Composite amplification system, kit and application of 28 short tandem repeats

Abstract

The present invention relates to the field of biotechnology, and relates to a multiplex amplification system, a kit and an application for 28 short tandem repeats. The multiplex amplification system includes 28 pairs of primers, which can simultaneously amplify 28 loci, and the primer sequences for the 28 loci are disclosed. Through the study of the genetic diversity of STR loci, the 28 loci selected by the present invention are located in the imprinted gene segments of the corresponding chromosomes. These loci have characteristics such as high individual discrimination power and high polymorphic information content, and can effectively and accurately detect the STR polymorphisms of the genome in a specific region. By comparing with the detection results of the parents, uniparental disomy in the corresponding segment can be indicated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and a multiplex amplification system for 28 short tandem repeats on human autosomes. Specifically, the system relates to a fluorescence detection kit for multiplex amplification by polymerase chain reaction using polymorphic genetic markers on chromosomes. The system can perform STR genotyping on specific regions of the human genome and, by comparing with the detection results of parents, can prompt uniparental disomy in the corresponding segments. Background Art

[0002] Uniparental Disomy (UPD): It refers to two homologous chromosomes being 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 chromosomes 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] UPD is usually caused by two non-disjunction events. The first occurs during meiosis, and the second occurs during mitosis. It is mainly divided into two categories: uniparental isodisomy and uniparental heterodisomy. Uniparental heterodisomy: Non-disjunction in meiosis I means that two homologous chromosomes cannot separate, resulting in an increased probability of two different homologous chromosomes from the same parent or uniparental heterodisomy. Uniparental isodisomy: Non-disjunction in meiosis II refers to the failure of sister chromatids to divide into daughter cells, resulting in uniparental isodisomy.

[0004] Trisomy rescue mechanism: A mutated disomic gamete during meiosis fertilizes with a normal gamete to form a trisomic zygote. Subsequently, during cleavage, chromosomal anaphase lag occurs and one of the chromosomes is lost, and the trisomy becomes disomic. The lost chromosome is the one from the normal gamete, and the two chromosomes retained in the zygote are isodisomic, and the trisomic zygote becomes uniparental disomy. If the lost chromosome is one of the disomic gametes, the two chromosomes retained in the zygote are heterodisomic, and the cells containing heterodisomy continue to divide to generate normal cells. Heterodisomy enables the embryo to grow and develop normally or significantly reduces the teratogenicity of the original trisomy, allowing the embryo to survive. Theoretically, the probability of uniparental diploidy occurring due to trisomy rescue is 1 / 3.

[0005] Monosomy rescue mechanism: A nullisomic gamete fertilizes with a normal gamete to form a haploid zygote. Subsequently, to maintain balance, during cleavage, a single chromosome replicates to become a diploid zygote, that is, the monosomy rescue after the appearance of a UPD zygote (rarer than trisomy rescue) will result in complete alleles of the same homolog, without a heterozygous region.

[0006] The most classic method for UPD detection is STR analysis. STR markers are very abundant throughout the genome, and many have a very high heterozygosity, which reflects the differences in allele frequencies in the population. CMA platforms with SNP probes can also be used for UPD detection. In the case of whole exome or whole genome sequencing, by analyzing the SNP distribution in pedigrees, algorithms can also be used to detect UPD. In addition, for imprinting gene diseases, methylation PCR and MLPA are of certain significance for etiological detection. The principle is to analyze the differential methylation regions or the methylation status of imprinting centers within larger chromosomal regions (usually several megabases).

[0007] Human genomic STR (short tandem repeat) is a DNA sequence that is relatively stable in genetic inheritance and is formed by tandem repeats with several bases as the core unit. The differentiation between different races, different populations, and even different individuals is through the 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, mostly existing 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 Mendel's law.

[0008] The detection method provided by the present invention can effectively and accurately detect the STR polymorphism of the genomic specific region. By comparing with the detection results of the parents, it can prompt the UPD of the corresponding section. In addition, it also has the detection characteristics of simple operation, strong specificity, high sensitivity, high throughput, strong reliability, and low cost. Summary of the Invention

[0009] The present invention provides a multiplex amplification system, a kit and its application for 28 short tandem repeats, with six-color fluorescence labeling and extremely high sensitivity. When using low-concentration DNA as a template, a complete genotyping map can be detected. The loci adopted by the present invention have extremely high individual recognition rates and polymorphisms. By comparing with the detection results of the parents, it can prompt the UPD of the corresponding section.

[0010] In order to achieve the above-mentioned invention purposes, the technical solutions adopted by the present invention are as follows:

[0011] Composite amplification system for 28 short tandem repeats, said composite amplification system comprising 28 pairs of primers, capable of simultaneously amplifying 28 STR loci: A6-1, A6-2, A6-3, A6-4, A7-1, A7-2, A7-3, A7-4, A11-1, A11-2, A11-3, A11-4, A14-1, A14-2, A14-3, A14-4, A15-1, A15-2, A15-3, A15-4, A20-1, A20-2, A20-3, A20-4, A20-5, A20-6, A20-7, A20-8.

[0012] The primers and their corresponding primer concentrations are as follows (Table 1):

[0013] Table 1: Primer sequences and concentration ratios of each locus in the composite amplification system

[0014]

[0015]

[0016] The amplified loci in the amplification system are respectively labeled with five colors of fluorescence. The same fluorescence label is regarded as the same group. The five groups of combinations are as follows:

[0017] FAM: A6-1, A6-2, A6-3, A6-4, A7-1;

[0018] HEX: A7-2, A7-3, A7-4, A11-1, A11-2, A11-3, A11-4;

[0019] TAMRA: A14-1, A14-2, A14-3, A14-4, A15-1;

[0020] ROX: A15-2, A15-3, A15-4, A20-1, A20-2;

[0021] PURPLE: A20-3, A20-4, A20-5, A20-6, A20-7, A20-8.

[0022] The six groups of fluorescence labels are FAM, HEX, TAMRA, ROX, PURP and ORG.

[0023] The first group is labeled with FAM label, the second group is labeled with HEX label, the third group is labeled with TAMRA label, the fourth group is labeled with ROX label, and the fifth group is labeled with PURP label. The internal standard for detecting with this composite amplification system is labeled with orange fluorescein label ORG, which is the sixth group of fluorescence label.

[0024] The amplification system also includes PCR Master Mix and template DNA.

[0025] The PCR Master Mix includes: 10 mM ammonium sulfate, 10 mM potassium chloride, 60 mM Tris-HCl with a pH of 8.3, 2.5 mM magnesium ions, 0.9 μg / μl BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na4P2O7, 0.25 mM Dntp, and 2 U Taq enzyme. It can be unfrozen at -20°C to prevent repeated freezing and thawing during use.

[0026] The amplification procedure of the multiplex amplification system is as follows: The first step is denaturation: 95°C for 5 minutes; thermal cycling: 94°C for 20 seconds; 60°C for 120 seconds, for a total of 30 cycles; final extension at 72°C for 10 minutes; incubation: 15°C. The amplification products of the present invention need to be subjected to capillary electrophoresis for fragment analysis.

[0027] The above amplification system or kit can be applied in the indication of uniparental disomy.

[0028] The reaction buffer (5*Master Mix S) used in the multiplex amplification system involved in the present invention has a special formula (Table 2)

[0029] Table 2: Formula of reaction buffer (5*Master Mix S)

[0030]

[0031]

[0032] The PCR amplification procedure (Table 3) used in the multiplex amplification system involved in the present invention.

[0033] Table 3: Amplification procedure of the multiplex amplification system of the present invention

[0034]

[0035] The DNA samples involved in the present invention can be derived from human blood, amniotic fluid, aborted tissues, etc. The DNA samples can be subjected to DNA extraction and treatment by the kit method, phenol-chloroform extraction method, Chelex-100 method, and magnetic bead method.

[0036] The amplification products of the present invention can be detected using ABI series genetic analyzers. The detection results can be analyzed using data analysis software such as Genemapper to obtain the corresponding STR typing maps and data.

[0037] The present invention statistically analyzed 1,523 samples from the Chinese Han population, calculated relevant data, and proved that the selected loci of the present invention have high polymorphism information and a relatively high individual recognition rate. The statistical results of the genetic data of 28 autosomal STRs used in the present invention (Table 4).

[0038] Table 4 Statistical results of the genetic data of 28 loci used in the present invention

[0039]

[0040]

[0041] Among them, HET: the abbreviation of Heterozygotie, heterozygosity, used to measure gene polymorphism; DP: the abbreviation of Power of Discrimination, individual recognition ability; PIC: the abbreviation of Polymorphism information content, polymorphism information content.

[0042] Beneficial effects

[0043] Through the study of the genetic diversity of STR loci, the present invention selected 28 loci: A6-1, A6-2, A6-3, A6-4, A7-01, A7-2, A7-3, A7-4, A11-1, A11-2, A11-3, A11-4, A14-1, A14-2, A14-3, A14-4, A15-1, A15-2, A15-3, A15-4, A20-1, A20-2, A20-3, A20-4, A20-5, A20-6, A20-7, A20-8. These loci have characteristics such as high individual recognition ability and high polymorphism information content. By judging the STR typing of the human specific region genome and comparing with the parental detection results, the UPD of the corresponding section can be prompted. In addition, it also has the detection characteristics of simple operation, strong specificity, high sensitivity, high throughput, strong reliability and low cost.

[0044] The reaction buffer (5*Master Mix S) used in the multiplex amplification system involved in the present invention has a special formula, does not freeze at -20°C, and has a short PCR amplification program time and high amplification efficiency. The DNA samples involved in the present invention can be derived from human blood, amniotic fluid, aborted tissues, etc.

[0045] The present invention statistically analyzed 1,523 samples from the Chinese Han population, calculated relevant data, and proved that the selected loci of the present invention have high polymorphism information and a relatively high individual recognition rate. Brief description of the drawings

[0046] Figure 1The amplification map obtained for the father sample using the amplification system of the present invention;

[0047] Figure 2 The amplification map obtained for the mother sample using the amplification system of the present invention;

[0048] Figure 3 The amplification map obtained for the child sample using the amplification system of the present invention; Detailed implementation manners

[0049] Example 1 Obtaining individual STR typing using a multiplex amplification system of 28 short tandem repeats

[0050] 1. Collection of blood and amniotic fluid samples (blood and amniotic fluid samples were donated by volunteers)

[0051] 2. DNA extraction

[0052] Genomic DNA was extracted using the Chelex-100 method (refer to "Forensic DNA Protocol". Humana Press, 1998). 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, 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. Reaction system

[0054] After oscillating and mixing each reaction reagent (buffer, primer mixture, genomic DNA, etc.), the PCR reaction mixture was prepared in the following manner. The total volume of the amplification system was 25 μl, including 5 μl of primer mixture (5*Primer mix), where the primer concentrations are shown in Table 2, 5 μl of reaction buffer (5*Master Mix S), 2 μl of genomic DNA (extracted template DNA, direct amplification sample, etc.), and 13 μl of ddH2O.

[0055] 4. PCR reaction program

[0056] The PCR amplification program used for the multiplex amplification system involved in the present invention (Table 5).

[0057] Table 5 Amplification program of the multiplex amplification system of the present invention

[0058]

[0059] 5. Capillary electrophoresis detection

[0060] Mix the Orange500 internal standard and formamide in a ratio of 2.5:100. Take 12.5 μl of the mixture and add it to a 96-well plate. Then add 1 μl of the amplification 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, centrifuge, and then place it on an ABI3500 sequencer for detection preparation.

[0061] 6. Data analysis

[0062] Import the original data. In the File menu on the main page, select Add sample to project, find the sample file, select the folder, click add to list, and then click add. The sample file will be displayed in the Project window. Select the analysis parameters. Define the analysis method, panel, and size standard. Browse the original data of the sample electrophoresis. Select the file name of a certain sample. Under the "sample" menu, select "Raw data". Move the tracking line to stop the cursor on the right side of the primer peak (before the first orange internal standard peak). Use the value displayed on the X-axis at the lower left corner of the window at this time as the starting point in the analysis parameters of the analysis method. Click the green analysis button. A save project dialog box will appear. After naming and saving, the software will start processing the data. After the analysis is completed, analysis completed will be displayed at the lower left corner. Analyze the data obtained using the GeneMapperRID-X software and generate a map, as Figures 1 to 3 .

[0063] Example 2 Detection of UPD using a multiplex amplification system of 28 short tandem repeats

[0064] In this test, a group of triploid families were subjected to UPD detection. The results are shown in Figures 1 to 3 . The genotyping of each individual in the triploid family obtained by identification using the system of the present invention is as follows (Table 6):

[0065] Table 6: STR genotyping of individuals in triploid families

[0066] father mother child A6-1 9，9.3 9 9，9.3 A6-2 10，11 12，13 11，13 A6-3 30，31 29，31 29，31 A6-4 16，21 17，18 16，18 A7-1 10，18 19 19 A7-2 10，11 11，12 11 A7-3 15，18 15，17 15 A7-4 9，10 8，10 8 A11-1 8，12 11 8，11 A11-2 9 11，12 9，12 A11-3 10，11 11，12 11，12 A11-4 10，12 8，9 9，12 A14-1 10，11 10，11 11 A14-2 14，16 18，19 14，19 A14-3 12，13 13，15 13 A14-4 8，11 8，11 8，11 A15-1 13，21 18 18，21 A15-2 14 13，15.2 14，15.2 A15-3 11，17 11，15 11，15 A15-4 17，24 23 17，23 A20-1 22，24 23 23，24 A20-2 17，18 14 14，18 A20-3 13，15 13 13 A20-4 13，16 13，17.3 13，17.3 A20-5 18,19 18,20 18 A20-6 13，14 13 13，14 A20-7 17 20 17，20 A20-8 18,21 21,22 21

[0067] Analyze the triploid family. Maternal UPD is suspected in the 7p13-q32 region, and other detection methods can be further used for further detection.

[0068] The above embodiments are only preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and equivalent replacements can be made. These technical solutions obtained by improving and equivalently replacing the claims of the present invention all fall within the protection scope of the present invention.

Claims

1. A multiplex amplification system for 28 short tandem repeats, characterized in that, The multiplex amplification system includes 28 pairs of primers, which can simultaneously amplify 28 loci: A6-1, A6-2, A6-3, A6-4, A7-1, A7-2, A7-3, A7-4, A11-1, A11-2, A11-3, A11-4, A14-1, A14-2, A14-3, A14-4, A15-1, A15-2, A15-3, A15-4, A20-01, A20-2, A20-3, A20-4, A20-5, A20-6, A20-7, A20-8; The 28 pairs of primers are respectively:

2. The composite amplification system according to claim 1, wherein The concentration of each primer is:

3. The composite amplification system according to claim 1, characterized in that, The amplified loci in the multiplex amplification system are respectively labeled with five colors of fluorescence. The same fluorescence label is regarded as the same group. The five groups of combinations are respectively: The first group: A6-1, A6-2, A6-3, A6-4, A7-1; The second group: A7-2, A7-3, A7-4, A11-1, A11-2, A11-3, A11-4; The third group: A14-1, A14-2, A14-3, A14-4, A15-1; The fourth group: A15-2, A15-3, A15-4, A20-1, A20-2; The fifth group: A20-3, A20-4, A20-5, A20-6, A20-7, A20-8.

4. The multiplex amplification system according to claim 3, wherein, The first group is labeled with FAM, the second group is labeled with HEX, the third group is labeled with TAMRA, the fourth group is labeled with ROX, and the fifth group is labeled with PURP.

5. The multiplex amplification system according to claim 1 or 2, characterized in that, The amplification system further includes PCR Master Mix and DNA template.

6. The multiplex amplification system according to claim 5, wherein The PCR Master Mix includes: 10 mM ammonium sulfate, 10 mM potassium chloride, 60 mM Tris-HCl with pH 8.3, 2.5 mM magnesium ion, 0.9 μg / μl BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na4P2O7 and 0.25 mM Dntp, and 2 U Taq enzyme.

7. The multiplex amplification system according to claim 5, wherein The DNA sample is derived from human blood, amniotic fluid or aborted tissue.

8. The multiplex amplification system according to claim 1, wherein The reaction conditions during the amplification of the amplification system are: Step 1: Denature at 95 °C for 5 minutes, Step 2: Denature at 94 °C for 20 seconds, Step 3: Anneal at 60 °C for 2 minutes. Repeat steps 2 to 3 for 30 times, and finally extend at 72 °C for 10 minutes.

9. A kit, comprising the multiplex amplification system according to any one of claims 1-8.

10. Use of the amplification system according to any one of claims 1-8 in the preparation of a uniparental disomy detection kit.

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