Compound Amplification System and Kit for 16 Mini Y-STR

Abstract

The present invention belongs to the field of biotechnology and relates to a multiplex amplification system and kit for 16 Mini Y-STRs, with the amplified fragment length less than 290 bp, which is used to supplement the detection of large fragment loci greater than 350 bp in conventional Y kits on the market, for samples with amplification failure in trace degraded forensic materials. The present invention involves simultaneously amplifying multiple short tandem repeats in a single PCR system. Specifically, the loci are 16 Y short tandem repeats with high genetic polymorphisms. Primers are designed and fluorescent groups are labeled respectively. This system has an extremely high individual recognition rate and non-paternity exclusion rate, a large amount of information, and good compatibility. The present invention fills the current gap in the Y-case market and can be used in combination with all high-locus Y-case kits on the market. When large fragment amplification fails in trace degraded forensic materials, this system can be used to complement the missing large fragments and obtain complete genotyping data of more than 35 loci as much as possible. The present invention has higher accuracy and better sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a multiplex amplification system and kit for 16 Mini Y-STRs. This system simultaneously amplifies the Y-chromosome short tandem repeat (Y-STR) of multiple human male DNAs. When large locus amplification fails in trace degraded samples, adding the Mini-Y system to the sample can complement the lost large fragments and obtain complete genotyping data of more than 35 loci as much as possible, achieving the purpose of screening male family systems through Y-typing data comparison. Background Art

[0002] So far, the Y database construction has completed the entry of 70 - 80 million male family Y DNA genotyping data, and the Y database construction is basically completed. With the gradual improvement of the Y database, in addition to individual identification of conventional STRs, family comparison in the Y database has gradually become an indispensable and effective means. Completing the Y database construction generally requires performing conventional and Y tests on case samples together; that is, adding Y-STR detection while performing autosomal STR detection. Adding Y-STR detection has become a trend in the current scientific development.

[0003] Family comparison of male samples is a very important link. As the most widely used product in the Y case market, Y Platinum has been upgraded based on the YFP kit of the same manufacturer to adapt to the needs of family screening. However, due to the limitations of six-color fluorescence technology, while meeting high loci, it cannot take into account the fragment length (such problems exist in both 38Y and 37 + 5). The fragment sizes of products from each manufacturer are all between 550bp - 600bp. In fact, in the process of processing trace and degraded samples, small fragments before 300bp are most likely to be detected, and large fragments after 300bp are extremely likely to fail to obtain accurate genotyping results due to amplification failure. Generally speaking, the actual use effect of Y Platinum is not better than that of YFP, and it is difficult to achieve the purpose of family screening. Moreover, the locus arrangement order of each manufacturer is in accordance with core loci < preferred loci < alternative loci, and it is impossible to increase the number of loci obtained by changing products or adding reactions in the detection of trace and degraded samples.

[0004] In view of the current situation that Y-STR case reagents on the market are prone to missing large fragments in trace detection, it is necessary to develop a multiplex amplification system for Mini Y-STR. Summary of the Invention

[0005] The present invention provides a 16 Mini Y-STR multiplex amplification system and a kit, which have six-color fluorescence labeling and extremely high sensitivity. A complete genotyping map can be obtained even with a DNA template of 0.0625 ng. The locus combination adopted in the present invention is for the large-fragment loci of more than 350 bp of Y Platinum and the main domestic products in the market (Yuewei, Zhongde, Jidian, Haier Shi, etc.), including the loci DYS533, DYS643, DYS645, DYS388, YGATAH4, DYS518, DYS456, DYS557, DYS549, DYS393, DYS444, DYS391, DYS593, DYS596, DYS447, DYS522. It has an extremely high individual recognition rate and non-paternity exclusion rate. The present invention can be used for forensic individual identification, paternity testing, and family line investigation and analysis, with higher accuracy and better sensitivity; especially for trace and degraded forensic samples, by adding Mini Y-STR detection, the lost large fragments can be supplemented to obtain complete genotyping data of more than 35 loci as much as possible.

[0006] The technical solution provided by the present invention is as follows:

[0007] In the first aspect, a 16 Mini Y-STR multiplex amplification system is provided. The multiplex amplification system includes 16 primer pairs, which can simultaneously amplify 16 Y-STR loci, namely DYS533, DYS643, DYS645, DYS388, YGATAH4, DYS518, DYS456, DYS557, DYS549, DYS393, DYS444, DYS391, DYS593, DYS596, DYS447, and DYS522. The 16 primer pairs are respectively:

[0008]

[0009]

[0010] In some embodiments, the detection loci in the multiplex amplification system are respectively labeled with five colors of fluorescence. The same fluorescence labeling is regarded as the same group. The five groups of combinations are: the first group is DYS533, DYS643, and DYS388; the second group is YGATAH4 and DYS518; the third group is DYS391, DYS549, DYS447, and DYS645; the fourth group is DYS393, DYS444, DYS557, and DYS522; the fifth group is DYS456, DYS596, and DYS593.

[0011] In some embodiments, the five groups of fluorescence labels are FAM, HEX, TAMRA, ROX, and PUP respectively.

[0012] Further, the labels of the first group are FAM labels, the labels of the second group are HEX labels, the labels of the third group are TAMRA labels, the labels of the fourth group are ROX labels, and the labels of the fifth group are PUP labels.

[0013] In some embodiments, the multiplex amplification system further includes a PCR buffer, template DNA, and Taq DNA polymerase.

[0014] The components of the PCR buffer include: 10 mM ammonium sulfate, 10 mM potassium chloride, 50 mM Tris-HCl with a pH of 8.3, 2 mM magnesium ions, and 0.2 mM dNTP. The amount of Taq DNA polymerase used is 2 U.

[0015] The reaction conditions during the amplification of the amplification system are as follows:

[0016] Step 1: Denaturation at 95°C for 5 minutes, Step 2: Denaturation at 94°C for 10 seconds, Step 3: Annealing and extension at 59°C for 90 s, repeat Steps 2 and 3 for 30 times, Step 4: Extension at 60°C for 10 minutes.

[0017] The template DNA is from human bone, saliva, blood, hair, or semen stain.

[0018] In a second aspect, a kit is provided, including the above multiplex amplification system.

[0019] In a third aspect, the application of the above multiplex amplification system or kit in individual identification, paternity testing, and family pedigree screening analysis is provided.

[0020] Through research on the loci of the main Y kit products used in the market and analysis of the actual needs of customers, the present invention constructs 16 Mini Y-STR multiplex amplification systems, which can simultaneously amplify 16 Y-STR loci: DYS533, DYS643, DYS645, DYS388, YGATAH4, DYS518, DYS456, DYS557, DYS549, DYS393, DYS444, DYS391, DYS593, DYS596, DYS447, and DYS522. The amplified fragments of these loci in the main kits used in the market are all greater than 350 bp, and it is very easy to fail to amplify in trace degraded samples.

[0021] The present invention adopts a six-color fluorescence labeling system, groups the above 16 loci and performs fluorescence labeling: FAM labels DYS533, DYS643 and DYS388; HEX labels YGATAH4 and DYS518; TAMRA labels DYS391, DYS549, DYS447 and DYS645; ROX labels DYS393, DYS444, DYS557 and DYS522; the PUP group labels DYS456, DYS596 and DYS593. A pair of primers is designed upstream and downstream of the core sequence of each locus, and the amplification products are separated according to the difference in molecular weight, and there should be no overlap between two loci. All primers are mixed for multiplex amplification experiments to ensure no non-specific amplification. At the same time, in the detection component of the present invention, the standard is labeled with Orange, which can clearly label and distinguish the sizes of each gene locus in the detection sample.

[0022] The PCR reaction of the present invention is carried out in a specific buffer environment, and the buffer components include: 10 mM ammonium sulfate, 10 mM potassium chloride, 50 mM Tris-HCl (pH 8.3, 25 °C), 2 mM magnesium ions and 0.2 mM dNTP.

[0023] Each reaction of the present invention requires 2 U of hot-start Taq DNA polymerase.

[0024] The amplification products of the present invention need to be subjected to capillary electrophoresis for fragment analysis.

[0025] The fluorescence-labeled multiplex amplification detection system of the present invention is applicable to the detection of DNA samples extracted from forensic materials such as bones, saliva, blood, hair, and semen stains of human males.

[0026] According to the requirements of the "Operation Instruction Manual for the Detection of Forensic Science Human Fluorescence-Labeled Y-STR Multiplex Amplification Detection Reagent" of the present invention, 16 loci are amplified at one time, which is applicable to forensic DNA analysis and pedigree investigation in all physical evidence cases involving human male cells.

[0027] The design concept of the technical solution of the present invention:

[0028] 1. Selection of loci

[0029] The most widely used Y-case kits on the market generally include more than 35 Y-STR loci, such as the Y Platinum Kit of ABI in the United States. However, due to the limitations of six-color fluorescence technology, while meeting the high loci, the fragment length cannot be taken into account at the same time. The fragment sizes of the products of each manufacturer are all between 550bp and 600bp. In the actual processing of trace and degraded samples, small fragments before 300bp are most likely to be detected. After 300bp, large segments are extremely likely to fail to obtain accurate genotyping results due to amplification failure. Generally speaking, the actual use effect is not better than YFP, and it is difficult to achieve the purpose of family investigation. And the locus arrangement order of the multi-locus products of each manufacturer is in accordance with core locus < preferred locus < alternative locus, and the number of loci obtained cannot be increased by changing products or adding additional reactions in the detection of trace and degraded samples.

[0030] The loci selected in the present invention are all large fragment loci greater than 350bp of Y Platinum and the main domestic products on the market (such as Zhongde, Jidian, Haier Shi, etc.). In the face of trace and degraded forensic materials, amplification failure is extremely likely to occur. Adding the Mini Y system to the sample can complement the lost large fragments and obtain complete genotyping data of more than 35 loci as much as possible, so as to achieve the purpose of systematically investigating male families through Y genotyping data comparison. The Y-STR loci used are DYS533, DYS643, DYS645, DYS388, YGATAH4, DYS518, DYS456, DYS557, DYS549, DYS393, DYS444, DYS391, DYS593, DYS596, DYS447 and DYS522.

[0031] The present invention can detect 16 Y-STR loci. The probability of excluding non-paternity obtained through this information is higher than 0.99999999, and the individual recognition rate is higher than 0.99999999, which can ensure that the possibility of two unrelated individuals having overlapping characteristics is less than 10 16 above.

[0032] 2. Primer design

[0033] The primers described are designed by using software such as Primer Premier5 and NCBI Blast. When designing primers, the Tm values of each primer should be ensured to be within the range of (60±3)°C as much as possible, the amplification efficiencies are similar, and the sizes of the amplification products of each pair of primers differ by more than 10bp. After the design is completed, use software such as AutoDimer to analyze primer dimers and the interactions between different primers. If there are interactions that can produce non-specific products or dimers, they need to be redesigned until primer sequences that meet the requirements are obtained.

[0034] Select a human male DNA template and perform single amplification using the above 16 primer pairs respectively. Place the amplification products on a 2.0% agarose gel for electrophoresis, and adjust the PCR system and amplification conditions according to the electrophoresis results to obtain the common amplification conditions for the 16 primer pairs. The final expected effect is that under the same system and amplification conditions, all primer pairs can produce bright and relatively single target bands. If a primer fails to meet the above conditions, redesign the primer.

[0035] 3. Establishment of fluorescence labeling system

[0036] Select FAM, HEX, TAMAR, ROX, and PUP to establish a five-color fluorescence multiplex amplification system. Divide the designed 16 primer pairs into five groups for fluorescence labeling. Each group uses one fluorescence label, namely FAM, HEX, TMR, ROX, and PUP respectively. After obtaining the fluorescence-labeled primers, use their paired non-fluorescent primer combinations, and then perform single amplification respectively. Place the amplification products on a 3500 genetic analyzer for capillary electrophoresis, and evaluate the amplification efficiency of each pair of primers according to the results of capillary electrophoresis detection. Subsequently, mix the primers with the same fluorescence label in the same tube for amplification, place the amplification products on a 3500 genetic analyzer for capillary electrophoresis, and determine the amplification efficiency of each pair of primers according to the results of capillary electrophoresis detection, as well as judge whether non-specific amplification is caused by the mixed amplification of this group of primers. Finally, preliminarily determine the addition amount of each primer pair according to the capillary electrophoresis results of single amplification and combined amplification. Mix the 16 primer pairs in the same tube for amplification, and then adjust their respective concentrations according to the electrophoresis results of multiplex amplification to make the amplification efficiency (reflected in the peak height of the electrophoresis results) of each primer pair basically the same. Finally, determine the sequences of the 16 primer pairs.

[0037] 4. Optimization of amplification reaction system

[0038] Determine the parameters of the 16-locus multiplex amplification system through multiple repeated experiments, including: selection of Taq DNA polymerase, Mg 2+ concentration, ionic strength of the buffer, amount of Taq DNA polymerase, and amount of DNA template.

[0039] 5. Optimization of reaction program

[0040] Through a large number of experiments, explore the temperature and time ranges of denaturation, annealing, and extension of the reaction program. It is considered that better results can be obtained under the following conditions (see Table 1):

[0041] Table 1 Polymerase chain reaction amplification conditions

[0042]

[0043] Beneficial effects:

[0044] 1. It contains 16 Y-STR loci, and when combined with the mainstream Y-case kit products in the market, it can ensure that the number of loci with a fragment length less than 350bp is more than 35, with a large amount of information and good compatibility.

[0045] 2. It has a wide template adaptation range and is applicable to forensic DNA analysis in all physical evidence cases involving male cells. All biological samples containing male cells, such as bloodstains, semen stains, saliva, hair, nails, cartilage, and other human tissues, can be identified.

[0046] 3. The system has good specificity and stability. After being repeatedly verified many times, there are no non-specific amplification products, and the signal intensity is stable.

[0047] 4. It has high sensitivity, and an accurate genotype can be obtained with a DNA template amount of 0.0625 ng.

[0048] 5. It has strong anti-inhibition ability: This multiplex amplification system can tolerate Humic Acid: 250 ng / μL, Tannic Acid: 150 ng / μL, Hematin porcine: 1000 μM, Indigocarmine: 14 mM, Ethanol: 6%, Calcium: 1 mM. Description of the Drawings

[0049] Figure 1 It is the amplification schematic diagram of Sample 1 (cell line) in the embodiment of the present invention; among them, (a) is the amplification effect of the quality control product, and (b) is the Mini Y genotyping standard (Ladder) map.

[0050] Figure 2 It is the direct amplification schematic diagram of Sample 2 (blood card) in the embodiment of the present invention.

[0051] Figure 3 It is the amplification schematic diagram of Sample 3 (extracted from oral swab cotton swab) in the embodiment of the present invention.

[0052] Figure 4 It is the direct amplification schematic diagram of Sample 4 (saliva card) in the embodiment of the present invention.

[0053] Figure 5 It is the annealing temperature gradient amplification schematic diagram of Sample 5 (9948 standard DNA) in the embodiment of the present invention.

[0054] Figure 6 It is the sensitivity amplification schematic diagram of Sample 6 (9948 standard DNA) in the embodiment of the present invention.

[0055] Figure 7Schematic diagram of amplification for anti-inhibition ability test in the embodiments of the present invention; wherein (a) is tannic acid, (b) is humic acid, (c) is heme, (d) is indigo, (e) is ethanol, and (f) is calcium ion.

[0056] Figure 8 Schematic diagram of amplification for cooperating with Y41 test in degrading case samples in the embodiments of the present invention; wherein (a) is Y41 amplification sample J0002, (b) is Mini Y amplification sample J0002, (c) is Y41 amplification sample J0008, and (d) is Mini Y amplification sample J0008. Specific implementation manners

[0057] Example 1

[0058] Detecting the locus typing of cell line samples by using 16 Mini Y-STR multiplex amplification kits

[0059] 1. Source of cell line samples (the cell line samples are from the DNA provided by Yuwei Gene)

[0060] 2. DNA dilution

[0061] The finished DNA is 2 ng / μL. Take 10 μL and put it into a 500 μL centrifuge tube, and add 10 μL of TE for dilution and standby. 3. Reaction system:

[0062] After oscillating and mixing each reaction reagent (Buffer, Primer Mix, dNTP, etc.), prepare the PCR reaction mixture according to the volume ratio in the table (except for the template), and dispense 9 μL into the PCR reaction tube. Finally, add 1 μL of template to each reaction tube, and centrifuge and then proceed to the next step. The composition of the reaction system is shown in Table 2.

[0063] Table 2 Standard amplification reaction system

[0064] Component Name Volume (μL) Sterile Deionized Water 2.8 2.5× Reaction Buffer 4 Primer Mix 2 Taq DNA Polymerase 0.2 Template 1 Total Volume 10 μL

[0065] 4. PCR reaction procedure:

[0066] Place the PCR reaction tube on the thermal cycler, and design and run the following program:

[0067] Step 1: Denature at 95 °C for 5 minutes, Step 2: Denature at 94 °C for 10 seconds, Step 3: Anneal and extend at 59 °C for 90 s, repeat steps 2 to 3 for 30 times, Step 4: Extend at 60 °C for 10 minutes. After the operation is completed, store the product in the refrigerator at 4 °C.

[0068] 5. Capillary electrophoresis detection

[0069] Mix the Orange 500 internal standard and formamide at 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 ABI5000xL sequencer for detection preparation.

[0070] 6. Data analysis

[0071] 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. With any sample file name, select "raw data" in the "sample" menu. Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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 GeneMapper software and generate a map, as shown in Figure 1 . It can be seen that the DNA typing map obtained through the multiplex amplification system of the present invention is clear and accurate.

[0072] 7. Experimental results

[0073] The standard DNA can be amplified well and accurately typed. The amplification map is as shown in Figure 1 . The DNA typing map is clear and accurate.

[0074] 8. The result analysis is shown in the following table:

[0075]

[0076]

[0077] Example 2

[0078] Detect the locus typing of Volunteer 2 using a 16-plex Mini Y-STR amplification kit

[0079] 1. Source of the blood card sample (The blood is coated on an FTA blood card and dried before use. Punch out a 1.2 mm diameter blood card and directly amplify it in a 10 μL system)

[0080] 2. Reaction system:

[0081] Oscillate and mix each reaction reagent (Buffer, Primer Mix, dNTP, etc.), and prepare a PCR reaction mixture according to the volume ratio in the table (except for the template). Aliquot 9 μL into PCR reaction tubes, and finally add 1 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 2.

[0082] 3. PCR reaction program:

[0083] Place the PCR reaction tubes on the thermal cycler, and design and run the following program:

[0084] Step 1: Denature at 95 °C for 5 minutes. Step 2: Denature at 94 °C for 10 seconds. Step 3: Anneal and extend at 59 °C for 90 s, repeat steps 2 and 3 for 30 times. Step 4: Extend at 60 °C for 10 minutes. After the run is completed, store the product in the refrigerator at 4 °C.

[0085] 4. Capillary electrophoresis detection

[0086] Mix Orange 500 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 amplified product sample or the allele standard. Mix and let stand for several minutes. Denature at 95 °C for 3 min, immediately ice-bath for 3 min, and after centrifugation, place it on an ABI5000xL sequencer for detection preparation.

[0087] 5. Data analysis

[0088] 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, rename a sample file, and select raw data in the "sample" menu. Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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, and a save project dialog box will appear. After naming and saving, the software will start processing the data, and analysis completed will be displayed at the lower left corner after the analysis is finished. Analyze the data obtained using GeneMapper software and generate a graph, see Figure 2 .

[0089] 6. Experimental results

[0090] The blood card samples can be well amplified and accurately genotyped, as Figure 2 shown.

[0091] 7. The results analysis is shown in the following table:

[0092]

[0093]

[0094] Example 3

[0095] Detecting the genotyping of 3 loci of volunteers using 16 Mini Y-STR multiplex amplification kits

[0096] 1. Collection of oral swab samples (the samples were donated by volunteers)

[0097] 2. DNA extraction

[0098] Genomic DNA was extracted using the Chelex-100 method (refer to <Forensic DNAdProtocol>>. HumanaPress, 1998). One oral swab cotton swab (sample 3) was placed in a 1500 μL centrifuge tube, and the Chelex solution was oscillated and mixed to fully suspend Chelex. 880 μL of Chelex-100 (5%) solution and 20 μL of proteinase K (20 mg / ml) were added to each tube and oscillated and mixed. After incubation at 56 °C for two hours or overnight, it was taken out and oscillated for 2 minutes, heated in boiling water for 10 minutes, and then centrifuged at 13000 rpm for 5 minutes. 150 μL of the supernatant was carefully transferred to a new centrifuge tube.

[0099] 3. Reaction system:

[0100] After oscillating and mixing each reaction reagent (Buffer, Primer Mix, dNTP, etc.), a PCR reaction mixture was prepared according to the volume ratio in the table (except for the template), and 9 μL was aliquoted into PCR reaction tubes. Finally, 1 μL of the template was added to each reaction tube, and after centrifugation, it proceeded to the next step. The composition of the reaction system is shown in Table 2.

[0101] 4. PCR reaction program:

[0102] The PCR reaction tubes were placed on the thermal cycler, and the following program was designed and run:

[0103] Step 1: Denaturation at 95 °C for 5 minutes, Step 2: Denaturation at 94 °C for 10 seconds, Step 3: Annealing and extension at 59 °C for 90 s, repeat steps 2 to 3 for 30 times, Step 4: Extension at 60 °C for 10 minutes. After the run was completed, the products were stored at 4 °C in the refrigerator.

[0104] 5. Capillary electrophoresis detection

[0105] Mix the Orange 500 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 the 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 ABI5000xL sequencer for preparation of detection.

[0106] 6. Data analysis

[0107] 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. Arbitrarily name a sample file and select "raw data" in the "sample" menu. Move the tracking line to stop the cursor on the right side of the primer peak (before the first red 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 GeneMapper software and generate a map, as shown in Figure 3 。

[0108] 7. Experimental results

[0109] The DNA extracted from the oral swab samples can be amplified well and accurately typed. The amplification map of the DNA extracted from oral swabs using the Chelex-100 method is as shown in Figure 3 shown.

[0110] 8. The result analysis is shown in the following table:

[0111]

[0112]

[0113] Example 4

[0114] Detect the genotyping of 4 loci of volunteers using a 16 Mini Y-STR multiplex amplification kit

[0115] 1. Source of saliva card samples (The saliva is coated on the saliva card and dried before use. Punch a 10 μL system of a blood card with a diameter of 1.2 mm for direct amplification)

[0116] 2. Reaction system:

[0117] Oscillate and mix each reaction reagent (Buffer, Primer Mix, dNTP, etc.), and prepare a PCR reaction mixture according to the volume ratio shown in the table (except for the template). Aliquot 9 μL into PCR reaction tubes, and finally add 1 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 2.

[0118] 3. PCR reaction program:

[0119] Place the PCR reaction tubes on the thermal cycler, and design and run the following program:

[0120] Step 1: Denature at 95 °C for 5 minutes. Step 2: Denature at 94 °C for 10 seconds. Step 3: Anneal and extend at 59 °C for 90 s. Repeat steps 2 to 3 for 30 times. Step 4: Extend at 60 °C for 10 minutes. After the run is completed, store the product in the refrigerator at 4 °C.

[0121] 4. Capillary electrophoresis detection

[0122] Mix Orange 500 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 amplified product sample or the allele standard. Mix and let stand for several minutes. Denature at 95 °C for 3 min, immediately ice-bath for 3 min, and after centrifugation, place it on the ABI5000xL sequencer for detection preparation.

[0123] 5. Data analysis

[0124] 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 a sample file name, and in the "sample" menu, select "raw data". Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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 GeneMapper software and generate a map, see Figure 2 。

[0125] 6. Experimental results

[0126] Saliva card samples can be amplified well and accurately typed, as Figure 4 shown.

[0127] 7. The results are analyzed in the following table:

[0128]

[0129]

[0130] Example 5

[0131] Annealing Temperature Test of 16 Mini Y-STR Multiplex Amplification Kits

[0132] 1. The cell line samples are from 9948DNA produced by Yuewei Gene, Suzhou

[0133] 2. Experimental Setup

[0134] Dilute 9948DNA produced by Yuewei Gene, Suzhou to 1 ng / μL as the template for PCR reaction.

[0135] 3. Reaction System:

[0136] After oscillating and mixing each reaction reagent (Buffer, Primer Mix, dNTP, etc.), prepare the PCR reaction mixture according to the volume ratio in the table (except for the template), dispense 9 μL into PCR reaction tubes, and finally add 1 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 2.

[0137] 4. PCR Reaction Program:

[0138] Place the PCR reaction tubes on the thermal cycler and design and run the following program:

[0139] Step 1: Denature at 95°C for 5 minutes, Step 2: Denature at 94°C for 10 seconds, Step 3: Anneal and extend at 59°C for 90 s, repeat steps 2 to 3 for 30 times, Step 4: Extend at 60°C for 10 minutes. After the run is completed, store the product in the refrigerator at 4°C.

[0140] 5. Capillary Electrophoresis Detection

[0141] Mix Orange 500 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 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 and then place it on the ABI5000xL sequencer for detection.

[0142] 6. Data Analysis

[0143] Import the original data. In the File menu on the main page, select Add sample to project, locate 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, arbitrarily name a sample file, and select raw data in the sample menu. Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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 GeneMapper software and generate a graph as shown in Figure 5 。

[0144] 7. Experimental results

[0145] 9948 DNA at 0.5 ng can be amplified well and accurately typed at a total of 5 annealing temperature gradients of 57 °C, 58 °C, 59 °C, 60 °C, and 61 °C. The amplification graph is as shown in Figure 5 shown. The sample typing is completely consistent, and there are no non-specific amplification peaks. This indicates that the 16 Mini Y-STR multiplex amplification kits have good stability and can tolerate an annealing temperature change of ±2 °C.

[0146] 7. The result analysis is shown in the following table:

[0147]

[0148]

[0149] Example 6

[0150] Sensitivity test of 16 Mini Y-STR multiplex amplification kits

[0151] 1. The cell line samples are derived from 9948 DNA produced by Suzhou Yuewei Gene

[0152] 2. Experimental settings

[0153] Dilute 9948 DNA produced by Suzhou Yuewei Gene to 2 ng / μL, 1 ng / μL, 0.5 ng / μL, 0.25 ng / μL, 0.125 ng / μL, 0.625 ng / μL, and 0.3125 ng / μL respectively as the templates for the PCR reaction.

[0154] 3. Reaction system:

[0155] Oscillate and mix each reaction reagent (Buffer, Primer Mix, dNTP, etc.), and prepare the PCR reaction mixture according to the volume ratio shown in the table (except for the template). Aliquot 9 μL into PCR reaction tubes, and finally add 1 μL of the template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 2.

[0156] 4. PCR reaction procedure:

[0157] Place the PCR reaction tubes on the thermal cycler, and design and run the following program:

[0158] Step 1: Denature at 95 °C for 5 minutes. Step 2: Denature at 94 °C for 10 seconds. Step 3: Anneal and extend at 59 °C for 90 s. Repeat steps 2 and 3 for 30 times. Step 4: Extend at 60 °C for 10 minutes. After the run is completed, store the product in the refrigerator at 4 °C.

[0159] 5. Capillary electrophoresis detection

[0160] Mix Orange 500 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 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 the ABI5000xL sequencer for detection preparation.

[0161] 6. Data analysis

[0162] 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. Rename a sample file name. In the "sample" menu, select "raw data". Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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 GeneMapper software and generate a map, see Figure 6 .

[0163] 7. Experimental results

[0164] Good amplification can be achieved when the input of DNA template is 0.125 ng or more. The amplification profiles are as shown in Figure 6 , and the sample genotyping is completely consistent, without non-specific amplification peaks. This indicates that the 16 Mini Y-STR multiplex amplification system has high sensitivity, and accurate genotyping can be obtained with an input DNA template amount of 0.0625 ng.

[0165] 8. The results analysis is shown in the following table:

[0166]

[0167] Example 7

[0168] Anti-inhibition ability test of 16 Mini Y-STR multiplex amplification kits

[0169] 3. The cell line samples are derived from 9948 DNA produced by Suzhou Yuewei Gene

[0170] 4. Experimental settings

[0171] Dilute the 9948 DNA produced by Suzhou Yuewei Gene to 1 ng / μL respectively as the template for PCR reaction. Add tannic acid to the amplification system to make its concentration 10 ng / μL, 50 ng / μL, 100 ng / μL, 150 ng / μL, 200 ng / μL, 250 ng / μL and 500 ng / μL respectively; add humic acid to the amplification system to make its concentration 10 ng / μL, 50 ng / μL, 100 ng / μL, 150 ng / μL, 200 ng / μL, 250 ng / μL and 500 ng / μL respectively; add heme to the amplification system to make its concentration 200 μM, 400 μM, 600 μM, 800 μM, 1000 μM and 2000 μM respectively; add indigo to the amplification system to make its concentration 4 mM, 6 mM, 8 mM, 10 mM, 12 mM and 14 mM respectively; add ethanol to the amplification system to make its volume ratio 0.5%, 1%, 2%, 4%, 6% and 8%; add calcium ions to the amplification system to make its concentration 1 mM, 2 mM, 3 mM, 4 mM and 5 mM.

[0172] 3. Reaction system:

[0173] Oscillate and mix each reaction reagent (Buffer, Primer Mix, dNTP, etc.), then mix according to the volume ratio in the table (except for the template), add the corresponding amount of inhibitor, prepare the PCR reaction mixture, aliquot 9 μL into PCR reaction tubes, and finally add 1 μL of template to each reaction tube. After centrifugation, proceed to the next step. The composition of the reaction system is shown in Table 3.

[0174] Table 3: Configuration of anti-inhibition test system

[0175] Component Name Volume (μL) Inhibitor 1 2.5× Reaction Buffer 4 Primer Mix 2 Tag DNA Polymerase 0.2 Template 1 Make up to 10 μL with water

[0176] 4. PCR Reaction Program:

[0177] Place the PCR reaction tube on the thermal cycler and design and run the following program:

[0178] Step 1: Denature at 95°C for 5 minutes. Step 2: Denature at 94°C for 10 seconds. Step 3: Anneal and extend at 59°C for 90 s. Repeat steps 2 and 3 for 30 times. Step 4: Extend at 60°C for 10 minutes. After the run, store the product at 4°C in the refrigerator.

[0179] 5. Capillary Electrophoresis Detection

[0180] Mix Orange 500 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 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, centrifuge, and then place it on the ABI5000xL sequencer for detection preparation.

[0181] 6. Data Analysis

[0182] Import the raw 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 raw data of the sample electrophoresis. Rename a sample file name. In the "sample" menu, select "raw data". Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red 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 GeneMapper software and generate a map, see Figure 7 。

[0183] 7. Experimental Results

[0184] From Figure 7It can be seen that the inhibition ability of the Mini Y-STR multiplex amplification kit decreases with the increase in the concentrations of tannic acid, humic acid, heme, and indigo. Among them, the large fragments show an obvious downward trend with the increase in concentration, while the decrease in small fragments is relatively weak. Therefore, the inhibitor has an obvious inhibitory effect on large fragments. The concentration of the system's anti-tannic acid inhibition ability is 150 ng / μL, the humic acid inhibition ability concentration is 250 ng / μL; the heme inhibition ability concentration is 1000 μM; the indigo inhibition ability concentration is 14 mM; the ethanol inhibition ability concentration is 6%; the calcium ion inhibition ability concentration is 2 mM.

[0185] Example 8

[0186] Testing of 16 degraded samples of the Mini Y-STR multiplex amplification kit

[0187] 1. The cell samples are from the degraded case DNA of Shenzhen Luohu Branch.

[0188] 2. Experimental setup

[0189] The degraded case DNA of Shenzhen Luohu Branch is used as the template for the PCR reaction. At the same time, Y41 of AB Company is tested to detect the detection effect of the Mini Y system on degraded forensic samples.

[0190] 3. Reaction system:

[0191] After oscillating and mixing each reaction reagent (Buffer, Primer Mix, dNTP, etc.), a PCR reaction mixture is prepared according to the volume ratio in the table (except for the template), and 9 μL is dispensed into PCR reaction tubes. Finally, 1 μL of the template is added to each reaction tube, and after centrifugation, the next step is carried out. The composition of the reaction system is shown in Table 2.

[0192] 4. PCR reaction program:

[0193] The PCR reaction tubes are placed on the thermal cycler, and the following program is designed and run:

[0194] Step 1: Denaturation at 95 °C for 5 minutes, Step 2: Denaturation at 94 °C for 10 seconds, Step 3: Annealing and extension at 59 °C for 90 s, repeat steps 2 to 3 for 30 times, Step 4: Extension at 60 °C for 10 minutes. After the run is completed, the products are stored at 4 °C in the refrigerator.

[0195] 5. Capillary electrophoresis detection

[0196] Mix Orange 500 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 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 after centrifugation, place it on the ABI5000xL sequencer for detection preparation.

[0197] 6. Data Analysis

[0198] Import the original data. In the File menu on the main page, select Add sample to project, locate 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. With any sample file name, select Raw data in the Sample menu. Move the tracking line so that the cursor stops on the right side of the primer peak (before the first red internal standard peak), and 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 GeneMapper software and generate a map, Figure 8 showing two of the samples, J0002 and J0008.

[0199] 7. Experimental Results

[0200] For the degraded case samples, except for the individual completely degraded ones, the Y41 kit can detect small fragments before 250bp, and most of the large fragments are lost. Using the Y mini kit for additional testing can reach more than 30 loci. The specific detection effects are shown in Table 4, Figure 8 showing two of the samples, J0002 and J0008.

[0201] Table 4: Effect of Supplementary Testing of Mini Y on Degraded Case Samples

[0202]

[0203] As can be seen from the above, the 16 Mini Y-STR multiplex amplification kits have good effects in testing degraded samples, meeting the design requirements.

Claims

1. A multiplex amplification system for 16 Mini Y-STRs, characterized in that, The multiplex amplification system includes 16 primer pairs, simultaneously amplifying 16 Y-STR loci: DYS533, DYS643, DYS645, DYS388, YGATAH4, DYS518, DYS456, DYS557, DYS549, DYS393, DYS444, DYS391, DYS593, DYS596, DYS447 and DYS522. The 16 primer pairs are respectively: The primer pair sequences for amplifying DYS533 are shown in SEQ ID NO.1 and SEQ ID NO.2; The primer pair sequences for amplifying DYS643 are shown in SEQ ID NO.3 and SEQ ID NO.4; The primer pair sequences for amplifying DYS388 are shown in SEQ ID NO.5 and SEQ ID NO.6; The primer pair sequences for amplifying YGATAH4 are shown in SEQ ID NO.7 and SEQ ID NO.8; The primer pair sequences for amplifying DYS518 are shown in SEQ ID NO.9 and SEQ ID NO.10; The primer pair sequences for amplifying DYS391 are shown in SEQ ID NO.11 and SEQ ID NO.12; The primer pair sequences for amplifying DYS549 are shown in SEQ ID NO.13 and SEQ ID NO.14; The primer pair sequences for amplifying DYS447 are shown in SEQ ID NO.15 and SEQ ID NO.16; The primer pair sequences for amplifying DYS645 are shown in SEQ ID NO.17 and SEQ ID NO.18; The primer pair sequences for amplifying DYS393 are shown in SEQ ID NO.19 and SEQ ID NO.20; The primer pair sequences for amplifying DYS444 are shown in SEQ ID NO.21 and SEQ ID NO.22; The primer pair sequences for amplifying DYS557 are shown in SEQ ID NO.23 and SEQ ID NO.24; The primer pair sequences for amplifying DYS522 are shown in SEQ ID NO.25 and SEQ ID NO.26; The primer pair sequences for amplifying DYS456 are shown in SEQ ID NO.27 and SEQ ID NO.28; The primer pair sequences for amplifying DYS596 are shown in SEQ ID NO.29 and SEQ ID NO.30; The primer pair sequences for amplifying DYS593 are shown in SEQ ID NO.31 and SEQ ID NO.32; The amplified loci in the multiplex amplification system are respectively fluorescently labeled with five colors. The same fluorescent label is regarded as the same group. The five groups of combinations are respectively: The first group: DYS533, DYS643 and DYS388; The second group: YGATAH4 and DYS518; The third group: DYS391, DYS549, DYS447 and DYS645; The fourth group: DYS393, DYS444, DYS557 and DYS522; The fifth group, DYS456, DYS596 and DYS593; The markers of the first group are FAM markers, the markers of the second group are HEX markers, the markers of the third group are TAMRA markers, the markers of the fourth group are ROX markers, and the markers of the fifth group are PUP markers.

2. The multiplex amplification system according to claim 1, wherein the amplification system further comprises a PCR buffer, template DNA and Taq DNA polymerase.

3. The composite amplification system according to claim 2, wherein the PCR buffer solution comprises: 10 mM ammonium sulfate, 10 mM potassium chloride, 50 mM Tris-HCl at pH 8.3, 2 mM magnesium ions and 0.2 mM dNTP, and the amount of Taq DNA polymerase used is 2 U.

4. The multiplex amplification system according to claim 2, wherein The template DNA is derived from the bones, saliva, blood, hair or semen stains of a human male.

5. The multiplex amplification system according to claim 1, wherein the reaction conditions during the amplification of the amplification system are: Step 1: Denaturation at 95 °C for 5 minutes, Step 2: Denaturation at 94 °C for 10 seconds, Step 3: Annealing and extension at 59 °C for 90 s, repeat steps 2 to 3 for 30 times, Step 4: Extension at 60 °C for 10 minutes.

6. A kit comprising the multiplex amplification system according to any one of claims 1-5.

7. Use of the amplification system according to any one of claims 1-5 or the kit according to claim 6 in individual identification analysis.

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