STR fluorescent labeling composite amplification system, kit and application thereof

Abstract

The application relates to the field of biotechnology and discloses a STR (short tandem repeat) fluorescence labeling compound amplification system, a kit and application of the STR fluorescence labeling compound amplification system to detection of uniparental disomy of chromosome 7. The STR fluorescence labeling compound amplification system comprises seven pairs of primers, each pair of primers carries a fluorescent dye label, and primer sequences for the seven sites are disclosed, and seven sites can be simultaneously amplified. The STR fluorescence labeling compound amplification system can effectively and accurately detect STR polymorphism of a specific region genome, can prompt uniparental disomy of a corresponding segment by comparison with a parent detection result, can significantly reduce a false positive rate, can effectively improve detection accuracy and reliability, and has important clinical application value and wide market prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a STR fluorescence labeling compound amplification system, a kit and application. BACKGROUND

[0002] Uniparental Disomy (UPD) refers to two homologous chromosomes being inherited from one parent. UPD can be a certain fragment on a chromosome or an entire chromosome. Whether UPD leads to the occurrence of a clinical phenotype depends on whether it exists on a chromosome affected by imprinting or whether it leads to the occurrence of a related recessive disease. The incidence of UPD of all chromosomes (not just chromosomes with imprinting regions) is 1 / 2000.

[0003] The main cause of maternal UPD (7) is the meiotic error of the mother, accounting for about 71%.

[0004] The existing UPD detection methods are: SNP probe detection, whole exon sequencing or whole genome sequencing, methylation PCR and MLPA. The CMA platform with SNP probes can be used for UPD detection, the SNP distribution is analyzed, and an algorithm is used to detect UPD, but it cannot distinguish UPD from ROH caused by close genetic inheritance, and needs to be combined with family verification. UPD can be judged by whole exon sequencing or whole genome sequencing, but the cost is high and the data analysis is complex. In addition, for imprinting gene diseases, methylation PCR and MLPA have certain significance for etiological detection, the principle is to analyze the differential methylation region or imprinting center methylation state in a larger region (usually several megabases) of the chromosome, but it needs to be combined with genotype analysis to distinguish UPD and methylation defects.

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

[0006] In view of the deficiencies existing in the prior art, the present application provides an STR fluorescently labeled composite amplification system, a kit and an application. The STR fluorescently labeled composite amplification system has two-color fluorescent labeling, and the sites used in the present application have extremely high individual identification rate and polymorphism, and can perform screening of 7-chromosome uniparental disomy (UPD) STR sites. By comparing with the detection results of parents, UPD of the 7-autosome can be prompted, and the specificity is high and the sensitivity is high.

[0007] To achieve the above-mentioned purpose, the embodiments of the present application adopt the following technical solutions:

[0008] An STR fluorescently labeled composite amplification system, the amplification system comprising 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 fluorescently labeled composite amplification system can simultaneously amplify seven STR sites: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. The primer sequences for amplifying the seven STR sites, the primer concentration range and the primer sequence numbers for amplifying the seven STR sites are SEQ ID NO: 1-14, as shown in Table 2.

[0009] According to one aspect of the present application, the sites in the STR fluorescently labeled composite amplification system are labeled by three kinds of fluorescence: the first group is P7-1, P7-2, P7-3, and the fluorescence label is FAM label; the second group is P7-4, P7-5, P7-6, P7-7, and the fluorescence label is HEX label; the third group is the internal standard primer used in the composite amplification system, and the fluorescence label is ORG label.

[0010] According to one aspect of the present application, the STR fluorescently labeled composite amplification system comprises a PCR mixture and a DNA template.

[0011] According to one aspect of the present application, the PCR mixture comprises ammonium sulfate, potassium chloride, Tris-HCl, magnesium ion, BSA, DMSO, ethylene glycol, Na4P2O7 and Taq enzyme, and specifically can be: 12 mM of ammonium sulfate, 8 mM of potassium chloride, 65 mM of Tris-HCl with pH 8.5, 2.5 mM of magnesium ion, 1 ug / ul of BSA, 5% of DMSO, 6% of ethylene glycol, 1 mM of Na4P2O7, 0.25 mM of dNTP and 2U of Taq enzyme, and the ingredients and corresponding concentrations are shown in Table 4.

[0012] According to one aspect of the present application, the reaction procedure of the STR fluorescent labeling compound amplification system is as follows: denaturation at 95 DEG C for 5 minutes; denaturation at 94 DEG C for 10 seconds, annealing at 59 DEG C for 1 minute and 30 seconds, and cycling for 25 times; and extension at 72 DEG C for 20 minutes.

[0013] According to one aspect of the present application, the DNA template used in the amplification system is derived from human blood, amniotic fluid or abortion tissue.

[0014] According to one aspect of the present application, the DNA template can be extracted by a kit method, a phenol chloroform extraction method, a Chelex-100 method or a magnetic bead method.

[0015] A kit comprising the above-mentioned STR fluorescent labeling compound amplification system.

[0016] The above-mentioned STR fluorescent labeling compound amplification system or the above-mentioned kit is used for detecting monoparental disomy of human chromosome 7.

[0017] Advantages of the present application: through the above technical solution, the genetic diversity of STR loci is researched, and seven selected sites are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. These sites have characteristics such as high individual identification and high polymorphism information content. Through STR typing of human specific region genome, compared with the detection result of parents, UPD of chromosome 7 can be prompted. The present application can effectively and accurately detect STR polymorphism of chromosome 7 genome, and compared with the detection result of parents, UPD can be prompted. The present application has the advantages of simple operation, strong specificity, high sensitivity, strong reliability and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Fig. 1 The amplification spectrum obtained by the amplification system of the present application for the child sample of family 1;

[0020] Fig. 2 The amplification spectrum obtained by the amplification system of the present application for the father sample of family 1;

[0021] Fig. 3 The amplification spectrum obtained by the amplification system of the present application for the mother sample of family 1;

[0022] Fig. 4An amplification map obtained by using the amplification system of the present application for the sample of the child of family 2;

[0023] Fig. 5 An amplification map obtained by using the amplification system of the present application for the sample of the father of family 2;

[0024] Fig. 6 An amplification map obtained by using the amplification system of the present application for the sample of the mother of family 2. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0026] The present application is to compare the typing of the samples of the parents to interpret the results. In normal inheritance, the chromosomes come from both parents, so the typing of the child has the typing from the father and the typing from the mother. If UPD occurs, the typing comes from one of the parents, i.e., if the typing of the child is completely consistent with that of the mother and there is a double peak at one or more sites, it is suggested that the child is a maternal heterodisomy; if all the sites of the child are single peaks and the corresponding typing can be found in the mother, it is suggested that the child is a maternal homodisomy; in the case of only the mother and the child, if all the sites are single peaks and there are two or more sites for which the corresponding typing cannot be found in the mother sample, it is suggested that the child is a paternal homodisomy; in the case of only the mother and the child, if there is a double peak at one or more sites and there are two or more sites for which the corresponding typing cannot be found in the mother sample, it is suggested that the child is a paternal heterodisomy; in the case of the samples of the parents and the child, if all the sites are single peaks and the corresponding typing can be found in the father, it is suggested that the child is a paternal homodisomy; in the case of the samples of the parents and the child, if there is a double peak at one or more sites and the typing is completely consistent with that of the father, it is suggested that the child is a paternal heterodisomy.

[0027] Embodiment I

[0028] A STR fluorescence labeling composite amplification system

[0029] The STR composite amplification system for screening UPD pathogenic related chromosomes comprises 7 pairs of primers, which can simultaneously amplify 7 sites: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7.

[0030] The primer names and the corresponding primer sequences and numbers are shown in Table 1.

[0031] Table 1

[0032]

[0033] The amplified sites in the amplification system are respectively marked by two colors of fluorescence, the same fluorescence marker is regarded as the same group, and the two groups are combined as follows:

[0034] The first group: P7-1, P7-2, P7-3, and the fluorescence marker is FAM marker;

[0035] The second group: P7-4, P7-5, P7-6, P7-7, and the fluorescence marker is HEX marker;

[0036] The third group is an internal standard for detecting the composite amplification system, which is marked by an orange fluorescein marker ORG.

[0037] The composite amplification system comprises a PCR mixture and a DNA template.

[0038] The PCR mixture comprises 0.008M of ammonium sulfate, 0.0025M of potassium chloride, 0.065M of Tris-HCl, magnesium ions, 1mg / mL of BSA, 5% of DMSO, 6% of ethylene glycol, 1mM of Na4P2O7 and 2U of Taq enzyme.

[0039] The amplification procedure of the composite amplification system is denaturation at 95℃ for 5 minutes, denaturation at 94℃ for 10 seconds, annealing at 59℃ for 1 minute and 30 seconds, and cycling for 25 times, and extension at 72℃ for 20 minutes.

[0040] The amplification product of the application needs to be subjected to capillary electrophoresis, so as to perform fragment analysis.

[0041] Example 2

[0042] A STR fluorescence marker composite amplification system kit

[0043] The STR fluorescence marker composite amplification system kit comprises the STR fluorescence marker composite amplification system described in Example 1.

[0044] Example 3

[0045] Selection of STR sites

[0046] The application carries out statistics on 1352 samples of Chinese Han population, calculates heterozygosity HET, individual identification DP and polymorphic information content PIC data, and selects sites with higher correlation. The STR fluorescence labeling composite amplification system provided in the embodiment comprises primer pairs for amplifying 7 STR sites on human chromosome 7. The 7 STR sites on human chromosome 7 are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. The primer sequences for amplifying the 7 STR sites, the primer concentration range and the primer sequence numbers for amplifying the 7 STR sites are SEQ ID NO: 1-14, as shown in Table 2 below.

[0047] Table 2

[0048]

[0049] The genetic data statistics results of the 7 selected autosomal STR sites on chromosome 7 include site, fluorescence labeling, chromosome location, heterozygosity HET, individual identification DP and polymorphic information content PIC, and the specific results are shown in Table 3 below.

[0050] Table 3

[0051]

[0052] The selected sites in the embodiment have higher polymorphic information and individual identification rate and higher heterozygosity.

[0053] The primer pairs of the amplified sites in the STR fluorescence labeling composite amplification system in the embodiment are divided into two groups, which are labeled with two colors of fluorescence, the first group: P7-1, P7-2, P7-3, each of which has one primer 5' labeled with FAM label; the second group: P7-4, P7-5, P7-6, P7-7, each of which has one primer 5' labeled with HEX label.

[0054] The PCR mixture in the STR fluorescence labeling composite amplification system of the application, characterized in that the PCR mixture comprises 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.

[0055] Table 4

[0056]

[0057] The amplification procedure of the STR composite amplification system is: denaturation at 95℃ for 5 minutes; denaturation at 94℃ for 10 seconds, annealing at 59℃ for 1 minute and 30 seconds, and cycling for 25 times; extension at 72℃ for 20 minutes.

[0058] The amplification product of the STR composite amplification system is mixed with QD550 internal standard and formamide at a ratio of 2.5:100, 9 μl of the mixture is added to a 96-well plate, 1 μl of the amplification product sample or allele standard is added, the mixture is mixed and left for several minutes, denaturation is performed at 95℃ for 3 min, then ice bath is performed for 3 min, and then the sample is placed on an ABI 3500 sequencer for detection.

[0059] The amplification product of the STR composite amplification system is detected by using an ABI series genetic analyzer. The detection result can be analyzed on a Genemapper or other data analysis software to obtain a corresponding STR typing map and data.

[0060] The STR composite amplification system and the kit provided by the application are suitable for various types of detection materials, including but not limited to DNA samples extracted from human blood, amniotic fluid, abortion tissue and the like. The DNA samples of one or both parents and the child are detected by using the kit provided by the application, and the sample results are analyzed and interpreted, so that a key hint can be given to human single-parent disomy.

[0061] Example Four

[0062] STR sites are screened from positions 10 Mb before and after the UPD target segment MEST (q32.2) (130486171-130506292) of chromosome 7, and the selected STR sites contain simple repeats of 3-5 bases.

[0063] Primers are designed by using Primer Premier 5 software before and after the core repeat region of the selected STR sites for amplification. It is required that the amplification product is a single band when a single locus is amplified, and no non-specific amplification product exists. After the primers are determined, DNA samples of 1352 samples of the Han population are tested, the heterozygosity, individual polymorphism and individual identification ability of the sites are calculated, and finally, 7 STR sites of chromosome 7 are selected: P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. The specific positions of the STR sites on the chromosome are shown in Table 2.

[0064] The amplification product lengths of the 7 STR sites in the application are in the range of 100-550 bp. According to the lengths of the amplification product fragments of each site, the same fluorescently labeled different amplification product fragment length sites are used, and the primers of the sites with similar fragment lengths are labeled with two different colors of fluorescence, so that multiple sites can be amplified in one tube in the same reaction system. The primers of the 7 sites in the application are labeled with FAM and HEX, which are two different colors of fluorescence.

[0065] FAM fluorescence label: P7-1, P7-2, P7-3 each has a primer 5' end with FAM fluorescence label;

[0066] HEX fluorescence label: P7-4, P7-5, P7-6, P7-7 each has a primer 5' end with HEX fluorescence label.

[0067] The application comprises one STR composite amplification system, which is a composite amplification system for detecting maternal and paternal chromosome 7 UPD, and comprises seven STR sites P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7.

[0068] Example five

[0069] Obtaining individual STR typing by using the STR fluorescence label composite amplification system constructed by the primer pair for amplifying seven short tandem repeat sequences

[0070] 3.1 Collection of blood and amniotic fluid samples (blood and amniotic fluid samples are donated by volunteers)

[0071] 3.2 DNA extraction

[0072] Chelex-100 method is used to extract genomic DNA, 0.5-5 μl of anticoagulated whole blood / amniotic fluid is placed in a 500 μl centrifuge tube, Chelex solution is mixed and shaken to fully suspend Chelex, 195 μl of Chelex-100 (5%) solution and 5 μl of protease K (20 mg / ml) are added to each tube and mixed and shaken, and then incubated at 56 degrees Celsius for two hours or overnight, then shaken for two minutes, heated in boiling water for 10 minutes, and then centrifuged at 13000 rpm for 5 minutes, and 150 μl of supernatant is carefully transferred to a new centrifuge tube.

[0073] 3.3 PCR reaction

[0074] After mixing and shaking the reaction reagents (buffer, primer mixture, genomic DNA, etc.), the PCR reaction mixture is prepared as follows, and the total volume of the amplification system is 25 μl. The specific components and volumes are shown in Table 5 below.

[0075] Table 5

[0076]

[0077] The primer components and concentrations are shown in Table 3, and the amplification reaction system is as follows: denaturation at 95 degrees Celsius for 5 minutes; denaturation at 94 degrees Celsius for 10 seconds, annealing at 59 degrees Celsius for 1 minute and 30 seconds, and cycling 25 times; and extension at 72 degrees Celsius for 20 minutes.

[0078] 3.4 Capillary electrophoresis detection

[0079] Mix QD550 internal standard and formamide in proportion of 2.5:100, take 9 μl of the mixture and add to 96-well plate, then add 1 μl of amplification product sample or allele standard, mix and stand for several minutes, denature at 95℃ for 3 min, immediately ice bath for 3 min, centrifuge and put on ABI 3500 sequencer for detection.

[0080] 3.5 Data analysis

[0081] Import raw data, analyze data obtained by using GeneMapper RID-X software and generate a map as shown in Figs. 1-6 .

[0082] Example 6

[0083] UPD detection was performed on a set of triad families by using the constructed STR fluorescence-labeled composite amplification system of 7 short tandem repeat sequences.

[0084] UPD detection was performed on a set of triad families in the test, and the results are shown in Figs. 1-3 . Each individual of the triad family was typed by using the system of the application, and the typing results are shown in Table 6.

[0085] Table 6

[0086]

[0087] The results show that the child typing is completely consistent with the mother, 4 of the 7 loci are double typed, excluding homozygosity, and P7-1 and P7-7 cannot be found in the father sample, suggesting maternal UPD, and in combination with the above results, it is thus suggested that maternal UPD of the 7q31-q32 segment is suspected, and further possibly maternal heterozygosity, which is consistent with the MS-PCR results.

[0088] UPD detection was performed on a set of triad families in the test, and the results are shown in Figs. 4-6 . Each individual of the triad family was typed by using the system of the application, and the typing results are shown in Table 7.

[0089] Table 7

[0090]

[0091] The results show that the child typing is from both parents, 6 of the 7 loci are double typed, and the 7 loci are neither completely consistent with the mother nor completely consistent with the father, which belongs to normal inheritance, and is consistent with the MS-PCR results.

[0092] The advantages of the embodiment of the present application are as follows: through the above technical scheme, the genetic diversity of the STR locus is researched, and the selected seven sites are P7-1, P7-2, P7-3, P7-4, P7-5, P7-6 and P7-7. These sites have characteristics such as high individual identification and high polymorphic information content. Through the STR typing of the specific region genome of human and the comparison with the detection result of the parents, the UPD of the No. 7 autosome can be prompted. The No. 7 autosome genome STR polymorphism can be effectively and accurately detected, and the UPD can be prompted through the comparison with the detection result of the parents. The present application has the advantages of simple operation, high specificity, high sensitivity, high reliability, low cost and the like.

[0093] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A STR fluorescent label multiplex amplification system, characterized in that, The STR fluorescence-labeled complex amplification system comprises seven primer pairs designed for seven STR loci on human chromosome 7, i.e. 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; and the upstream and downstream primer sequences for amplifying P7-7 are shown in SEQ ID NO. 13 and SEQ ID NO.

14. The primer pairs of the amplified loci in the STR fluorescence-labeled complex amplification system are labeled by three colors of fluorescence, and the same fluorescence label is regarded as the same group, and the three groups are combined as follows: The first group: P7-1, P7-2 and P7-3, and the fluorescence label is FAM label; The second group: P7-4, P7-5, P7-6 and P7-7, and the fluorescence label is HEX label; The third group: the internal standard primer used in the complex amplification system, and the fluorescence label is ORG label.

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 is 0.01-0.1 μM; the concentration range of the primer pair corresponding to the P7-2 locus is 0.05-0.1 μM; the concentration range of the primer pair corresponding to the P7-3 locus is 0.05-0.2 μM; the concentration range of the primer pair corresponding to the P7-4 locus is 0.05-0.15 μM; the concentration range of the primer pair corresponding to the P7-5 locus is 0.15-0.3 μM; the concentration range of the primer pair corresponding to the P7-6 locus is 0.2-0.7 μM; and the concentration range of the primer pair corresponding to the P7-7 locus is 0.5-0.9 μM. 3.The STR fluorescent labeling complex amplification system according to claim 1, characterized in that, The STR fluorescence-labeled complex amplification system comprises a PCR mixture and a DNA template.

4. The STR fluorescent labeling complex amplification system according to claim 3, characterized in that, The PCR mixture comprises ammonium sulfate, potassium chloride, Tris-HCl, magnesium ions, BSA, DMSO, ethylene glycol, Na4P2O7 and Taq enzyme.

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

6. The STR fluorescent labeling complex amplification system according to claim 5, characterized in that, The DNA template is derived from human blood, amniotic fluid or abortion tissue.

7. A kit characterized in that, The kit comprises the STR fluorescence-labeled complex amplification system according to any one of claims 1-6.

8. Use of the STR fluorescent labeling multiplex amplification system according to any one of claims 1-6 in the preparation of a kit for indicating uniparental disomy.

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