation product with the PCR premix and perform PCR amplification. At this time, the ligation product will be amplified under the action of the primers;
[0085] 4. The amplified PCR product is deformed and subjected to capillary electrophoresis. At this time, amplified fragments of different lengths and different fluorescence signals can be separated, so that the fluorescence information and length information of each amplified fragment can be obtained;
[0086] 5. The copy number of each target site can be calculated based on the acquired fluorescence information and length information. Specifically, the copy number calculation method can be to standardize the fluorescence peak height value of the amplified fragment corresponding to the detection probe group in each probe group, compare the data of the standardized detection sample with the data of the normal reference sample, and calculate the copy number of the detection sample based on the copy number of the normal reference sample.
[0087] The specific calculation process can be as follows:
[0088] The R value of the detection probe group a is obtained by dividing the fluorescence peak height value of the amplified fragment corresponding to a detection probe group a in the probe group by the fluorescence peak height value of the amplified fragment corresponding to a reference probe group b in the same probe group;
[0089] The R value of the detection probe group a in the test sample is divided by the R value of the detection probe group a in n normal male reference samples, and then multiplied by the copy number of the detection probe group a in the normal reference sample to obtain n corrected copy values of the detection probe group a for the reference probe group b, wherein the test sample is a sample of the Y chromosome copy number to be detected, and the normal male reference sample is a male sample with a known normal Y chromosome copy number;
[0090] Based on n corrected copy number values, the relative copy number of detection probe set a relative to reference probe set b is calculated;
[0091] Repeat the above steps to calculate the relative copy numbers of the detection probe group a for all reference probe groups in the same probe group, and obtain i relative copy numbers of the detection probe group a;
[0092] Based on the i relative copy numbers, the copy number of the amplified fragment corresponding to the detection probe group a is obtained.
[0093] The step of calculating the relative copy number of the detection probe set a to the reference probe set b based on the n correction copy number values comprises:
[0094] When n is greater than 1, determining whether the coefficient of variation of the n corrected copy numbers is less than or equal to a first threshold, and if so, taking the average of the n corrected copy numbers as the relative copy number of the detection probe set a relative to the reference probe set b;
[0095] If the coefficient of variation of the n corrected copy values is greater than the first threshold, the corrected copy values farthest from the median of the n corrected copy values are sequentially eliminated until the coefficient of variation of the remaining m corrected copy values is less than the second threshold, where m is greater than or equal to 2n / 3, and the average of the m corrected copy values is taken as the relative copy number of the detection probe group a relative to the reference probe group b;
[0096] If there are no remaining m corrected copy number values whose coefficient of variation is less than the second threshold, the median of the n corrected copy number values is taken as the relative copy number of the detection probe set a to the reference probe set b.
[0097] Wherein, based on i relative copy numbers, the step of obtaining the copy number of the amplified fragment corresponding to the detection probe group a comprises:
[0098] Determine whether the coefficient of variation of the i relative copy numbers is less than or equal to a third threshold value, and if so, take the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe group a;
[0099] If the coefficient of variation of the i relative copy numbers is greater than the third threshold, the relative copy numbers farthest from the median of the i relative copy numbers are removed in sequence until the coefficient of variation of the remaining j relative copy numbers is less than the fourth threshold, where j is greater than or equal to 2n / 3, and the average of the j relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a;
[0100] If there are no remaining j relative copy numbers whose coefficient of variation is less than the fourth threshold, the median of the i relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a.
[0101] In one embodiment, the first threshold and the third threshold may be 10%, and the second threshold and the fourth threshold should be lower than the first threshold and the third threshold, respectively. For example, the second threshold and the fourth threshold may be 5%.
[0102] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.
[0103] Example 1: Probe and primer design
[0104] In this embodiment, 118 target sites were selected for 61 genes on the Y chromosome for probe design. In order to ensure the accuracy of the test structure, 32 target sites were selected on the reference genes on the autosomes and 5 target sites were selected on the X chromosome for probe design.
[0105] Specifically, the location information of the target site and the corresponding gene in this embodiment are shown in Table 1 below:
[0106] Table 1 Target site information table
[0107]
[0108]
[0109]
[0110] A probe set is designed for each of the above target sites, each probe set includes a 5' end probe and a 3' end probe, wherein the 5' end probe includes a first universal primer binding site and a first connecting sequence arranged in sequence; the 3' end probe includes a second connecting sequence and a second universal primer binding site arranged in sequence.
[0111] Specifically, the first connection sequences of 118 probe sets targeting 118 target sites on chromosome Y are shown in SEQ ID NOs. 1 to 118, and the second connection sequences are shown in SEQ ID NOs. 119 to 236.
[0112] The first connection sequences of the five probe sets for chromosome X are shown in SEQ ID NOs. 237 to 241, and the second connection sequences are shown in SEQ ID NOs. 242 to 246.
[0113] The first connection sequences of the 32 probe sets for the reference gene are shown in SEQ ID NOs. 247-278, and the second connection sequences are shown in SEQ ID NOs. 279-310.
[0114] The first universal primer binding site of all the above probe sets adopts four common designs, as shown in the following table. The first universal primer binding site of each 5' end probe selects one of the sequences shown in Table 2 below:
[0115] Table 2 The first universal primer binding site sequence
[0116]
[0117] Accordingly, four first universal primers were designed, and four different fluorescent groups PET, FAM, VIC, and NED were used to label the four first universal primers respectively and connected to the 5' end to label the four first universal primers respectively. Specifically, the sequence information of the four first universal primers is shown in Table 3 below:
[0118] Table 3 The first universal primer sequence
[0119]
[0120]
[0121] The second universal primer binding site of all the above probe sets adopts two common designs, as shown in the following table. The second universal primer binding site of each 3' end probe selects one of the sequences shown in Table 4 below:
[0122] Table 4 Second universal primer binding site sequence
[0123]
[0124] Accordingly, two second universal primers are designed, and the 5' ends of the two second universal primers are connected to filling sequences of different lengths. Specifically, the sequence information of the two second universal primers is shown in Table 5 below:
[0125] Table 5 Second universal primer sequence
[0126]
[0127] Specifically, the correspondence between the target site position, the first universal primer binding site, the second universal primer binding site, the first connection sequence, and the second connection sequence of each probe in this embodiment is shown in Table 6 below, where the target site number refers to the target site number in Table 1.
[0128] Table 6 Probe sequence information and target site information
[0129]
[0130]
[0131] Example 2: Kit Design
[0132] Based on the primers and probes designed above, a kit was constructed.
[0133] Specifically, the kit includes a first ligation premix, a second ligation premix, a ligation reaction termination solution and a PCR premix.
[0134] The first ligation premix solution includes ddH2O, 4×GC Solution, and a probe mixture solution. The probe mixture solution is a mixed solution of the probes designed in the above-mentioned Example 1, and the concentration is 0.01 μM.
[0135] The second ligation premix solution includes ddH2O, 10×Taq ligation buffer, and Taq ligase 6.5 U / μL.
[0136] The ligation reaction termination solution includes EDTA 72mM.
[0137] The PCR premix solution includes ddH2O, 10×PCR buffer, dNTP0.4mM, MgCl23.5mM, primer mixture concentration 2μM, and Taq DNA polymerase 0.05U / μL.
[0138] Example 3: Detection of samples to be tested
[0139] 1. Sample preparation: Take blood from three men as test samples, and take blood from a male volunteer with known normal copy number as a normal reference sample. Use phenol:chloroform method to extract genomic DNA as samples, and quantitatively dilute all four samples to 50ng / μL;
[0140] In other embodiments, the sample to be tested may also be other body fluid samples, biopsy tissue samples or paraffin-embedded tissue samples of animals, such as blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural effusion, nipple aspirate fluid, lymph fluid, fluid from the respiratory, intestinal or urogenital system, tears, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, internal organ system fluid, ascites, tumor cyst fluid, amniotic fluid, or a combination thereof, derived from an animal (such as a human).
[0141] 2. Ligation reaction: Take 5 μL of the first ligation premix and 1 μL of the sample DNA to make a total volume of 6 μL; then take 6 μL of the second ligation premix and add it to the above mixture and shake to mix well, and perform the ligation procedure at 98℃ for 6min; 5x (96℃ for 20s, 60℃ for 3h); 94℃ for 2min; 72℃ Forever;
[0142] 3. Termination reaction: After the ligation reaction is completed, remove the ligation product and add the ligation reaction termination solution on the ice box;
[0143] 4. PCR reaction: Take 18 μL of PCR premix and 2 μL of the terminated ligation product, mix them, shake and centrifuge, and perform PCR program at 95℃ for 2 min; 5x (94℃ for 20 s, 62℃ for 40 s / -1℃ per cycle, 72℃ for 1.5 min); 27x (94℃ for 20 s, 57℃ for 40 s, 72℃ for 1.5 min); 68℃ for 30 min; 16℃ forever;
[0144] 5. Capillary electrophoresis: dilute the PCR product 10 times, take 1 μL and mix it with 8.9 μL Hidi and 0.1 μL Liz500 to a total volume of 10 μL. Denature at 95°C for 5 minutes and perform capillary electrophoresis on the ABI3730XL sequencer to separate amplified fragments of different lengths and different fluorescence signals, and obtain the fluorescence information and position information of each fragment, such as Figure 2 As shown, Figure 2 It is a chromatogram of each fragment after PCR amplification of each sample in Example 3 of the present application, wherein each peak represents an amplification product corresponding to each target site.
[0145] 6. Copy number calculation: Based on the method of the above embodiment, the ratio of the fluorescence peak height of each target site on the Y chromosome to the fluorescence peak height of the reference gene is compared with the normal reference sample to calculate the relative copy number of each target site. Figures 3 to 5 , Figure 3 is a data diagram of the copy number of each target site after PCR amplification of the sample 1 to be tested in Example 3 of the present application, Figure 4 is a data diagram of the copy number of each target site after PCR amplification of the sample 2 to be tested in Example 3 of the present application, Figure 5This is a data diagram of the copy number of each target site after PCR amplification of the sample 3 to be tested in Example 3 of the present application, and the positions of the sites in the figure are abbreviated. Figures 3 to 5 The data of Table 7 are obtained. In the copy number calculation process of this embodiment, the first threshold and the third threshold are 10%, and the second threshold and the fourth threshold are 5%.
[0146] Table 7 Copy number of target sites in each sample of Example 3
[0147]
[0148]
[0149]
[0150]
[0151] See also Figures 3 to 5 As shown in Table 7, the three samples to be tested were all normal male samples, and the copy numbers of all probes were normal; the CV values of the entire copy number calculation results of all samples were around 4%, and the data were accurate and reliable.
[0152] It is obvious to those skilled in the art that the present application is not limited to the details of the exemplary embodiments described above, and that the present application can be implemented in other specific forms without departing from the spirit or essential features of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present application. Any reference numeral in a claim should not be regarded as limiting the claim to which it relates.
[0153] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A probe and primer for detecting Y chromosome copy number variation, characterized in that: The probe comprises: 118 probe sets covering a total of 118 target sites on 61 genes on chromosome Y, the positions of the 118 target sites are shown in Table 1 of the specification from No. 1 to No. 118; at least one of the probe sets covering at least one target site located on a reference gene; Wherein, each of the probe sets comprises a 5' end probe and a 3' end probe which are at least partially complementary to the 5' end and the 3' end of the target site, respectively; The primers include a forward primer and a reverse primer for the probe set.
2. The probe and primer according to claim 1, characterized in that: Each of the 5' end probes comprises a first universal primer binding site and a first linker sequence arranged in sequence, and each of the 3' end probes comprises a second linker sequence and a second universal primer binding site arranged in sequence, wherein the first linker sequence and the second linker sequence are at least partially complementary to the 5' end and the 3' end of the target site, respectively; The primers include: a first universal primer set targeting the first universal primer binding site, comprising at least one first universal primer, each of the first universal primers being at least partially complementary to the first universal primer binding site of at least one of the 5' end probes; The second universal primer set targeting the second universal primer binding site includes at least one second universal primer, and each of the second universal primers is at least partially complementary to the second universal primer binding site of at least one of the 3' end probes.
3. The probe and primer according to claim 2, characterized in that: The first connection sequences of the 118 probe sets for the 118 target sites on the Y chromosome are shown in SEQ ID NOs. 1 to 118, and the second connection sequences are shown in SEQ ID NOs. 119 to 236.
4. The probe and primer according to claim 2, characterized in that: The probe further comprises: Five probe sets covering five target sites on chromosome X, wherein the positions of the five target sites on chromosome X are shown in Table 1 of the specification as NO.119 to NO.123, the first connection sequences of the five probe sets for chromosome X are shown in SEQ ID NOs.237 to 241, and the second connection sequences are shown in SEQ ID NOs.242 to 246.
5. The probe and primer according to claim 2, characterized in that: The probe includes 32 probe groups covering 32 target sites located on the reference gene, the positions of the 32 target sites of the reference gene are shown in NO.124 to NO.155 in Table 1 of the specification, the first connection sequence of the 32 probe groups for the reference gene is shown in SEQ ID NO.247~278, and the second connection sequence is shown in SEQ ID NO.279~310.
6. The probe and primer according to any one of claims 2 to 5, characterized in that: The first universal primer set includes four first universal primers, the sequences of which are shown in SEQ ID NOs.311 to 314, and the 5' ends of the four first universal primers are respectively labeled with different fluorescent groups, and the second universal primer set includes two second universal primers, the sequences of which are shown in SEQ ID NOs.315 to 316; The first universal primer binding site of the 5' end probe of each probe set is selected from one of the sequences shown in SEQ ID NOs. 317-320, and the second universal primer binding site of the 3' end probe of each probe set is selected from one of the sequences shown in SEQ ID NOs. 321-322.
7. A kit for detecting Y chromosome copy number variation, characterized in that: include: A first ligation premix, comprising 4×GC Solution and a probe mixture, wherein the probe mixture comprises the probe according to any one of claims 1 to 6; The second ligation premix includes 10× Taq buffer and Taq ligase; Ligation reaction termination solution, including EDTA; A PCR premix comprises 10×PCR buffer, dNTP, MgCl2, a primer mix and Taq DNA polymerase, wherein the primer mix comprises the primers according to any one of claims 1 to 6.
8. A method for using the kit according to claim 7, characterized in that: include: The first ligation premix solution and the sample DNA are mixed, and then the second ligation premix solution is added to perform a ligation reaction so that the probe is connected to the target site; Adding the ligation reaction termination solution; Add PCR premix solution and perform PCR amplification; The amplified PCR product is denatured and then subjected to capillary electrophoresis to separate the amplified fragments, obtain the information of each amplified fragment and calculate the copy number.
9. The method of use according to claim 8, characterized in that: The probe comprises a plurality of probe groups, each of which comprises a plurality of probe groups, the plurality of probe groups comprising at least one detection probe group for chromosome Y and at least one reference probe group for a reference gene, and the plurality of probe groups in each of which correspond to the same forward primer and reverse primer; The step of obtaining information of each amplified fragment and calculating the copy number comprises: The R value of the detection probe group a is obtained by dividing the fluorescence peak height value of the amplified fragment corresponding to a detection probe group a in the probe group by the fluorescence peak height value of the amplified fragment corresponding to a reference probe group b in the same probe group; The R value of the detection probe group a in the test sample is divided by the R value of the detection probe group a in n normal male reference samples, and then multiplied by the copy number of the detection probe group a in the normal reference sample to obtain n corrected copy values of the detection probe group a for the reference probe group b, wherein the test sample is a sample of the Y chromosome copy number to be detected, and the normal male reference sample is a male sample with a known normal Y chromosome copy number; Based on the n corrected copy number values, the relative copy number of the detection probe group a relative to the reference probe group b is calculated; Repeat the above steps to respectively calculate the relative copy numbers of the detection probe group a for all reference probe groups in the same probe group, and obtain i relative copy numbers of the detection probe group a; Based on the i relative copy numbers, the copy number of the amplified fragment corresponding to the detection probe group a is obtained.
10. The method of use according to claim 9, characterized in that: The step of calculating the relative copy number of the detection probe set a relative to the reference probe set b based on the n correction copy number values comprises: When n is greater than 1, determining whether the coefficient of variation of the n corrected copy numbers is less than or equal to a first threshold value, and if so, taking the average value of the n corrected copy numbers as the relative copy number of the detection probe group a relative to the reference probe group b; If the coefficient of variation of the n corrected copy values is greater than a first threshold, the corrected copy values farthest from the median of the n corrected copy values are sequentially eliminated until the coefficient of variation of the remaining m corrected copy values is less than a second threshold, wherein m is greater than or equal to 2n / 3, and the average value of the m corrected copy values is taken as the relative copy number of the detection probe group a relative to the reference probe group b; If there are no remaining m correction copy number values whose coefficient of variation is less than the second threshold, taking the median of the n correction copy number values as the relative copy number of the detection probe group a to the reference probe group b; The step of obtaining the copy number of the amplified fragment corresponding to the detection probe group a based on the i relative copy numbers comprises: Determine whether the coefficient of variation of the i relative copy numbers is less than or equal to a third threshold value, and if so, take the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe group a; If the coefficient of variation of the i relative copy numbers is greater than the third threshold, the relative copy numbers farthest from the median of the i relative copy numbers are sequentially eliminated until the coefficient of variation of the remaining j relative copy numbers is less than the fourth threshold, wherein j is greater than or equal to 2n / 3, and the average value of the j relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a; If there are no remaining j relative copy numbers whose coefficient of variation is less than the fourth threshold, the median of the i relative copy numbers is taken as the copy number of the amplified fragment corresponding to the detection probe group a.