STR fluorescence labeling composite amplification system, kit and application of STR fluorescence labeling composite amplification system

Abstract

The invention relates to the technical field of biology, and discloses an STR fluorescence labeling composite amplification system, a kit and an application of the STR fluorescence labeling composite amplification system to detection of chromosome 7 monophilic disome.The STR fluorescence labeling composite amplification system comprises seven pairs of primers, each primer pair carries a fluorescent dye marker, and the invention discloses primer sequences aiming at seven sites and capable of amplifying the seven sites at the same time. The STR fluorescence labeling composite amplification system provided by the invention can be used for effectively and accurately detecting the STR polymorphism of the genome in a specific area, can prompt the monoparental disomes in a corresponding section by comparing with a parent detection result, can obviously reduce the false positive rate, effectively improves the accuracy and reliability of detection, and has a wide application prospect. The traditional Chinese medicine composition has important clinical application value and wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a STR fluorescence labeling multiplex amplification system, a kit and an application thereof. Background Art

[0002] Uniparental Disomy (UPD) refers to the situation where two homologous chromosomes are both inherited from one parent. UPD can be a segment on a chromosome or an entire chromosome. Whether UPD causes a clinical phenotype depends on whether it exists on a chromosome affected by genetic imprinting or whether it leads to the occurrence of related recessive diseases. The incidence of UPD for all chromosomes (not just chromosomes with imprinted regions) is 1 / 2000.

[0003] Maternal meiotic errors are the main cause of maternal UPD(7), accounting for about 71%.

[0004] Existing detection methods for UPD include: SNP probe detection, whole exome sequencing or whole genome sequencing, methylation PCR and MLPA. The CMA platform with SNP probes can be used for the detection of UPD. By analyzing the SNP distribution and using algorithms to detect UPD, it is unable to distinguish UPD from ROH caused by consanguineous inheritance and requires family verification. UPD can be judged by whole exome sequencing or whole genome sequencing, but the cost is high and the data analysis is complex. In addition, for imprinted gene diseases, methylation PCR and MLPA have certain significance for the detection of the cause. The principle is to analyze the differentially methylated regions or the methylation status of imprinting centers within a relatively large region of the chromosome (usually several megabases), but it is necessary to combine genotype analysis to distinguish UPD from methylation defects.

[0005] DNA-based polymorphic markers are classic methods for studying UPD, and the most commonly used is STR sequence markers. Human genome STR (short tandem repeat sequence) is a DNA sequence that is relatively stable in genetic inheritance and is formed by tandem repeats with several bases as the core unit. Different populations and even different individuals are distinguished by differences in the core unit sequence and the number of repeats, which also constitutes the genetic polymorphism of STR. In the genome, there is an STR locus on average every 15 - 20 kb, accounting for 10% of the genome. It mostly exists in non-coding regions and introns. The repeat unit is 2 - 6 bp, the number of repeats is 10 - 60 times, the fragment size is 70 - 500 bp, and it shows co-dominant inheritance according to Mendelian laws. The STR multiplex amplification technology is time-saving and simple to operate, and has the advantages of low cost, strong specificity, high sensitivity, high throughput and strong reliability. It can cover the whole genome and is suitable as a common means for UPD detection. Summary of the Invention

[0006] In view of the deficiencies existing in the current prior art, the present invention provides a STR fluorescent labeling multiplex amplification system, a kit and an application. The STR fluorescent labeling multiplex amplification system has two-color fluorescent labeling, and the loci adopted by the present invention have extremely high individual recognition rates and polymorphisms, and can be used for screening STR loci of uniparental disomy on chromosome 7. By comparing with the detection results of the parents, the UPD of autosome 7 can be prompted, with strong specificity and high sensitivity.

[0007] To achieve the above object, the embodiments of the present invention adopt the following technical solutions: A STR fluorescent labeling multiplex amplification system, the amplification system includes seven primer pairs: P7-1-F, P7-1-R; P7-2-F, P7-2-R; P7-3-F, P7-3-R; P7-4-F, P7-4-R; P7-5-F, P7-5-R; P7-6-F, P7-6-R and P7-7-F, P7-7-R. The STR fluorescent labeling multiplex amplification system can simultaneously amplify 7 STR loci: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. The primer sequences, primer concentration ranges for amplifying the 7 STR loci and the primer sequence numbers for amplifying the 7 STR loci are SEQ ID NO: 1-14, as shown in Table 2.

[0008] According to one aspect of the present invention, the loci in the STR fluorescent labeling multiplex amplification system are labeled with three kinds of fluorescence: the first group is P7-1, P7-2, P7-3, and the fluorescent label is FAM label; the second group is P7-4, P7-5, P7-6, P7-7, and the fluorescent label is HEX label; the third group is the internal standard primer adopted by the multiplex amplification system, and the fluorescent label is ORG label.

[0009] According to one aspect of the present invention, the STR fluorescent labeling multiplex amplification system includes a PCR mixture and a DNA template.

[0010] According to one aspect of the present invention, the PCR mixture includes ammonium sulfate, potassium chloride, Tris-HCl, magnesium ions, BSA, DMSO, ethylene glycol, Na4P2O7 and Taq enzyme, specifically: 12 mM ammonium sulfate, 8 mM potassium chloride, 65 mM Tris-HCl with a pH of 8.5, 2.5 mM magnesium ions, 1 ug / ul BSA, 5% DMSO, 6% ethylene glycol, 1 mM Na4P2O7, 0.25 mM dNTP and 2 U Taq enzyme. The components and corresponding concentrations are shown in Table 4.

[0011] According to one aspect of the present invention, the reaction procedure of the STR fluorescence-labeled multiplex amplification system is as follows: denaturation at 95°C for 5 minutes; denaturation at 94°C for 10 seconds, annealing at 59°C for 1 minute and 30 seconds, with 25 cycles; extension at 72°C for 20 minutes.

[0012] According to one aspect of the present invention, the DNA template used in the amplification system is derived from human blood, amniotic fluid or aborted tissue.

[0013] According to one aspect of the present invention, the DNA template can be extracted and processed by the kit method, phenol-chloroform extraction method, Chelex-100 method or magnetic bead method.

[0014] A kit, which includes the above-mentioned STR fluorescence-labeled multiplex amplification system.

[0015] Application of the above-mentioned STR fluorescence-labeled multiplex amplification system or the above-mentioned kit in detecting uniparental disomy of human chromosome 7.

[0016] Advantages of the implementation of the present invention: Through the above technical solutions, for the study of the genetic diversity of STR loci, the 7 selected loci are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7. These loci have characteristics such as high individual discrimination power and high polymorphic information content. By judging the STR typing of the human specific region genome and comparing with the parental detection results, UPD of chromosome 7 can be indicated. It can effectively and accurately detect the STR polymorphism of chromosome 7 genome, and can indicate UPD by comparing with the parental detection results. The operation of the present invention is simple, with strong specificity, high sensitivity, strong reliability and low cost. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the amplification map obtained by using the amplification system of the present invention for the child sample of Family 1; Figure 2 It is the amplification map obtained by using the amplification system of the present invention for the father sample of Family 1; Figure 3 It is the amplification map obtained by using the amplification system of the present invention for the mother sample of Family 1; Figure 4 It is the amplification map obtained by using the amplification system of the present invention for the child sample of Family 2; Figure 5 The amplification map obtained by using the amplification system of the present invention for the father sample of pedigree 2; Figure 6 The amplification map obtained by using the amplification system of the present invention for the mother sample of pedigree 2. Detailed implementation manners

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] The present invention interprets the results by comparing the genotypes of the parental samples. In normal inheritance, chromosomes come from both parents. Therefore, the genotype of the child contains both the genotype from the father and the genotype from the mother. If UPD occurs, the genotype comes from one of the parents, that is: if the genotype of the child is completely consistent with that of the mother and there are double peaks at one or more loci, it indicates maternal heterodisomy; if all loci of the child are single peaks and corresponding genotypes can be found in the mother, it indicates maternal isodisomy; in the case of only mother and child samples, all loci are single peaks and there are two or more loci for which corresponding genotypes cannot be found in the mother sample, it indicates paternal isodisomy; in the case of only mother and child samples, there are double peaks at one or more loci and there are two or more loci among all loci for which corresponding genotypes cannot be found in the mother sample, it indicates paternal heterodisomy; in the case of father, mother and child samples, all loci are single peaks and corresponding genotypes can be found in the father, it indicates paternal isodisomy; in the case of father, mother and child samples, there are double peaks at one or more loci and it is completely consistent with the father, it indicates paternal heterodisomy.

[0021] Example 1

[0022] An STR fluorescent labeling multiplex amplification system Screen an STR multiplex amplification system related to the onset of UPD. The multiplex amplification system includes 7 pairs of primers, which can simultaneously amplify 7 loci: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7.

[0023] The primer names, corresponding primer sequences, and numbers are shown in Table 1 below.

[0024] Table 1

[0025] The amplified loci in the amplification system are respectively labeled with two colors of fluorescence. The same fluorescence label is regarded as the same group. The two groups of combinations are: Group 1: P7-1, P7-2, P7-3, fluorescently labeled with FAM; Group 2: P7-4, P7-5, P7-6, P7-7, fluorescently labeled with HEX; Group 3 is the internal standard for this multiplex amplification system, labeled with the orange fluorophore ORG.

[0026] The multiplex amplification system includes a PCR mixture and a DNA template.

[0027] The PCR mixture includes 0.008 M ammonium sulfate, 0.0025 M potassium chloride, 0.065 M Tris-HCl, magnesium ions, 1 mg / mL BSA, 5% DMSO, 6% ethylene glycol, 1 mM Na4P2O7, and 2 U Taq polymerase.

[0028] The amplification program of the multiplex amplification system is denaturation at 95 °C for 5 minutes; denaturation at 94 °C for 10 seconds, annealing at 59 °C for 1 minute and 30 seconds, with 25 cycles; extension at 72 °C for 20 minutes.

[0029] The amplification products of the present invention need to be subjected to capillary electrophoresis for fragment analysis.

[0030] Example 2

[0031] A kit for an STR fluorescent-labeled multiplex amplification system The kit for the STR fluorescent-labeled multiplex amplification system includes the STR fluorescent-labeled multiplex amplification system described in Example 1.

[0032] Example 3

[0033] Selection of STR loci The present invention statistically analyzed 1352 samples of the Chinese Han population, calculated the heterozygosity HET, discrimination power DP, and polymorphic information content PIC data, and selected loci with relatively high correlations. The STR fluorescent-labeled multiplex amplification system provided in this example includes primer pairs for amplifying 7 STR loci on human chromosome 7. The 7 STR loci on human chromosome 7 are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, and P7-7. The primer sequences, primer concentration ranges, and primer sequence numbers for amplifying the 7 STR loci are SEQ ID NO: 1-14, as shown in Table 2 below.

[0034] Table 2

[0035] The statistical results of the genetic data of the 7 STR loci on chromosome 7 selected in the present invention include locus, fluorescent label, chromosomal position, heterozygosity (HET), discrimination power (DP) for individual identification, and polymorphism information content (PIC). The specific results are shown in Table 3 below.

[0036] Table 3

[0037] The loci selected in this example have high polymorphism information, high individual identification rate, and high heterozygosity.

[0038] The primer pairs of the amplified loci in the STR fluorescent label multiplex amplification system in this example are divided into two groups, each fluorescently labeled with two colors. The first group: P7-1, P7-2, P7-3, in which one primer at the 5' end of each is fluorescently labeled with FAM; the second group: P7-4, P7-5, P7-6, P7-7, in which one primer at the 5' end of each is fluorescently labeled with HEX.

[0039] The PCR mixture in the STR fluorescent label multiplex amplification system of the present invention is characterized in that the PCR mixture includes ammonium sulfate, potassium chloride, Tris-HCl, magnesium ions, BSA, DMSO, ethylene glycol, Na4P2O7, and Taq enzyme, and the components and corresponding concentrations are shown in Table 4.

[0040] Table 4

[0041] The amplification program of the STR multiplex amplification system is: denaturation at 95°C for 5 minutes; denaturation at 94°C for 10 seconds, annealing at 59°C for 1 minute and 30 seconds, for 25 cycles; extension at 72°C for 20 minutes.

[0042] The amplified product of the STR multiplex amplification system mixes the QD550 internal standard and formamide in a ratio of 2.5:100. Take 9 μl of the mixture and add it to a 96-well plate, then add 1 μl of the amplified product sample or allelic standard, mix and let stand for several minutes, denature at 95°C for 3 min, immediately ice-bath for 3 min, and centrifuge and then place it on an ABI3500 sequencer for preparation of detection.

[0043] The amplified product of the STR multiplex amplification system is detected using a genetic analyzer of the ABI series. The detection results can be analyzed using data analysis software such as Genemapper to obtain the corresponding STR typing maps and data.

[0044] The STR multiplex amplification system and kit provided by the present invention are applicable to a variety of sample types, including but not limited to DNA samples extracted from human blood, amniotic fluid, aborted tissues, etc. By using the kit provided by the present invention to detect the DNA samples of one or both parents and the DNA sample of the child, and analyzing and interpreting the sample results, key hints can be made for human uniparental disomy.

[0045] Example 4

[0046] STR loci were screened from positions 10 Mb before and after the MEST (q32.2) (130486171 - 130506292) of the UPD target region on chromosome 7. The selected STR loci contain simple repeats of 3 - 5 bases.

[0047] Using Primer Premier 5 software, primers were designed for amplification before and after the core repeat region of the selected STR loci. It was required that the amplification product be a single band during single - locus amplification, and there should be no non - specific amplification products. After the primers were determined, DNA samples were extracted from 1352 samples of the Han population for testing. The heterozygosity, individual polymorphism, and individual discrimination ability of the loci were calculated. Combining the amplification situation and locus efficiency, finally 7 STR loci were selected on chromosome 7: P7 - 1, P7 - 2, P7 - 3, P7 - 4, P7 - 5, P7 - 6, and P7 - 7. The specific positions of the STR loci on the chromosome are shown in Table 2.

[0048] The amplification product lengths of the 7 STR loci in the present invention are in the range of 100 - 550 bp. According to the fragment lengths of the amplification products of each locus, loci with different amplification product lengths were labeled with the same fluorescence, and primers of loci with similar fragment lengths were labeled with two different colors of fluorescence, ensuring that multiple loci can be amplified simultaneously in one tube in the same reaction system. The primers of the 7 loci in the present invention were labeled with two different colors of fluorescence, FAM and HEX, respectively.

[0049] FAM fluorescence labeling: One primer of each of P7 - 1, P7 - 2, and P7 - 3 has a FAM fluorescence label at the 5' end; HEX fluorescence labeling: One primer of each of P7 - 4, P7 - 5, P7 - 6, and P7 - 7 has a HEX fluorescence label at the 5' end.

[0050] The present invention includes 1 STR multiplex amplification system, which is a multiplex amplification system for detecting maternal and paternal UPD of chromosome 7, and includes 7 STR loci: P7 - 1, P7 - 2, P7 - 3, P7 - 4, P7 - 5, P7 - 6, and P7 - 7.

[0051] Example 5

[0052] Obtaining individual STR genotyping using a STR fluorescence-labeled multiplex amplification system constructed with primer pairs for amplifying 7 short tandem repeats 3.1 Collection of blood and amniotic fluid samples (blood and amniotic fluid samples were donated by volunteers) 3.2 DNA extraction Genomic DNA was extracted using the Chelex-100 method. Take 0.5 - 5 μl of anticoagulated whole blood / amniotic fluid and place it in a 500 μl centrifuge tube. Oscillate and mix the Chelex solution to fully suspend Chelex. Add 195 μl of Chelex-100 (5%) solution and 5 μl of proteinase K (20 mg / ml) to each tube, and oscillate and mix well. After incubating at 56 °C for two hours or overnight, take it out and oscillate for 2 minutes, heat in boiling water for 10 minutes, then centrifuge at 13000 rpm for 5 minutes. Carefully transfer 150 μl of the supernatant to a new centrifuge tube.

[0053] 3.3 PCR reaction After oscillating and mixing each reaction reagent (buffer, primer mixture, genomic DNA, etc.), prepare the PCR reaction mixture in the following manner. The total volume of the amplification system is 25 μl, and the specific components and volumes are shown in Table 5 below.

[0054] Table 5

[0055] Among them, the primer components and concentrations are shown in Table 3. The amplification reaction system is: denaturation at 95 °C for 5 minutes; denaturation at 94 °C for 10 seconds, annealing at 59 °C for 1 minute and 30 seconds, for 25 cycles; extension at 72 °C for 20 minutes.

[0056] 3.4 Capillary electrophoresis detection Mix the QD550 internal standard and formamide in a ratio of 2.5:100. Take 9 μl of the mixture and add it to a 96-well plate. Then add 1 μl of the amplified product sample or allele standard, mix and let stand for several minutes. Denature at 95 °C for 3 min, immediately ice-bath for 3 min, and centrifuge, then place it on an ABI3500 sequencer for preparation of detection.

[0057] 3.5 Data analysis Import the original data, analyze the data obtained using the GeneMapperRID-X software and generate a map, as Figure 1-6 shown.

[0058] Example 6

[0059] Perform UPD detection using a STR fluorescence-labeled multiplex amplification system constructed with primer pairs for amplifying 7 short tandem repeats.

[0060] In this test, UPD detection was performed on a group of trios, and the results are shown in Figure 1-3As shown. Using the system of the present invention for identification, the genotyping of each individual in the triad family is shown in Table 6 below.

[0061] Table 6

[0062] The results show that: the child's genotype is exactly the same as the mother's. Among the 7 loci, 4 loci are double-typed, excluding isodisomy. Moreover, no corresponding genotypes of P7-1 and P7-7 can be found in the father's sample, indicating maternal UPD. Combining the above results, it is thus suggested that maternal UPD is suspected in the 7q31-q32 region, and further it may be maternal heterodisomy, which is consistent with the MS-PCR results.

[0063] In this test, UPD detection was performed on a group of triad families, and the results are shown in Figure 4-6 As shown. Using the system of the present invention for identification, the genotyping of each individual in the triad family is shown in Table 7 below.

[0064] Table 7

[0065] The results show that: the child's genotype comes from both parents. Among the 7 loci, 6 loci are double-typed. The 7 loci are neither exactly the same as the mother's nor exactly the same as the father's, which belongs to normal inheritance and is consistent with the MS-PCR results.

[0066] Advantages of the implementation of the present invention: Through the above technical solution, for the study of the genetic diversity of STR loci, the 7 selected loci are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7. These loci have characteristics such as high individual discrimination power and high polymorphic information content. By judging the STR genotyping of the human specific region genome and comparing with the parental detection results, it is possible to suggest UPD of chromosome 7. It can effectively and accurately detect the STR polymorphism of the chromosome 7 genome, and by comparing with the parental detection results, it is possible to suggest UPD. The operation of the present invention is simple, with strong specificity, high sensitivity, strong reliability and low cost.

[0067] As mentioned above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A STR fluorescence-labeled multiplex amplification system, characterized in that, It includes seven primer pairs designed for seven STR loci on human chromosome 7, and the seven STR loci on human chromosome 7 are: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7; Each primer pair carries a fluorescent dye label; the upstream and downstream primer sequences for amplifying P7-1 are shown in SEQ ID NO.1 and SEQ ID NO.2; the upstream and downstream primer sequences for amplifying P7-2 are shown in SEQ ID NO.3 and SEQ ID NO.4; the upstream and downstream primer sequences for amplifying P7-3 are shown in SEQ ID NO.5 and SEQ ID NO.6; the upstream and downstream primer sequences for amplifying P7-4 are shown in SEQ ID NO.7 and SEQ ID NO.8; the upstream and downstream primer sequences for amplifying P7-5 are shown in SEQ ID NO.9 and SEQ ID NO.10; the upstream and downstream primer sequences for amplifying P7-6 are shown in SEQ ID NO.11 and SEQ ID NO.12; the upstream and downstream primer sequences for amplifying P7-7 are shown in SEQ ID NO.13 and SEQ ID NO.

14.

2. The STR fluorescent labeling multiplex amplification system according to claim 1, characterized in that The concentration range of the primer pair corresponding to the P7-1 locus for amplification is 0.01 - 0.1 μM; the concentration range of the primer pair corresponding to the P7-2 locus for amplification is 0.05 - 0.1 μM; the concentration range of the primer pair corresponding to the P7-3 locus for amplification is 0.05 - 0.2 μM; the concentration range of the primer pair corresponding to the P7-4 locus for amplification is 0.05 - 0.15 μM; the concentration range of the primer pair corresponding to the P7-5 locus for amplification is 0.15 - 0.3 μM; the concentration range of the primer pair corresponding to the P7-6 locus for amplification is 0.2 - 0.7 μM; the concentration range of the primer pair corresponding to the P7-7 locus for amplification is 0.5 - 0.9 μM.

3. The STR fluorescent labeling multiplex amplification system according to claim 1, wherein In the STR fluorescence-labeled multiplex amplification system, the primer pairs of the amplified loci are respectively labeled with three colors of fluorescence. The same fluorescence label is regarded as the same group, and the three groups of combinations are: The first group: P7-1, P7-2, P7-3; The second group: P7-4, P7-5, P7-6, P7-7; The third group: the internal standard primer used in the multiplex amplification system.

4. The STR fluorescent labeling multiplex amplification system according to claim 3, wherein For the fluorescence label, the first group is labeled with FAM, the second group is labeled with HEX, and the third group is labeled with ORG.

5. The STR fluorescent labeling multiplex amplification system according to claim 1, characterized in that The STR fluorescence-labeled multiplex amplification system includes a PCR mixture and a DNA template.

6. The PCR mixture according to claim 5, wherein The PCR mixture includes ammonium sulfate, potassium chloride, Tris-HCl, magnesium ions, BSA, DMSO, ethylene glycol, Na4P2O7 and Taq enzyme.

7. The STR fluorescent labeling multiplex amplification system according to claim 1, characterized in that, The reaction conditions of the STR fluorescence-labeled multiplex amplification system: denaturation at 95 °C for 5 minutes; denaturation at 94 °C for 10 seconds, annealing at 59 °C for 1 minute and 30 seconds, cycling 25 times; extension at 72 °C for 20 minutes.

8. The STR fluorescent labeling multiplex amplification system according to claim 5, wherein The DNA template is derived from human blood, amniotic fluid or aborted tissue.

9. A kit, characterized in that, The kit includes the STR fluorescent-labeled multiplex amplification system according to any one of claims 1-8.

10. Use of the STR fluorescent-labeled multiplex amplification system according to any one of claims 1-8 in the preparation of a kit for indicating uniparental disomy.

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