CN117660650A - Primers and probes for detecting copy number variation of BRCA1 and 2 genes - Google Patents
Primers and probes for detecting copy number variation of BRCA1 and 2 genes Download PDFInfo
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Abstract
The application discloses a primer and a probe for detecting copy number variation of BRCA1 and 2 genes, wherein the probe comprises 43 probe groups covering 43 target sites on 23 exons of the BRCA1 genes; 55 probe sets covering 55 target sites on 27 exons of the BRCA2 gene; each probe set 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, each 5 'end probe comprises a first universal primer binding site and a first connecting sequence which are sequentially arranged, and each 3' end probe comprises a second connecting sequence and a second universal primer binding site which are sequentially arranged; the primer includes a first universal primer set directed to a first universal primer binding site; a second universal primer set directed to a second universal primer binding site. The method has the advantages of high detection flux, high detection efficiency, high readiness, reduced cost and high resolution.
Description
Technical Field
The application relates to the field of molecular biology detection, in particular to a primer and a probe for detecting copy number variation of BRCA1 and 2 genes.
Background
BRCA1 and BRCA2 genes (BRCA 1/2 genes) are two excellent genes capable of inhibiting malignant tumor occurrence, and play an important role in damage repair and normal growth of cells. The BRCA1/2 gene is the most main susceptibility gene of familial hereditary breast cancer and ovarian cancer; the risk of suffering from breast cancer and ovarian cancer of individuals with BRCA1 gene mutation is 50% -85% and 15% -45%, respectively, and the risk of suffering from breast cancer and ovarian cancer of individuals with BRCA2 gene mutation is 50% -85% and 10% -20%, respectively.
There are two pathogenic mutant forms of the BRCA1/2 gene, one of which is represented by a frameshift mutation, nonsense mutation and missense mutation caused by single or several base changes. Another form of pathogenic mutation involves changes of hundreds to millions of base pairs, often changes in one or more exons, including base deletions, duplications, insertions, inversions, ectopic, etc., known as large fragment rearrangements (large genomic rearrangements, LGRs). The incidence rate of the BRCA1/2 gene LGRs of breast cancer patients is 2.2%, which accounts for 10.8% of all pathogenic gene mutations of BRCA 1/2. Although occurring at a relatively low frequency, LGRs can lead to abnormal peptide chain structure and protein function of mutations, once they occur, LGRs are often pathogenic, and BRCA1/2 germ line LGRs are transmitted in the family, leading to the continued occurrence of breast cancer ovarian cancer patients in the family, with a great deal of harm. Therefore, for families with family history of breast cancer and ovarian cancer, the mutation state of BRCA1/2 in the families is determined through gene detection, and the method is very important for the treatment of tumor patients in the families and the prevention and screening of tumors of healthy carriers.
The deletion and repetition of large segments of genes, also known as gene Copy Number Variation (CNVs), are currently detected by methods directed to the CNV variation of BRCA1/2 genes, mainly comprising: multiplex Ligation Probe Amplification (MLPA), real-time fluorescent quantitative PCR (RT-qPCR), microarray comparative gene hybridization (array-CGH), affymetrix CNV chip technology, second generation gene sequencing technology, and the like. The arrate-CGH, affymetrix CNV chip technology and the second generation gene sequencing technology are mainly used for detecting copy number variation of the whole genome, are accurate and efficient, but have relatively high cost; the RT-qPCR technology is simple and easy to operate, but has low flux, and is difficult to realize simultaneous detection of a plurality of fragments; MLPA is a mode of detecting and comparing the copy number of specific gene fragments to be mature so far, and is realized by a first generation sequencing platform, however, the method also has certain limitations, such as higher requirements on sample DNA, lower detection flux, easy occurrence of false positive as a result, and the like. Up to now, there is no formed copy number detection kit for BRCA1/2 gene on the market.
The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide a primer and a probe for detecting copy number variation of BRCA1 and 2 genes, which are used for solving various problems existing in CNVs detection of BRCA1 and BRCA2 genes in the prior art and providing a rapid, comprehensive, accurate and low-cost technology for detecting copy number variation of BRCA1 and 2 genes.
In order to achieve the above purpose, a technical scheme adopted in the application is as follows:
a primer and a probe for detecting copy number variation of BRCA1 and 2 genes, wherein the probe comprises:
covering 43 probe groups with 43 target sites on 23 exons of BRCA1 gene, wherein 1 target site is respectively arranged on 1, 2, 11, 17 and 23 exons of BRCA1 gene, 4 target sites are arranged on 10 exons of BRCA1 gene, and 2 target sites are arranged on each other exon;
covering 55 probe groups which are positioned on 27 exons of the BRCA2 gene and have 55 target sites, wherein 1 target site is respectively arranged on the No. 6 and the No. 18 exons of the BRCA2 gene, 5 target sites are arranged on the No. 11 exons of the BRCA2 gene, and 2 target sites are arranged on each other exons;
wherein each probe set 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 a target site respectively, each 5 'end probe comprises a first universal primer binding site and a first connecting sequence which are sequentially arranged, each 3' end probe comprises a second connecting sequence and a second universal primer binding site which are sequentially arranged, the first connecting sequence and the second connecting sequence are at least partially complementary to the 5 'end and the 3' end of the target site respectively, the first connecting sequences of 43 probe sets aiming at the exon of the BRCA1 gene are shown as SEQ ID NO. 1-43, and the second connecting sequences are shown as SEQ ID NO. 44-86; the first connecting sequence of the 55 probe groups aiming at the BRCA2 gene exon is shown as SEQ ID NO. 87-141, and the second connecting sequence is shown as SEQ ID NO. 142-196;
the primer comprises:
a first universal primer set for said first universal primer binding sites comprising at least one first universal primer, each said first universal primer being at least partially complementary to said first universal primer binding site of one said 5' end probe;
a second universal primer set for said second universal primer binding sites comprising at least one second universal primer, each said second universal primer being at least partially complementary to said second universal primer binding site of one said 3' end probe.
In one or more embodiments, the probe further comprises:
3 probe sets covering 2 target sites positioned at 8Kp upstream of the BRCA1 gene and 1 target site positioned at 200bp upstream of the BRCA1 gene, wherein a first connecting sequence of the 3 probe sets aiming at the upstream of the BRCA1 gene is shown as SEQ ID NO. 197-199, and a second connecting sequence is shown as SEQ ID NO. 200-202;
5 probe sets covering 5 target sites respectively located at 10Kb, 8Kb, 5Kb, 3Kb and 500bp of the upstream of the BRCA2 gene, wherein the first connecting sequence of the 5 probe sets aiming at the upstream of the BRCA2 gene is shown as SEQ ID NO. 203-207, and the second connecting sequence is shown as SEQ ID NO. 208-212.
In one or more embodiments, the probe further comprises:
1 probe group covering 1 target site of the untranslated region at the 5 'end of the BRCA1 gene, wherein a first connecting sequence of the 1 probe group aiming at the untranslated region at the 5' end of the BRCA1 gene is shown as SEQ ID NO.213, and a second connecting sequence is shown as SEQ ID NO. 214;
4 probe groups covering 4 target sites positioned in the 3 '-end untranslated region of the BRCA1 gene, wherein the first connecting sequence of the 4 probe groups aiming at the 3' -end untranslated region of the BRCA1 gene is shown as SEQ ID NO. 215-218, and the second connecting sequence is shown as SEQ ID NO. 219-222;
3 probe sets covering 3 target sites positioned in the 3 '-end untranslated region of the BRCA2 gene, wherein the first connecting sequence of the 3 probe sets aiming at the 3' -end untranslated region of the BRCA2 gene is shown as SEQ ID NO. 223-225, and the second connecting sequence is shown as SEQ ID NO. 226-228.
In one or more embodiments, the probe further comprises:
6 probe sets covering 6 target sites on introns 2, 10, 12, 16 and 22 of the BRCA1 gene, wherein the introns 12 of the BRCA1 gene have 2 target sites, the introns 2, 10, 16 and 22 have 1 target site, the first connecting sequence of the 6 probe sets aiming at the introns of the BRCA1 gene is shown as SEQ ID NO. 229-234, and the second connecting sequence is shown as SEQ ID NO. 235-240;
covering 1 probe group at 1 target site of X chromosome, wherein a first connecting sequence of the 1 probe group aiming at the X chromosome is shown as SEQ ID NO.241, and a second connecting sequence is shown as SEQ ID NO. 242;
1 probe set covering 1 target site of Y chromosome, the first ligation sequence of 1 probe set for Y chromosome is shown as SEQ ID NO.243, and the second ligation sequence is shown as SEQ ID NO. 244.
In one or more embodiments, the probe further comprises:
29 probe sets covering 29 target sites on the reference gene, wherein the first connecting sequences of the 29 probe sets aiming at the reference gene are shown as SEQ ID NO. 245-273, and the second connecting sequences are shown as SEQ ID NO. 274-302.
In one or more embodiments, the first universal primer set includes a plurality of first universal primers labeled with different fluorophores, and the second universal primer set includes a plurality of second universal primers linked with different lengths of filling sequences.
In one or more embodiments, the first universal primer set comprises four first universal primers, the sequences of which are shown in SEQ ID NO. 303-306, and the 5' ends of the four first universal primers are respectively marked by different fluorophores; the second universal primer group comprises two second universal primers, and the sequences of the second universal primers are shown as SEQ ID NO. 307-308;
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 No. 309-312, 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 No. 313-314.
In order to achieve the above purpose, another technical scheme adopted in the application is as follows:
provided is a kit for detecting copy number variation of BRCA1 and 2 genes, comprising:
a first ligation pre-mix comprising a 4 XGC Solution and a probe mix comprising the probe of any of the embodiments described above;
a second ligation pre-mix comprising 10 XTaq buffer and Taq ligase;
a ligation reaction terminating solution comprising EDTA;
PCR premix comprising 10 XPCR buffer, dNTPs, mgCl 2 A primer mix comprising the primer of any one of claims 1 to 7, and Taq DNA polymerase.
In order to achieve the above purpose, another technical scheme adopted in the application is as follows:
there is provided a method of using the kit of any one of the above embodiments, comprising:
mixing the first ligation pre-mix with the sample DNA, and then adding the second ligation pre-mix to perform ligation reaction to ligate the probe to a target site;
adding the connection reaction stopping solution;
adding a PCR premix to carry out PCR amplification;
and (3) carrying out capillary electrophoresis after denaturing the amplified PCR products, separating amplified fragments, obtaining information of each amplified fragment, and carrying out copy number calculation.
In one or more embodiments, the probe comprises a plurality of probe groups, each of the probe groups comprising a plurality of probe sets including at least one detection probe set for a BRCA1 or BRCA2 gene and at least one reference probe set for a reference gene, and the plurality of probe sets of each of the probe groups corresponding to the same forward and reverse primers;
the step of obtaining information of each amplified fragment and performing copy number calculation comprises the steps of:
dividing the fluorescence peak height value of an amplified fragment corresponding to a detection probe set a in the probe group by the fluorescence peak height value of an amplified fragment corresponding to a reference probe set b in the same probe group to obtain an R value of the detection probe set a;
dividing the R value of the detection probe set a in a detection sample by the R value of the detection probe set a in n normal reference samples, and multiplying the R value by the copy number of the detection probe set a in the normal reference samples to obtain n corrected copy numbers of the detection probe set a for the reference probe set b, wherein the detection samples are samples of which the BRCA1 and BRCA2 gene copy numbers are to be detected, and the normal reference samples are samples of which the BRCA1 and BRCA2 gene copy numbers are known to be normal;
when n is greater than 1, calculating the average value, standard deviation and variation coefficient of the n correction copy values, and taking the average value of the n correction copy values as the relative copy number of the detection probe set a to the reference probe set b if the variation coefficient of the n correction copy values is less than 10%;
if the variation coefficient of the n corrected copy values is greater than 10%, sequentially eliminating corrected copy values farthest from the median of the n corrected copy values until the variation coefficient of the remaining m corrected copy values is less than 5%, wherein m is greater than or equal to 2n/3, and taking the average value of the m corrected copy values as the relative copy number of the detection probe set a to the reference probe set b;
if the variation coefficient of the m correction copy values which are not remained is smaller than 5%, taking the median of the n correction copy values as the relative copy number of the detection probe set a to the reference probe set b;
repeating the steps, and respectively calculating the relative copy numbers of the detection probe group a aiming at all reference probe groups in the same probe group to obtain i relative copy numbers of the detection probe group a;
calculating the average value, standard deviation and variation coefficient of the i relative copy numbers, and taking the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a if the variation coefficient of the i relative copy numbers is smaller than 10%;
if the variation coefficient of the i relative copy numbers is greater than 10%, sequentially eliminating the relative copy numbers farthest from the median of the i relative copy numbers until the variation coefficient of the remaining j relative copy numbers is less than 5%, wherein j is greater than or equal to 2n/3, and taking the average value of the j relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a;
if the variation coefficient of the j relative copy numbers which are not remained is smaller than 5%, taking the median of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a.
The beneficial effect of this application is, in contrast to prior art:
the primer and the probe design can cover all exons of the BRCA1 gene and the BRCA2 gene, each exon is covered with 1 or more probe sets, the deletion or repetition of a large fragment of each exon can be detected at one time, and the detection flux is high;
the application method of the kit is simple in flow, only comprises connection reaction and PCR reaction, and the whole flow is not more than 24 hours in total, so that the detection efficiency is high;
the kit adopts the universal primer to amplify the connection product of each detection area, the amplification efficiency is basically consistent, and meanwhile, a plurality of reference gene loci are introduced to perform data correction calculation, the average detection difference CV value is less than 10%, and the readiness of the detection result can be ensured;
the kit can finish detection based on a first generation sequencing platform, and compared with a second generation sequencing platform detection technology, the cost is effectively reduced;
the method can accurately quantify the sites within 6 copies, and has high resolution.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the present application;
FIG. 2 is a schematic diagram of the chromatogram of each fragment after PCR amplification of the sample in example 3 of the present application.
Detailed Description
The applicant develops a primer, a probe and a kit for detecting copy number variation of 23 exons of BRCA1 genes and 27 exons of BRCA2 genes simultaneously, aiming at various problems existing in the current detection methods of Copy Number Variation (CNV) of BRCA1 and BRCA2 genes, and can detect the copy number variation of the BRCA1 and BRCA2 genes rapidly, comprehensively, accurately and at low cost.
Referring to fig. 1, fig. 1 is a schematic diagram of the detection principle of the present application. As shown in fig. 1, a change in copy number of a nucleic acid sequence containing 2 or more nucleotides in a sample can be detected simultaneously in a single detection.
Designing a probe set for each target site, wherein each probe set can comprise a 5' end probe and a 3' end probe, and the 5' end probe comprises a first universal primer binding site X, a first filling sequence A1 and a first specific sequence L1 which are sequentially arranged; the 3' end probe comprises a second specific sequence L2, a second filling sequence A2 and a second universal primer binding site Y which are sequentially arranged.
Wherein the first specific sequence L1 and the second specific sequence L2 can be respectively complementary to the 5 'end and the 3' end of the target site.
The first filling sequence A1 and the second filling sequence A2 are used for increasing the length difference of the subsequent amplification products, and the length of the first filling sequence A1 and the second filling sequence A2 is changed, so that the multiplexing of analysis is improved.
Each group of probes is respectively connected with a corresponding target site, and no gap exists between the 5 'end of the 5' end probe and the 3 'end of the 3' end probe of each probe group, so that a connection product is obtained.
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 set, and one of the probes may be extended to close the gap, thereby obtaining a ligation product, which can achieve the effects of this embodiment.
The ligation product may be amplified based on a forward primer complementary to the first universal primer binding site X and a reverse primer complementary to the second universal primer binding site Y.
By performing electrophoretic separation on the amplified fragments, information of each amplified fragment can be obtained, thereby performing copy number calculation.
To facilitate copy number calculation while further increasing the multiplicity of detection methods, in one embodiment, different fluorescent sequences may also be labeled on the primer sequences, and third stuffer sequences of different lengths may also be ligated to the primer sequences.
Further, the first universal primer binding site X of at least part of the probe sets may be identical for all probe sets, whereby at least part of the ligation products may be amplified based on the same universal primer.
Based on the above design, by analyzing the length and fluorescence amount of the amplified fragment, information of the amplified fragment is obtained and compared with a standard value to perform copy number calculation.
In some embodiments, a probe set covering the reference gene may also be introduced, and the fluorescence peak of the amplified fragment of the target gene is compared with the fluorescence peak of the amplified fragment of the reference gene, so that the fluorescence peak of the amplified fragment is normalized to determine the copy number change.
Specifically, the probe set covering the reference gene may correspond to the same forward primer and reverse primer as the partial probe set covering the target site, so that correction may be made based on the fluorescence peak of the reference gene, and finally the copy number of amplified fragments for each target site is calculated based on the copy number of the normal reference sample.
In one embodiment, all probes may be divided into a plurality of probe groups, each of which may correspond to the same forward primer and reverse primer, and each of which may include a plurality of probe sets, wherein the plurality of probe sets may include at least one detection probe set for a target gene and at least one reference probe set for a reference gene.
In the subsequent calculation process, the fluorescence peak values of the amplified fragments corresponding to all the detection probe groups in each probe group can be normalized based on the fluorescence peak values of the amplified fragments corresponding to the reference probe group in the probe group.
In particular, in the subsequent data processing process, a normal reference sample with a known normal copy number can be introduced, and meanwhile, the data of the detection sample after the standardized processing and the data of the normal reference sample are compared, so that the data of the detection sample are corrected, and the data accuracy is ensured.
Based on the primer and probe combination designed as above, a kit for copy number variation detection of each site of BRCA1/2 gene can be constructed.
In one embodiment, the kit may include a first ligation pre-mix, a second ligation pre-mix, a ligation reaction termination solution, and a PCR pre-mix.
Wherein the first connection pre-mixture comprises 4 XGC Solution and a probe mixture, and the probe mixture comprises the probes designed in the above embodiments;
the second ligation pre-mix comprises 10×Taq buffer and Taq ligase.
Ligation reaction termination solution, including EDTA.
The PCR premix comprises 10 XPCR buffer, dNTPs, mgCl 2 The primer mixture comprises the primers designed in each embodiment.
Specifically, the method for using the kit can comprise the following steps:
1. mixing the first ligation pre-mixture with the sample DNA, and then adding the second ligation pre-mixture, wherein at the moment, two probes of each probe set are hybridized with the 5 'end and the 3' end of a target site respectively and are ligated under the action of ligase to obtain a ligation product;
2. adding a connection reaction stopping solution into the connection product to stop the connection reaction;
3. mixing the terminated connection product with the PCR premix to carry out PCR amplification, wherein the connection product is amplified under the action of a primer;
4. performing capillary electrophoresis after deforming the amplified PCR products, wherein the amplified fragments with different lengths and different fluorescent signals can be separated, so that fluorescent information and length information of each amplified fragment can be obtained;
5. the copy number calculation of each target site can be performed based on the acquired fluorescence information and length information, specifically, the copy number calculation method can be that after the fluorescence peak height value of the amplified fragment corresponding to the detection probe group in each probe group is subjected to standardization processing, the data of the detection sample after the standardization processing is compared with the data of the normal reference sample, and the copy number of the detection sample is calculated based on the copy number of the normal reference sample.
The specific calculation process can be as follows:
dividing the fluorescence peak value of the amplified fragment corresponding to one detection probe set a in the probe group by the fluorescence peak value of the amplified fragment corresponding to one reference probe set b in the same probe group to obtain an R value of the detection probe set a;
dividing the R value of a detection probe set a in a detection sample by the R value of the detection probe set a in n normal reference samples, and multiplying the R value by the copy number of the detection probe set a in the normal reference samples to obtain n corrected copy numbers of the detection probe set a for the reference probe set b, wherein the detection sample is a sample of BRCA1 and BRCA2 gene copy numbers to be detected, and the normal reference sample is a sample of known BRCA1 and 2 gene copy numbers;
when n is greater than 1, calculating the average value, standard deviation and variation coefficient of n correction copy values, and taking the average value of n correction copy values as the relative copy number of the detection probe set a to the reference probe set b if the variation coefficient of n correction copy values is less than 10%;
if the variation coefficient of the n correction copy values is greater than 10%, sequentially eliminating correction copy values farthest from the median of the n correction copy values until the variation coefficient of the remaining m correction copy values is less than 5%, wherein m is greater than or equal to 2n/3, and taking the average value of the m correction copy values as the relative copy number of the detection probe set a to the reference probe set b;
if the variation coefficient of the m correction copy values which are not remained is smaller than 5%, taking the median of the n correction copy values as the relative copy number of the detection probe set a to the reference probe set b;
repeating the steps, and respectively calculating the relative copy numbers of the detection probe set a aiming at all the reference probe sets in the same probe group to obtain i relative copy numbers of the detection probe set a;
calculating the average value, standard deviation and variation coefficient of the i relative copy numbers, and taking the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a if the variation coefficient of the i relative copy numbers is smaller than 10%;
if the variation coefficient of the i relative copy numbers is greater than 10%, sequentially eliminating the relative copy numbers farthest from the median of the i relative copy numbers until the variation coefficient of the rest j relative copy numbers is less than 5%, wherein j is greater than or equal to 2n/3, and taking the average value of the j relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a;
if the variation coefficient of the j relative copy numbers which are not remained is smaller than 5%, taking the median of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
Example 1: probe and primer design
The probes and primers of this example were designed for the BRCA1 gene and BRCA2 gene, and for the reference gene. In other embodiments, the probe and primer design may be performed on only one of the genes, and thus copy number variation detection may be performed on a single gene.
Specifically, the target sites of probes for the above genes were selected as follows:
43 target sites are arranged on 23 exons of the BRCA1 gene, wherein 1 target site is arranged on each of the 1, 2, 11, 17 and 23 exons of the BRCA1 gene, 4 target sites are arranged on 10 exons of the BRCA1 gene, and 2 target sites are arranged on each of the other exons;
55 target sites are arranged on 27 exons of the BRCA2 gene, wherein 1 target site is arranged on each of the No. 6 and No. 18 exons of the BRCA2 gene, 5 target sites are arranged on the No. 11 exons of the BRCA2 gene, and 2 target sites are arranged on each of the other exons;
2 target sites located 8Kp upstream of the BRCA1 gene, and 1 target site located 200bp upstream of the BRCA1 gene;
5 target sites respectively located at 10Kb, 8Kb, 5Kb, 3Kb and 500bp upstream of BRCA2 gene;
1 target site located in the 5 '-end untranslated region of the BRCA1 gene and 4 target sites located in the 3' -end untranslated region of the BRCA1 gene;
3 target sites located in the 3' -terminal untranslated region of the BRCA2 gene;
the gene is positioned at 6 target sites on introns 2, 10, 12, 16 and 22 of the BRCA1 gene, wherein the intron 12 of the BRCA1 gene has 2 target sites, and the introns 2, 10, 16 and 22 have 1 target site.
To distinguish sample sexes, 1 target site on the X chromosome and 1 target site on the Y chromosome were also selected.
To ensure the accuracy of the test results, 29 target sites on the reference gene were also selected.
Designing a probe set for each target site, wherein each probe set comprises a 5' end probe and a 3' end probe, and the 5' end probe comprises a first universal primer binding site and a first connecting sequence which are sequentially arranged; the 3' end probe includes a second ligation sequence and a second universal primer binding site in sequence.
Specifically, the first connecting sequences of 43 probe sets aiming at the BRCA1 gene exons are shown in SEQ ID NO. 1-43, and the second connecting sequences are shown in SEQ ID NO. 44-86; the first connecting sequence of the 55 probe groups aiming at the BRCA2 gene exon is shown as SEQ ID NO. 87-141, and the second connecting sequence is shown as SEQ ID NO. 142-196.
The first connecting sequences of the 3 probe groups on the upstream of the BRCA1 gene are shown in SEQ ID NO. 197-199, and the second connecting sequences are shown in SEQ ID NO. 200-202.
The first connecting sequence of the 5 probe groups on the upstream of the BRCA2 gene is shown as SEQ ID NO. 203-207, and the second connecting sequence is shown as SEQ ID NO. 208-212.
The first connecting sequence of the 1 probe set aiming at the untranslated region of the 5' end of the BRCA1 gene is shown as SEQ ID NO.213, and the second connecting sequence is shown as SEQ ID NO. 214.
The first connecting sequence of the 4 probe sets aiming at the untranslated region of the 3' end of the BRCA1 gene is shown as SEQ ID NO. 215-218, and the second connecting sequence is shown as SEQ ID NO. 219-222.
The first connecting sequence of the 3 probe sets aiming at the 3' -end untranslated region of the BRCA2 gene is shown as SEQ ID NO. 223-225, and the second connecting sequence is shown as SEQ ID NO. 226-228.
The first connecting sequence of the 6 probe groups aiming at the BRCA1 gene intron is shown as SEQ ID NO. 229-234, and the second connecting sequence is shown as SEQ ID NO. 235-240.
The first ligation sequence for the 1 probe set for the X chromosome is shown as SEQ ID NO.241 and the second ligation sequence is shown as SEQ ID NO. 242.
The first ligation sequence for the 1 probe set for the Y chromosome is shown as SEQ ID NO.243 and the second ligation sequence is shown as SEQ ID NO. 244.
The first connecting sequence of 29 probe groups aiming at the reference gene is shown as SEQ ID NO. 245-273, and the second connecting sequence is shown as SEQ ID NO. 274-302.
The first universal primer binding sites of all the above probe sets were designed in four general ways, as shown in the following table, and the first universal primer binding site of each 5' probe selected one of the sequences shown in the following table:
TABLE 1 first Universal primer binding site sequence
Accordingly, four first universal primers were designed and labeled with four different fluorophores PET, FAM, VIC, NED attached to the 5' end. Specifically, the sequence information of the four first universal primers is shown in the following table:
TABLE 2 first Universal primer sequences
The second universal primer binding sites of all the above probe sets were designed in two general ways, as shown in the following table, and the second universal primer binding site of each 3' end probe selected one of the sequences shown in the following table:
TABLE 3 second general primer binding site sequence
Correspondingly, two second universal primers are designed, and the 5' ends of the two second universal primers are connected with filling sequences with different lengths. Specifically, the sequence information of the two second universal primers is shown in the following table:
TABLE 4 second general primer sequences
Specifically, the correspondence relationship among the target site position, the first universal primer binding site, the second universal primer binding site, the first ligation sequence, and the second ligation sequence of each probe in this example is as follows:
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TABLE 5 Probe sequence information and target site information Table
Example 2: kit design
Based on the primers and probes designed as described above, a kit was constructed.
Specifically, the kit comprises a first ligation pre-mix, a second ligation pre-mix, a ligation reaction termination solution, and a PCR pre-mix.
The first connecting premix includes ddH 2 O, 4 XGC Solution, and a probe mixture, the probe mixture was the probe mixture designed in example 1, and the concentration was 0.01. Mu.M.
The second connecting premix comprises ddH 2 O, 10 XTaq ligation buffer, taq ligase 2U/. Mu.L.
The ligation reaction-terminated solution included EDTA20mM.
The PCR premix includes ddH 2 O, 10 XPCR buffer, 2.5mM dNTPs, 25mM MgCl 2 The concentration of the primer mixture was 3. Mu.M, and the concentration of Taq DNA polymerase was 0.04U/. Mu.L.
Example 3: sample detection to be tested
1. Sample preparation: blood from six volunteers was collected using phenol: extracting genome DNA by a chloroform method, taking the genome DNA as a sample, and quantitatively diluting six samples to 50 ng/mu L, wherein 2 of six volunteers are men, and 4 of the six volunteers are women; blood from a volunteer with a known normal copy number of BRCA1 and BRCA2 genes was taken as a normal reference sample.
In other embodiments, the sample to be tested may also be other body fluid samples of animals, biopsy tissue samples or paraffin embedded tissue samples. For example, it may be blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirate fluid, lymph fluid, respiratory, intestinal or genitourinary fluid, tears, saliva, breast milk, lymphatic fluid, semen, cerebrospinal fluid, organ internal fluid, ascites, tumor cyst fluid, amniotic fluid, or a combination thereof, from an animal (e.g., human).
2. Ligation reaction: the method comprises the steps of carrying out a first treatment on the surface of the The PCR plate was placed in an ice box, and after centrifugation, the ligation procedure was performed by shaking and mixing up Kong Jiaru ul of the first ligation mix, 2 ul of sample, and 10 ul of the second ligation mix: 98 ℃ for 6min;5x (96 ℃ C. 20s,60 ℃ C. 3 h); 94 ℃ for 2min; forever at 72 ℃;
3. terminating the reaction: after the ligation reaction was completed, the ligation product was removed, and 20. Mu.L of 20mM EDTA was added to the ice bin;
4. and (3) PCR reaction: adding 19 mu LPCR premix and 1 mu L of the above connection product into a PCR plate, mixing, vibrating and centrifuging, and carrying out PCR procedure at 95 ℃ for 2min;5x (94 ℃ C. 20s,62 ℃ C. 40s/-1 ℃ C. Per cycle,72 ℃ C. 1.5 min); 27x (94 ℃ 20s,57 ℃ 40s,72 ℃ 1.5 min); 30min at 68 ℃; forever at 16 ℃;
5. capillary electrophoresis: after diluting the product 5 times, taking 1 μl and mixing with 8.9 μl Hidi and 0.1 μl Liz500 to obtain a total volume of 10 μl, denaturing at 95deg.C for 5min, performing capillary electrophoresis on ABI3730XL sequencer, and separating amplified fragments of different lengths and different fluorescent signals to obtain fluorescence information and positional information of each fragment, as shown in FIG. 2, FIG. 2 is a schematic chromatographic diagram of each fragment after PCR amplification of the sample in example 3 of the present application, wherein each peak represents one amplification product corresponding to each target site.
6. Copy number calculation: comparing the ratio of the fluorescence peak height of each target site to the fluorescence peak height of the reference gene with a normal reference sample based on the method of the embodiment, and calculating to obtain the relative copy number of each target site to obtain the data in the following table.
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As shown in the data of the table, the copy number of each site of the 6 samples is unchanged, the result is negative, the CV value is between 6.1 and 9.4 percent, and the data is accurate and reliable.
The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the present application and its practical application to thereby enable one skilled in the art to make and utilize the present application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and the equivalents thereof.
Claims (10)
1. A primer and a probe for detecting copy number variation of BRCA1 and 2 genes are characterized in that,
the probe includes:
covering 43 probe groups with 43 target sites on 23 exons of BRCA1 gene, wherein 1 target site is respectively arranged on 1, 2, 11, 17 and 23 exons of BRCA1 gene, 4 target sites are arranged on 10 exons of BRCA1 gene, and 2 target sites are arranged on each other exon;
covering 55 probe groups which are positioned on 27 exons of the BRCA2 gene and have 55 target sites, wherein 1 target site is respectively arranged on the No. 6 and the No. 18 exons of the BRCA2 gene, 5 target sites are arranged on the No. 11 exons of the BRCA2 gene, and 2 target sites are arranged on each other exons;
wherein each probe set 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 a target site respectively, each 5 'end probe comprises a first universal primer binding site and a first connecting sequence which are sequentially arranged, each 3' end probe comprises a second connecting sequence and a second universal primer binding site which are sequentially arranged, the first connecting sequence and the second connecting sequence are at least partially complementary to the 5 'end and the 3' end of the target site respectively, the first connecting sequences of 43 probe sets aiming at the exon of the BRCA1 gene are shown as SEQ ID NO. 1-43, and the second connecting sequences are shown as SEQ ID NO. 44-86; the first connecting sequence of the 55 probe groups aiming at the BRCA2 gene exon is shown as SEQ ID NO. 87-141, and the second connecting sequence is shown as SEQ ID NO. 142-196;
the primer comprises:
a first universal primer set for said first universal primer binding sites comprising at least one first universal primer, each said first universal primer being at least partially complementary to said first universal primer binding site of one said 5' end probe;
a second universal primer set for said second universal primer binding sites comprising at least one second universal primer, each said second universal primer being at least partially complementary to said second universal primer binding site of one said 3' end probe.
2. The primer and probe of claim 1, wherein the probe further comprises:
3 probe sets covering 2 target sites positioned at 8Kp upstream of the BRCA1 gene and 1 target site positioned at 200bp upstream of the BRCA1 gene, wherein a first connecting sequence of the 3 probe sets aiming at the upstream of the BRCA1 gene is shown as SEQ ID NO. 197-199, and a second connecting sequence is shown as SEQ ID NO. 200-202;
5 probe sets covering 5 target sites respectively located at 10Kb, 8Kb, 5Kb, 3Kb and 500bp of the upstream of the BRCA2 gene, wherein the first connecting sequence of the 5 probe sets aiming at the upstream of the BRCA2 gene is shown as SEQ ID NO. 203-207, and the second connecting sequence is shown as SEQ ID NO. 208-212.
3. The primer and probe of claim 1, wherein the probe further comprises:
1 probe group covering 1 target site of the untranslated region at the 5 'end of the BRCA1 gene, wherein a first connecting sequence of the 1 probe group aiming at the untranslated region at the 5' end of the BRCA1 gene is shown as SEQ ID NO.213, and a second connecting sequence is shown as SEQ ID NO. 214;
4 probe groups covering 4 target sites positioned in the 3 '-end untranslated region of the BRCA1 gene, wherein the first connecting sequence of the 4 probe groups aiming at the 3' -end untranslated region of the BRCA1 gene is shown as SEQ ID NO. 215-218, and the second connecting sequence is shown as SEQ ID NO. 219-222;
3 probe sets covering 3 target sites positioned in the 3 '-end untranslated region of the BRCA2 gene, wherein the first connecting sequence of the 3 probe sets aiming at the 3' -end untranslated region of the BRCA2 gene is shown as SEQ ID NO. 223-225, and the second connecting sequence is shown as SEQ ID NO. 226-228.
4. The primer and probe of claim 1, wherein the probe further comprises:
6 probe sets covering 6 target sites on introns 2, 10, 12, 16 and 22 of the BRCA1 gene, wherein the introns 12 of the BRCA1 gene have 2 target sites, the introns 2, 10, 16 and 22 have 1 target site, the first connecting sequence of the 6 probe sets aiming at the introns of the BRCA1 gene is shown as SEQ ID NO. 229-234, and the second connecting sequence is shown as SEQ ID NO. 235-240;
covering 1 probe group at 1 target site of X chromosome, wherein a first connecting sequence of the 1 probe group aiming at the X chromosome is shown as SEQ ID NO.241, and a second connecting sequence is shown as SEQ ID NO. 242;
1 probe set covering 1 target site of Y chromosome, the first ligation sequence of 1 probe set for Y chromosome is shown as SEQ ID NO.243, and the second ligation sequence is shown as SEQ ID NO. 244.
5. The primer and probe of claim 1, wherein the probe further comprises:
29 probe sets covering 29 target sites on the reference gene, wherein the first connecting sequences of the 29 probe sets aiming at the reference gene are shown as SEQ ID NO. 245-273, and the second connecting sequences are shown as SEQ ID NO. 274-302.
6. The primer and probe of any one of claims 1 to 5, wherein the first universal primer set comprises a plurality of first universal primers labeled with different fluorophores and the second universal primer set comprises a plurality of second universal primers linked with filling sequences of different lengths.
7. The primer and probe according to claim 6, wherein the first universal primer set comprises four first universal primers, the sequences of which are shown in SEQ ID NOS.303-306, and the 5' ends of the four first universal primers are respectively marked by different fluorophores; the second universal primer group comprises two second universal primers, and the sequences of the second universal primers are shown as SEQ ID NO. 307-308;
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 No. 309-312, 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 No. 313-314.
8. A kit for detecting copy number variation of BRCA1 and 2 genes, comprising:
a first ligation pre-mix comprising a 4 xgc Solution and a probe mix comprising the probe of any one of claims 1 to 7;
a second ligation pre-mix comprising 10 XTaq buffer and Taq ligase;
a ligation reaction terminating solution comprising EDTA;
PCR premix comprising 10 XPCR buffer, dNTPs, mgCl 2 A primer mix comprising the primer of any one of claims 1 to 7, and Taq DNA polymerase.
9. A method of using the kit of claim 8, comprising:
mixing the first ligation pre-mix with the sample DNA, and then adding the second ligation pre-mix to perform ligation reaction to ligate the probe to a target site;
adding the connection reaction stopping solution;
adding a PCR premix to carry out PCR amplification;
and (3) carrying out capillary electrophoresis after denaturing the amplified PCR products, separating amplified fragments, obtaining information of each amplified fragment, and carrying out copy number calculation.
10. The method of claim 9, wherein the probes comprise a plurality of probe groups, each probe group comprising a plurality of probe groups, the plurality of probe groups comprising at least one detection probe group for BRCA1 or BRCA2 genes and at least one reference probe group for reference genes, and the plurality of probe groups of each probe group corresponding to the same forward and reverse primers;
the step of obtaining information of each amplified fragment and performing copy number calculation comprises the steps of:
dividing the fluorescence peak height value of an amplified fragment corresponding to a detection probe set a in the probe group by the fluorescence peak height value of an amplified fragment corresponding to a reference probe set b in the same probe group to obtain an R value of the detection probe set a;
dividing the R value of the detection probe set a in a detection sample by the R value of the detection probe set a in n normal reference samples, and multiplying the R value by the copy number of the detection probe set a in the normal reference samples to obtain n corrected copy numbers of the detection probe set a for the reference probe set b, wherein the detection samples are samples of which the BRCA1 and BRCA2 gene copy numbers are to be detected, and the normal reference samples are samples of which the BRCA1 and BRCA2 gene copy numbers are known to be normal;
when n is greater than 1, calculating the average value, standard deviation and variation coefficient of the n correction copy values, and taking the average value of the n correction copy values as the relative copy number of the detection probe set a to the reference probe set b if the variation coefficient of the n correction copy values is less than 10%;
if the variation coefficient of the n corrected copy values is greater than 10%, sequentially eliminating corrected copy values farthest from the median of the n corrected copy values until the variation coefficient of the remaining m corrected copy values is less than 5%, wherein m is greater than or equal to 2n/3, and taking the average value of the m corrected copy values as the relative copy number of the detection probe set a to the reference probe set b;
if the variation coefficient of the m correction copy values which are not remained is smaller than 5%, taking the median of the n correction copy values as the relative copy number of the detection probe set a to the reference probe set b;
repeating the steps, and respectively calculating the relative copy numbers of the detection probe group a aiming at all reference probe groups in the same probe group to obtain i relative copy numbers of the detection probe group a;
calculating the average value, standard deviation and variation coefficient of the i relative copy numbers, and taking the average value of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a if the variation coefficient of the i relative copy numbers is smaller than 10%;
if the variation coefficient of the i relative copy numbers is greater than 10%, sequentially eliminating the relative copy numbers farthest from the median of the i relative copy numbers until the variation coefficient of the remaining j relative copy numbers is less than 5%, wherein j is greater than or equal to 2n/3, and taking the average value of the j relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a;
if the variation coefficient of the j relative copy numbers which are not remained is smaller than 5%, taking the median of the i relative copy numbers as the copy number of the amplified fragment corresponding to the detection probe set a.
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