Capillary electrophoresis-based mixture syndrome DIP-STR examination and analysis method

Abstract

The invention discloses a mixture syndrome DIP-STR examination and analysis method based on capillary electrophoresis. The invention provides the application of the five DIP-STR markers in any one of the following applications: (A1) mixed DNA (Deoxyribose Nucleic Acid) detection; (A2) identifying mixed spots; and the five DIP-STR markers are respectively SDD7 (rs34666822-STR), SDD8 (rs138646306-STR), SDD30 (rs11279377-STR), SDI7 (rs10652632-STR) and DSI7 (rs56113354-STR), and the five DIP-STR markers are respectively used for marking the five DIP-STR markers. Compared with the prior art, the five screened DIP-STR markers have obvious advantages in analysis of unbalanced mixed DNA samples, and have wide application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a mixture DIP-STR test analysis method based on capillary electrophoresis. Background Art

[0002] In forensic evidence science, DNA technology is widely used in the examination of common degraded, diluted or mixed biological evidence (hair, cigarette butts, blood samples, semen stains, etc.). For mixed biological evidence, a phenomenon often occurs. For example, in a mixed biological evidence, when the suspect's DNA is a component of the DNA mixed sample, it is not the main component (it is a secondary component DNA). Especially when the DNA of the victim or a person unrelated to the crime accounts for more than 90%-95% of the total DNA (i.e., the main component DNA), it greatly affects the detection of the suspect's DNA, and even makes it impossible to obtain the suspect's DNA data. With the improvement of the sensitivity and accuracy of forensic DNA testing, the rapid development of contact DNA evidence detection technology has made contact biological evidence play an increasingly important role. However, in some cases of property infringement and violent crimes with blood stains or semen stains at the scene, the proportion of the detection and analysis workload of contact DNA in unbalanced mixed stains is increasing. However, a large number of cases have confirmed that the existing DNA analysis methods have certain limitations for unbalanced mixed stain tests, and it is not easy to accurately detect trace DNA in mixed DNA samples. As a result, the DNA test results of some samples show a single DNA typing, which cannot provide more powerful court evidence or investigative clues.

[0003] It can be seen that forensic DNA analysis of mixed samples is very common in actual inspections, and its application value is relatively high, but the inspection of mixed DNA samples, especially unbalanced mixed DNA samples, is still a difficult problem. Studies have shown that the application of composite genetic markers (such as DIP-STR) may be a solution to this problem. The DIP-STR technology using composite genetic markers can be used to perform targeted and specific amplification of individual DNA with different DIP typing, so as to achieve the purpose of distinguishing the DNA of different individuals. However, to date, there is a lack of effective verification for DIP-STR markers reported in foreign literature; research on DIP-STR markers is limited, and the number of sites used for mixed sample inspection is insufficient, which limits its resolution ability and fails to play its due practical value. Summary of the invention

[0004] The purpose of the present invention is to provide a mixture DIP-STR test analysis method based on capillary electrophoresis.

[0005] In a first aspect, the present invention claims the use of five DIP-STR markers in any of the following:

[0006] (A1) Mixed DNA detection;

[0007] (A2) Mixed spot identification;

[0008] The five DIP-STR markers are respectively designated as SDD7, SDD8, SDD30, SDI7, and DSI7;

[0009] The SDD7 is rs34666822-STR, located at chr4:151527517-151527583;

[0010] The SDD8 is rs138646306-STR, located at chr5:12558525-12558561;

[0011] The SDD30 is rs11279377-STR, located at chr21:29846396-29846520;

[0012] The SDI7 is rs10652632-STR, located at chr2:13668609-13668658;

[0013] The DSI7 is rs56113354-STR, located at chr1:44553371-44553412.

[0014] Detailed information of the five DIP-STR markers is shown in Table 4.

[0015] In a second aspect, the present invention claims the use of a substance for detecting five DIP-STR markers in any of the following:

[0016] (A1) Mixed DNA detection;

[0017] (A2) Mixed spot identification;

[0018] The five DIP-STR markers are the same as those described in the first aspect above.

[0019] In the first aspect and the second aspect, the mixed DNA detection and the mixed stain identification may specifically include: determining which individuals the mixed stains / mixed DNA come from, and determining which individual(s) the secondary component DNA in the mixed stains / mixed DNA comes from.

[0020] In a third aspect, the present invention claims a set of primers for detecting mixed DNA, which consists of primer set A, primer set B, primer set C, primer set D and primer set E.

[0021] The primer set A is used to type the SDD7 in the first aspect of the above text, and consists of a specific primer A1 and a specific primer A2 targeting the DIP site, and a universal primer A3 targeting the STR site; the primer A1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.1; the primer A2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.2; and the primer A3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.3.

[0022] The primer group B is used to type the SDD8 in the first aspect of the above text, and consists of a specific primer B1 and a specific primer B2 targeting the DIP site, and a universal primer B3 targeting the STR site; the primer B1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.4; the primer B2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.5; the primer B3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.6.

[0023] The primer set C is used to type the SDD30 in the first aspect of the above text, and consists of a specific primer C1 and a specific primer C2 targeting the DIP site, and a universal primer C3 targeting the STR site; the primer C1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.7; the primer C2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.8; and the primer C3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.9.

[0024] The primer set D is used to type the SDI7 in the first aspect of the above text, and consists of a specific primer D1 and a specific primer D2 targeting the DIP site, and a universal primer D3 targeting the STR site; the primer D1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.10; the primer D2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.11; and the primer D3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.12.

[0025] The primer set E is used to type the DSI7 in the first aspect above, and consists of a specific primer E1 and a specific primer E2 targeting the DIP site, and a universal primer E3 targeting the STR site; the primer E1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.13; the primer E2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.14; and the primer E3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.15.

[0026] In a specific embodiment of the present invention, in the primer set A, the universal primer A3 is fluorescently labeled. In the primer set B, the universal primer B3 is fluorescently labeled. In the primer set C, the universal primer C3 is fluorescently labeled. In the primer set D, the universal primer D3 is fluorescently labeled. In the primer set E, the universal primer E3 is fluorescently labeled.

[0027] In a fourth aspect, the present invention claims a reagent or a kit containing the set of primers described in the third aspect above.

[0028] In a fifth aspect, the present invention claims the use of the primer set described in the third aspect above or the reagent or kit described in the fourth aspect above in any of the following:

[0029] (A1) Mixed DNA detection;

[0030] (A2) Mixed stain identification.

[0031] The mixed DNA detection and the mixed stain identification may specifically include: determining which individuals the mixed stains / mixed DNA come from, and determining which individual(s) the secondary component DNA in the mixed stains / mixed DNA comes from.

[0032] In a sixth aspect, the present invention claims a method for determining which individuals a mixed stain originates from.

[0033] The method for determining which individuals the mixed spots originate from, as claimed in the present invention, may include the following steps:

[0034] (a1) extracting DNA from the mixed stain to be identified to obtain mixed DNA; sampling each individual in the comparison group to obtain single individual DNA of known origin; the comparison group is composed of each individual from which the mixed stain is suspected to originate;

[0035] (a2) using the primer set described in the third aspect or the reagent or kit described in the fourth aspect to detect the mixed DNA and the single individual DNA of known origin in (a1), respectively, to obtain typing results of the mixed DNA at the five DIP-STR loci, and typing results of the single individual DNA of known origin at the five DIP-STR loci;

[0036] (a3) Determining which individuals in the comparison group the DNA in the mixed stain comes from by comparing the typing results of the mixed DNA obtained in (a2) with the typing results of the single individual DNA whose source is known.

[0037] Furthermore, in step (a2), when the mixed DNA and the single individual DNA of known origin are detected, two reaction systems are set for each primer set: one is a reaction system for amplifying using a specific primer targeting the DIP site and a universal primer targeting the STR site, and the other is a reaction system for amplifying using another specific primer targeting the DIP site and a universal primer targeting the STR site.

[0038] Furthermore, in step (a3), in the typing results of the mixed DNA, if the typing results of all the successfully detected sites in the 5 DIP-STR sites are consistent with the typing results of the corresponding sites of a single individual with a known origin, the mixed spot is considered to be derived from the corresponding known individual (Note: if for the same known individual, the mixed DNA test results show that some sites match while other sites do not match, the mixed spot cannot be considered to be derived from the known individual).

[0039] Furthermore, the detection can be performed by means of fluorescent labeling capillary electrophoresis technology.

[0040] In the above aspects, the mixed stain is an unbalanced mixed stain, that is, the mixed DNA extracted from the unbalanced mixed stain is composed of major donor DNA (with a relatively high content) and minor donor DNA (with a relatively low content).

[0041] In the above aspects, the mixed stains may be one or more of mixed blood stains, mixed saliva stains and contact marks of two or more individuals.

[0042] Furthermore, the mass ratio of the primary donor DNA to the secondary donor DNA in the mixed DNA is less than or equal to 1000:1.

[0043] The results of the minimum detection threshold of the present invention show that the technology can meet the detection needs of conventional and even contact and trace DNA samples. The experimental results show that the minimum detection threshold can reach 0.005ng, which is sufficient to meet the detection needs of contact DNA and trace DNA (DNA amount less than 0.1ng) commonly seen in current cases.

[0044] The present invention can be used as a powerful supplement to the current mixed DNA test. Comparing the results of the unbalanced mixed DNA sample detection of DIP-STR markers and autosomal STR markers, the autosomal STR markers commonly used in current forensic laboratories have certain defects when detecting mixed DNA samples, such as DNA masking effect, peak loss caused by amplification imbalance, stutter peak of the main donor DNA, and interference of noise signals after the mixing ratio increases. However, the DIP-STR markers with allele-specific primers can break through the bottleneck of autosomal STR marker detection when the secondary donor DNA has a DIP allele opposite to the main donor DNA, and obtain typing by targeting the secondary donor DNA. The results show that compared with the autosomal STR markers, most DIP-STR markers can still target the secondary donor DNA without interference when the DNA mixing ratio is as high as 1000:1. However, when there is no effective information gene at the DIP-STR site (the secondary donor DNA does not have a DIP allele opposite to the main donor DNA), the resolution of this method is the same as that of the conventional STR markers. The present invention breaks this limitation by using a relatively large DIP-STR marker group to increase the chance of the secondary donor DNA information genotype. The present invention uses the five DIP-STR markers with better performance selected in combination. The five DIP-STR markers selected in the present invention have obvious advantages in analyzing unbalanced mixed DNA samples and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the design principle of DIP-STR primers. A and B both show that the secondary donor DNA has different alleles from the primary donor DNA at the DIP site (marked by the box). Therefore, the reverse fluorescent allele-specific primer (Reverse-L-Primer) in the amplification system can target the haploid of the secondary donor DNA, while the forward universal STR primer combines with the upstream of the locus to amplify the STR site. The figure only shows two representative cases where the DIP sites in the primary donor DNA are all S-DIP. When the DIP sites in the primary donor DNA are all L-DIP, and the secondary donor DNA has an allele S-DIP at the DIP site that is different from the primary donor DNA, the principle is the same as above.

[0046] Figure 2 is the probability of the informative genotype. s and l are used to represent the gene frequencies of allele deletion (Short) and insertion (Long). The secondary donor DNA typing can be detected only when and only when the primary donor DNA is homozygous and the secondary donor DNA haplotype has a haplotype opposite to that of the primary donor DNA haplotype. According to the above figure, I = 2s 2 l2 +2s 3 l+2sl 3 .

[0047] Figure 3 is the detection threshold of SDD7.

[0048] Figure 4 is the detection threshold of SDD8.

[0049] Figure 5 is the detection threshold of SDD30.

[0050] Figure 6 is the detection threshold of SDI7.

[0051] Figure 7 is the detection threshold of DSI7.

[0052] Figure 8 This is the sensitivity test of SDD7.

[0053] Fig. 9 Sensitivity test for SDD8.

[0054] Fig.10 It is the sensitivity test of SDD30.

[0055] Fig.11 Sensitivity test for SDI7.

[0056] Fig.12 For sensitivity detection of DSI7. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] Example 1: Application of five DIP-STR markers in mixed DNA samples

[0060] 1. Experimental Materials

[0061] 1. Biological samples

[0062] (1) DNA standard: human genomic DNA 007 with known sequence (2 ng / μL) (GlobalFiler PCR Amplification Kit, Catalog No. 4476135, Thermo Fisher Scientific).

[0063] (2) Sources of single samples and simulated mixed DNA samples: 20 blood samples from unrelated individuals in the healthy adult population of China.

[0064] (3) Population sample: 200 blood samples from healthy adults in China.

[0065] 2. Reagents

[0066] DNA Blood Midi Kit (Qiagen, Germany); BK-WD-WD72 ultra-micro magnetic bead DNA extraction kit (Bokun Biotechnology, China); Qubit TM dsDNA HS Assay Kit (Invitrogen, USA); AmpliTaq Gold TM DNA Amplification Kit (Applied Biosystems, USA); dNTP Mix (2.5 mM) (Takara, Japan); Invitrogen TM Primers (10 μM, common primers and fluorescent primers) (Invitrogen, USA); DNATyper TM 19 kit internal standard and ladder (Public Security Bureau Forensic Identification Center, China); DNATyper21STR kit (Public Security Bureau Forensic Identification Center, China); Hi-Di TM Formamide (Thermo Fisher Scientific, USA); Agarose H (Sangon Biotechnology, China); 6×DNA Loading Buffer (Solaibao, China); D2000DNAMarker (Tiangen, China); Gelred dye (BIOTIU, USA); 5×TBE buffer (Biyuntian, China); anhydrous ethanol (analytical grade), etc.

[0067] 2. Experimental Methods

[0068] 1. Extraction of DNA samples

[0069] 1. DNA Blood Midi Kit is used to extract whole genomic DNA from blood samples

[0070] (1) Take the previously collected blood sample with anticoagulant added from the -80°C refrigerator to room temperature and mix it. Take 2 mL for later use (note that the blood sample container taken out at -80°C is prone to bursting due to temperature changes); prepare sufficient ultrapure water for later use.

[0071] (2) Preparation of reagents. Add 125 mL of anhydrous ethanol to Buffer AW1 and mix thoroughly; add 150 mL of anhydrous ethanol to Buffer AW2 and mix thoroughly; add 5.5 mL of deionized water to QIAGEN protease and mix thoroughly.

[0072] (3) Pipette 200 μL of QIAGEN protease into the bottom of a 15 mL centrifuge tube.

[0073] (4) Add 2 mL of blood sample and mix quickly to allow the protease to fully mix with the blood sample and accelerate the protease's decomposition of protein. Note: If the sample volume is less than 2 mL, use PBS buffer to make up the volume.

[0074] (5) Add 2.4 mL of Buffer AL to the sample and mix by inverting 15 times for at least 1 min.

[0075] (6) Incubate at 70°C for 10 min to fully lyse the cell membrane and nuclear membrane and release DNA.

[0076] (7) Take out the centrifuge tube, add 2 mL of anhydrous ethanol, and invert vigorously 10 times.

[0077] (8) Transfer half of the mixture obtained in the previous step to the QIAamp Midi centrifuge column (in a new 15 mL centrifuge tube), taking care not to wet the ring on the edge. Close the lid and centrifuge at 1850×g (3000 rpm) for 3 min.

[0078] (9) Remove the centrifuge column, discard the filtrate, and wipe off the spillage on the side of the tube to avoid cross contamination. Replace the centrifuge column in a 15 mL centrifuge tube, add the remaining liquid from step (6) to the QIAamp Midi centrifuge column, close the lid, and centrifuge at 1850 × g (3000 rpm) for 3 min.

[0079] (10) Remove the QIAamp Midi centrifuge column, discard the filtrate, wipe off the spillage on the side of the tube, place it in a 15 mL centrifuge tube, add 2 mL of Buffer AW1, tighten the cap, and centrifuge at 4500 × g (5000 rpm) for 1 min.

[0080] (11) Open the lid, add 2 mL of Buffer AW2 to the QIAamp Midi centrifuge column, tighten the lid, and centrifuge at 4500 × g (5000 rpm) for 15 min.

[0081] (12) Transfer the QIAamp Midi centrifuge column to a new 15 mL centrifuge tube and discard the filtrate and the used collection tube.

[0082] (13) At room temperature (15-25°C), pipette 300 μL of double-distilled water onto the membrane of the QIAamp Midi centrifuge column, close the lid, incubate for 5 min, and centrifuge at 4500 × g (5000 rpm) for 2 min.

[0083] (14) For high concentration DNA, repeat step (12) and elute again.

[0084] (15) Transfer the eluted DNA to a 1.5 mL centrifuge tube for later use or storage.

[0085] 2. WD-ultra-micro magnetic beads method to extract DNA from blood card samples

[0086] (1) Take a blood card sample and cut it into a size of about 2×2 mm.

[0087] (2) DNA was extracted using the WD-ultra-micro magnetic bead method using a BK-TQ-001E fully automatic 96-channel micro-DN extraction workstation (Bokun Biotechnology, China), and the elution volume was set to 50 μL.

[0088] (3) Random samples were taken from the 200 eluted DNA templates and quantified using a Qubit3.0 fluorescence quantification instrument. The quantitative results showed that the concentration of the randomly sampled samples was between 0.9 ng / μL and 1.2 ng / μL.

[0089] (4) The 200 DNA samples extracted from unrelated individuals were numbered and stored in a -20°C refrigerator for future use.

[0090] (II) DNA concentration determination

[0091] The pre-prepared buffer mixture containing dye was mixed with standard 1, standard 2, and sample in the ratio of 190μL:10μL, 190μL:10μL, and 199μL:1μL respectively, and then allowed to stand for 3 minutes. Select "dsDNA High sensitivity" on the Qubit3.0 fluorescence quantifier, set the sample volume to 1μL, and finally measure the concentration of the extracted DNA sample. After marking, freeze it in a -20℃ refrigerator for later use. Among them, the buffer mixture containing dye and standard 1 and standard 2 are all Qubit TM Products in dsDNA HS Assay Kit (Invitrogen, USA).

[0092] (III) Screening of target DIP-STR loci

[0093] According to the characteristics of the DIP-STR allele loci with excellent specificity and sensitivity that have been screened abroad, the screening conditions of the DIP-STR locus database are determined as follows:

[0094] (1) To prevent the amplification product from being too long, the interval between DIP and STR is less than 200 bp. The DIP locus can be located on the upstream or downstream flank of the autosomal STR locus, and the allele fragment range of the DIP-STR candidate gene locus is kept as small as possible;

[0095] (2) the DIP sequence length was 3–10 bp, and the minimum allele frequency in the screened DIP loci was higher than 0.15;

[0096] (3) Tri-, tetra-, penta-, and hexanucleotide STR repeat sequences are preferred because their stutter products have fewer repeats than dinucleotide STRs, which is beneficial for the analysis of mixed samples;

[0097] (4) The DIP-STR loci are located in the non-coding regions on different chromosomes or on different chromatin arms of the same chromosome, ensuring complete linkage equilibrium between the loci.

[0098] According to the above criteria, autosomal STR loci with good polymorphism in the Chinese population were screened from the STR database (http: / / www.cstl.nist.gov / strbase / ), and then DIP loci that met the above screening criteria on the upstream / downstream flanks of the STR loci were screened through UCSC (http: / / genome.ucsc.edu / ), and finally the complete linkage equilibrium between the DIP-STR loci was ensured. According to the comprehensive screening conditions, 15 loci with the highest ranking were selected and named SDD7 (STR-DIP-Deletion-7), SDD8 (STR-DIP-Deletion-8), SDD10 (STR-DIP-Deletion-10), SDD12 (STR-DIP-Deletion-12), and SDD13 (STR-DIP-Deletion-14). DD25(STR-DIP-Deletion-25), SDD30(STR-DIP-Deletion-30), SDD31(STR-DIP-Deletion-31), SDI7(STR-DIP-Insertion-7), SDI14(STR-DIP-Insertion-14), SDI15(STR- DIP-Insertion-15), DSD1 (DIP-STR-Deletion-1), DSD7 (DIP-STR-Deletion-7), DSD8 (DIP-STR-Deletion-8), DSI3 (DIP-STR-Insertion-3), DSI6 (DIP-STR-Insertion-6).

[0099] (IV) Primer design and verification

[0100] 1. Primer design

[0101] By designing bidirectional allele-specific primers and utilizing the adaptation of alleles at the DIP site between the components of the mixed DNA sample, the DIP-STR marker can be targeted to the relatively small amount of DNA in the mixed sample. The bidirectional primers corresponding to a DIP-STR site include: two specific primers (L / S primers) for amplifying the insertion / deletion region (L / S-DIP), and a universal primer for amplifying the STR region at the other end. If and only if the main component in the mixed sample is homozygous, the specific primers may be able to target the alleles in the minor component that are different from the main component for amplification. Figure 1 , taking the case where the primary donor DNA is SS homozygous (when the primary donor DNA is LL homozygous, the principle is the same), the secondary donor DNA is LL homozygous (such as Figure 1 A) or LS heterozygous (as shown in Figure 1The main donor DNA is amplified by targeting the different alleles L in the secondary donor DNA (shown in B), which contains a different allele L from the main donor DNA. A set of allele-specific primers (the forward primer is a universal STR primer, and the reverse primer is an L-DIP primer) is used to amplify the different alleles L in the secondary donor DNA. At this time, the DIP-STR site of the main donor DNA cannot be effectively amplified because it does not contain a complementary sequence to the specific primer, thereby eliminating the masking effect of the main donor DNA amplification in PCR and obtaining accurate typing of the secondary component. Based on the DIP-STR marker sequence screened by the database, the primer design software primer3 is used to design amplification primers. The design process needs to comprehensively consider factors such as the length and annealing temperature of the candidate marker site, and considering that the subsequent experiment needs to be performed with fluorescent electrophoresis, the length of the amplified fragments of the same fluorescent marker cannot overlap.

[0102] 2. PCR reaction and agarose gel electrophoresis

[0103] Leveraging AmpliTaq Gold TM DNA Amplification Kit, Invitrogen TM Primers (10 μM) and dNTP Mix (2.5 mM) were used to prepare the Master MIX mixture for DNA amplification. Sample DNA (human genomic DNA 007 with a known sequence at a concentration of 2 ng / μL) was added in different volume gradients (1 μL, 2 μL, 3 μL, 4 μL, 5 μL), and ultrapure water was added to make up the final volume to 20 μL (see Table 1 for details). PCR System 9700 performed a single PCR reaction for each DIP-STR marker: denaturation at 95°C for 5 min; amplification at 94°C for 30 s, 58°C / 55°C for 1 min, and 72°C for 1 min, for a total of 30 cycles; and finally extension at 72°C for 30 min.

[0104] Table 1. PCR reaction system (20 μL)

[0105] Element Volume(μ) Sample DNA (5 gradients in total) 1-5 One DIP end primer (10 μM) 0.4 STR end primer (10μM) 0.4 <![CDATA[10×PCR Buffer (without Mg 2+ )]]> 1.6 <![CDATA[Mg 2+ (25mM)]]> 2.0 dNTP (2.5mM each 1.0 Gold-labeled enzyme 0.2 Ultrapure water Make up to 20 μL

[0106] Note: “Gold standard enzyme” is AmpliTaq Gold TM DNA polymerase in DNA amplification kit (Applied Biosystems, USA). Two reaction systems were set up for each DIP-STR site: one was a DIP end primer and a universal STR end primer amplification system, and the other was another DIP end primer and a universal STR end primer amplification system.

[0107] Take a 1% agarose gel, mix 1μL Loading buffer with 5μL Tiangen D2000 by dot staining, and then load it to the first gel well. Then, mix 1μL Loading buffer with 5μL of the above PCR product and load it. Put the loaded film into the electrophoresis tank, with the gel well close to the cathode, add the prepared 1×TBE buffer, make sure that the buffer does not pass the gel, turn on the electrophoresis instrument, set 120V / 100mA, and observe and count the agarose gel electrophoresis band results under ultraviolet light after 45 minutes of electrophoresis. The agarose gel electrophoresis results of human genome DNA 007 samples with different gradients and known sequences show that the bands are obvious when the DNA sample amount in the 20μL system is about 6ng. Based on this and combined with the DNA concentration of 20 blood samples extracted in advance, calculate its dilution multiple and addition amount, and repeat the amplification and agarose electrophoresis operations for these samples.

[0108] 3. PCR product sequencing and analysis

[0109] The bands appearing in the agarose gel electrophoresis results of the 20 samples were marked, and the corresponding PCR amplification products were sent to the Thermo Fisher sequencing service platform for reverse sequencing (since the specific primer is a reverse primer, if the forward primer is a specific primer, forward sequencing is selected), and the sequencing feedback results were put into the alignment tool BLAST (basic local alignment search tool, NCBI sequence database website https: / / www.ncbi.nlm.nih.gov / ) to perform sequence similarity analysis on the product sequences of each genetic marker, to check whether the product sequences amplified by PCR with specific primers can match, and whether the obtained amplification products meet the experimental expectations. If they match, the correctness of the primer design can be verified, and the specific primers can be used for amplification inspection of the corresponding sites, and fluorescent labels can be added for subsequent experiments.

[0110] 4. Fluorescence PCR reaction and capillary electrophoresis

[0111] The fluorescently labeled STR end primer (10 μM), DIP end primer (10 μM), gold-labeled enzyme and pre-prepared Mix were mixed according to the proportions in Table 2 to prepare a MasterMix mixture for DNA amplification. Then, 1 μL of human genomic DNA 007 samples with known sequences in different concentration gradients (0.025 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.2 ng / μL, 0.4 ng / μL) were added respectively, and water was added to make up the final volume to 10 μL system (see Table 2 for details). A single PCR reaction was performed for each DIP-STR marker according to the amplification conditions mentioned above. Each single PCR reaction was performed using a PCR System 9700 thermal cycler.

[0112] Table 2. Fluorescence PCR reaction system (10 μL)

[0113] Element Volume(μ) Sample DNA 1 One DIP end primer (10 μM) 0.2 STR end primer (10 μM, fluorescent label) 0.2 <![CDATA[10×PCR Buffer (without Mg 2+ )]]> 0.8 <![CDATA[Mg 2+ (25mM)]]> 1.0 dNTP (2.5 mM each) 0.5 Gold-labeled enzyme 0.1 Ultrapure water Make up to 10 μL

[0114] Note: “Gold standard enzyme” is AmpliTaq Gold TM DNA polymerase in DNA amplification kit (Applied Biosystems, USA). Two reaction systems were set up for each DIP-STR site: one was a DIP end primer and a universal STR end primer amplification system, and the other was another DIP end primer and a universal STR end primer amplification system.

[0115] After mixing 10 μL of the prepared loading buffer and 1 μL of the amplified product and centrifuging, capillary electrophoresis was performed using an ABI3500xL genetic analyzer, and the PCR amplified products after electrophoresis were analyzed using Gene Mapper RID-X (version 1.4) software (Life Technologies), and the analysis threshold was set to 50 relative fluorescence units (RFU) (specifically: ≥50RFU is considered positive, and <50RFU is considered negative). The capillary electrophoresis results of human genomic DNA 007 (2ng / μL) samples with known sequences at different gradients showed that a good typing result could be obtained when the DNA sample amount in the 10 μL system was 0.1ng. Accordingly, the DNA extracted from 20 blood samples was diluted to 0.1ng / μL, and the above amplification and capillary electrophoresis operations were repeated to obtain the amplified fragment lengths of different sites in the 20 samples.

[0116] 5. Population data and statistical analysis

[0117] 1. Polymorphism test

[0118] DNA from 200 blood card samples extracted using the WD-ultra-micro magnetic bead method was taken out, PCR amplification was performed according to the amplification conditions and amplification system in Table 3, capillary electrophoresis was performed using an ABI3500xL genetic analyzer, and Gene ID-X software was used to analyze the PCR amplification products after electrophoresis, and the analysis threshold was set to 50 relative fluorescence units (RFU) (the positive and negative judgment criteria were the same as before).

[0119] 2. Haplotype naming method

[0120] The sequence results of the amplified products obtained by sequencing were analyzed, and the haplotypes of different fragments were named with reference to the insertion and deletion sequences of the DIP-STR sites and the number of repetitions of the STR repeat units. For example, L19 means that the haplotype DIP-STR site is an insertion site and the number of repetitions of the STR repeat unit is 19 times; or it means that the haplotype DIP-STR site is a non-deletion site and the number of repetitions of the STR repeat unit is 19 times. S9 means that the haplotype DIP-STR site is a non-insertion site and the number of repetitions of the STR repeat unit is 9 times; or it means that the haplotype DIP-STR site is a deletion site and the number of repetitions of the STR repeat unit is 9 times.

[0121] 3. Gene ID-X Software Settings

[0122] Design of panels and bins: Open Gene Mapper RID-X, find the Panel Manager tab in Tools, set NewKit and name it "DIP-STR"; set two NewPanels under Kit and name them "S" and "L" respectively; set NewMarkers under S and L respectively and name them as the names of each site according to the fragment length interval and fluorescence color obtained by different amplification systems in polymorphism test; set BinSet named "dip-str" in the newly set "DIP-STR", add bins under each Marker respectively, and the name and position interval of the bin should be set and adjusted theoretically according to the average value of the population data plus or minus three standard deviations. Analysis parameter setting: Open Gene Mapper RID-X, find the Panel Manager tab in Tools, set NewKit and name it "DIP-STR"; set two NewPanels under Kit and name them "S" and "L" respectively; set NewMarkers under S and L respectively and name them as the names of each site according to the fragment length interval and fluorescence color obtained by different amplification systems in polymorphism test; set BinSet named "dip-str" in the newly set "DIP-STR", add bins under each Marker respectively, and the name and position interval of the bin should be set and adjusted theoretically according to the average value of the population data plus or minus three standard deviations. ID-X, add sample data, add a new method in AnalysisMethod and name it MIX. In MIX, select Bin Set as "dip-str"; add Typer500 in LIZ, and set the size to 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 300bp, 350bp, 400bp, 490bp, and 500bp.

[0123] 4. Statistical analysis

[0124] According to the DIP-STR typing obtained from the electrophoresis results after amplification of 200 samples, the number of haplotypes (N) of each DIP-STR genetic marker site, the gene frequency corresponding to each haplotype, and the individual discrimination power (DP) of these samples were counted, and the observed heterozygosity (Obs.Het) was calculated using PowerstatsV12 (Promega) software. There are two situations in which DIP-STR can separate two mixed DNA samples by targeting the secondary donor DNA with specific primers (see Figure 2There are shaded marks in the figure): one is that the primary donor and secondary donor DNA are homozygous for different DIP types (SS&LL or LL&SS); the other is that the primary donor DNA is homozygous for DIP, and the secondary donor DNA is heterozygous for DIP (SS&SL or LL&SL) (only when the secondary donor DNA contains a haplotype that the primary donor DNA does not have can the secondary donor DNA be specifically amplified and distinguished, so the primary donor DNA cannot be heterozygous and cannot contain both haplotypes). From this, we can know that the formula for calculating the probability of the informative genotype is I=2s 2 l 2 +2s 3 l+2sl 3 , where s and l are the gene frequencies of alleles S and L. The DIP-STR allele frequencies and the corresponding random match probabilities of detecting DIP-STR genotypes were used to evaluate DIP-STR polymorphism and discrimination.

[0125] (VI) DIP-STR site performance analysis

[0126] 1. Research on detection threshold

[0127] For the DIP-STR loci obtained in the initial screening, multiple high-concentration DNA samples with heterozygous DIP typing at the loci were selected and gradient dilution (gradient dilution of a single sample) was performed, so that the amount of DNA template in the PCR reaction was: 1ng, 0.5ng, 0.25ng, 0.1ng, 0.05ng, 0.025ng, 0.01ng, 0.005ng. Each DIP-STR locus was amplified, detected and analyzed with the above DNA concentration gradient to explore the minimum input amount of DNA template for the locus in the DIP-STR experimental system, that is, the minimum detection threshold of the locus. The final concentration of the PCR amplification system was 10μL, the amplification conditions, capillary electrophoresis and annealing temperature were consistent with the previous ones, and the analysis threshold of the Gene Mapper RID-X software was set to 50RFU (the positive and negative judgment criteria were the same as before). In the three repeated experiments, if no detection peak appeared during the analysis, it means that the amount of DNA in the gradient cannot meet the requirements of the DIP-STR experimental system, so the gradient is excluded, otherwise it is retained. And so on, find out the minimum detection threshold corresponding to each DIP-STR site.

[0128] 2. Research on detection sensitivity

[0129] The sensitivity of the DIP-STR loci obtained in the initial screening was explored separately, and simulated unbalanced mixed DNA samples were prepared to verify the resolution and discrimination of the loci in unbalanced mixed DNA samples. To facilitate the analysis and statistics of the experiment, the DNA samples with homozygous DIP typing were used as the main donor, and the DNA samples with heterozygous DIP typing were used as the secondary donor (the homozygous and heterozygous here are for DIP, and the linked STR genotype differences are not considered). The two were mixed in gradient multiples, and the amount of the main donor DNA template to the amount of the secondary donor DNA template was designed to be 10:1, 20:1, 50:1, 100:1, 500:1, 1000:1 (mass ratio, see Table 3 for details). It should be noted that the amount of the secondary donor DNA sample in the 10μL PCR reaction system is not less than 0.1ng to ensure that there is no false negative result due to low sample size.

[0130] The PCR system, amplification conditions, capillary electrophoresis and analytical experimental conditions for each locus were consistent with the previous experiment. The samples were mixed according to the required unbalanced DNA mixed sample ratio and repeated three times. The maximum multiple ratio of the secondary donor DNA sample typing in the DNA mixed sample was counted and compared with the maximum multiple ratio of the mixed sample that could be distinguished by ordinary STR primers.

[0131] Table 3. Preparation of mixed DNA samples

[0132]

[0133] 3. Experimental Results and Analysis

[0134] 1. DIP-STR site pre-screening and primer design

[0135] After consulting the literature to determine the screening conditions, the marker loci were searched and pre-screened using databases such as UCSC. This study selected 15 DIP-STR loci that have not been reported and meet the screening conditions described in this article. For the convenience of explanation, different loci are named in this article (see Table 4 for details). Primer sequence information designed using primer3 software. The primer design and related information for the pre-screening loci are shown in Table 4.

[0136] Table 4. Pre-screening DIP-STR markers and primer sequence information

[0137]

[0138]

[0139] Note: F stands for forward primer; RS stands for reverse S primer, and RL stands for reverse L primer. R stands for reverse primer; FS stands for forward S primer, and FL stands for forward L primer. When initially testing the specificity of primers, use primers without fluorescence for verification. For further exploration, primers with fluorescence are required for capillary electrophoresis detection. Naming (taking SDD7 and SDI15 as examples): SDD7 (STR-DIP-Deletion-7) means that the gene locus screened out is that the STR sequence is ranked before the DIP sequence, and the DIP site is a missing GAGAGG sequence, and 7 is the site number during the screening process; SDI15 (STR-DIP-insertion-15) means that the gene locus screened out is that the STR sequence is ranked before the DIP sequence, and the DIP site is an inserted AGAT sequence, and 15 is the site number during the screening process. The "Chromosome: Physical Position" in the table is determined based on the alignment of the human whole genome sequence (version number GRCh38).

[0140] 2. Verification of pre-screening sites

[0141] The corresponding agarose gel electrophoresis results of 20 unrelated individual DNA samples are shown in Table 5. S and L refer to the bands that appear after agarose gel electrophoresis of the product amplified by a DIP end primer (S primer or L primer) and a universal primer at the STR end. Among the 15 pre-screened loci, SDD10, SDD12, SDD25, SDD31, DSD7 and DSI3 could not amplify PCR products, and the agarose gel electrophoresis results were all negative, so the above 6 loci were discarded.

[0142] The sequencing results of PCR products of 9 sites, namely SDD7, SDD8, SDD30, SDI7, SDI14, SDI15, DSD1, DSI6 and DSI7, were counted, and the alignment results in the NCBI database were compared with the original sequence information. It was found that 4 sites, namely SDI14, SDI15, DSD1 and DSI6, could not be used for subsequent experiments due to reasons such as inconsistency between the sequencing results and the target fragment sequence, no matching human genome sequence, and sequencing failure. Therefore, the above 4 sites were discarded.

[0143] Table 5. Agarose gel electrophoresis results of 20 samples

[0144]

[0145]

[0146] 3. Group data statistics and analysis

[0147] (1) Allele frequency

[0148] The allele frequencies corresponding to the five DIP-STR markers of 200 unrelated individual samples are shown in Table 6. According to the capillary electrophoresis typing results, the number of haplotypes (N) were 3, 4, 17, 13 and 9, respectively. The smaller number of haplotypes in SDD7 and SDD8 was due to the smaller number of STR alleles linked to DIP; the frequencies of S-type and L-type haplotypes were both above 0.20, and the haplotype number and frequency distribution of SDD30 and SDI7 loci were relatively ideal. The observed heterozygosity (Obs.Het) of all loci was ideal (see Table 7 for details). In addition, according to the statistical individual recognition power (DP) in Table 7, the combined discrimination power (the combined power of discrimination value, CDP) of these five DIP-STR markers was obtained, CDP = 1-(1-DP SDD7 )×(1-DP SDD8 )×(1-DP SDD30 )×(1-DP SDI7 )×(1-DP DSI7 )=0.9997. It can be seen that the five DIP-STR loci screened by the present invention have a high comprehensive identification ability, and the combined use of the screened DIP-STR loci can have a high resolution and individual identification ability, which further confirms its strong advantage in mixed DNA testing.

[0149] Table 6. Haplotype and gene frequencies of five DIP-STR markers in the Chinese population

[0150]

[0151]

[0152] (2) Informative genotype probability

[0153] The informative genotype probability (I value) was used to estimate the probability of detecting the secondary donor DNA using DIP-STR markers in a mixed DNA sample of two people. The allele frequencies were estimated based on 200 unrelated Chinese population samples, using the formula I = 2s 2 l 2 +2s 3 l+2sl 3 It was calculated that these five DIP-STR genetic markers had a high probability of informative genotypes (I values), with an average informative genotype frequency of approximately 0.37 (see Table 7 for details), all above 0.32. Compared with the previously reported data (0.32), the newly screened five loci had a higher I value, indicating that they have a high application value in the Chinese population.

[0154] Table 7. Forensic genetic parameters of five DIP-STR genetic markers from the Chinese population

[0155]

[0156] Note: The number of haplotypes N and haplotype frequencies were obtained from the statistics in Table 6. The observed heterozygosity, effective information gene frequency and individual recognition ability of the corresponding loci were obtained by entering the population data using Powerstats V12 (Promega) software.

[0157] The average probability of the genotype information of these five DIP-STR markers is 0.37, in other words, at least 37 minor donor DNAs can be detected in 100 mixed DNA samples. For these five DIP-STR markers, independent repeated experiments were performed on each mixed sample, and the experimental results were consistent with the binomial distribution. Therefore, the "BINOM.DIST.RANGE" function was used to calculate the expected estimated value of the site discrimination power in the Excel table. The results showed that when the five sites screened in this study were used to detect mixed DNA samples, at least one effective DIP-STR marker was observed in 90% of the mixed samples, which means that there is a 90% chance that different components in the mixed DNA can be successfully distinguished using only these five sites (Table 8).

[0158] Table 8. Detection of information marks

[0159]

[0160] 4. DIP-STR site performance analysis

[0161] (1) Research on detection threshold

[0162] By serial dilution of DNA samples with heterozygous DIP typing corresponding to each genetic marker (serial dilution of a single sample), the DNA sample amounts in the PCR reaction were: 1ng, 0.5ng, 0.25ng, 0.1ng, 0.05ng, 0.025ng, 0.01ng, 0.005ng. The threshold detection results of SDD7, SDD8, SDD30, SDI7 and DSI7 are shown in Figure 2. Figures 3 to 7 The peak height data after electrophoresis are shown in Table 9 (the results shown in the table are the averages of more than three repetitions). It can be seen that the minimum detection threshold that the primers at most sites can reach is 0.005 ng.

[0163] Table 9. Peak height data after electrophoresis

[0164]

[0165] Note: “-” means not detected.

[0166] (2) Investigation of detection sensitivity

[0167] When the five DIP-STR-labeled allele-specific primers were used to amplify the mixed DNA samples, all loci were able to detect the minor donor DNA in the sample with a ratio of 1000:1 (see Table 10, Figures 8 to 12 From the data in the chart, it can be found that the allele-specific primers of the five DIP-STR markers of the present invention have a higher sensitivity when amplifying a mixed DNA sample.

[0168] Table 10. Summary of detectable minor donor DNA in simulated mixed DNA samples

[0169] DIP-STR loci SDD7 SDD7 SDD8 SDD8 SDD30 SDD30 SDI7 SDI7 DSI7 DSI7 Detection of target genes S L S L S L S L S L Detection limit 1000:1 1000:1 1000:1 1000:1 1000:1 1000:1 1000:1 1000:1 1000:1 1000:1

[0170] Note: The detection limit refers to the maximum ratio of the primary donor DNA to the secondary donor DNA template in a mixed DNA sample that can detect the target gene after amplification using specific primers.

[0171] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of 5 DIP-STR markers in any of the following: (A1) Mixed DNA detection; (A2) Mixed spot identification; The five DIP-STR markers are respectively designated as SDD7, SDD8, SDD30, SDI7, and DSI7; The SDD7 is rs34666822-STR; The SDD8 is rs138646306-STR; The SDD30 is rs11279377-STR; The SDI7 is rs10652632-STR; The DSI7 is rs56113354-STR.

2. Use of a substance for detecting five DIP-STR markers in any of the following: (A1) Mixed DNA detection; (A2) Mixed spot identification; The five DIP-STR markers are respectively designated as SDD7, SDD8, SDD30, SDI7, and DSI7; The SDD7 is rs34666822-STR; The SDD8 is rs138646306-STR; The SDD30 is rs11279377-STR; The SDI7 is rs10652632-STR; The DSI7 is rs56113354-STR.

3. A set of primers for detecting mixed DNA, consisting of primer set A, primer set B, primer set C, primer set D and primer set E; The primer set A is used for typing the SDD7 in claim 1 or 2, and is composed of a specific primer A1 and a specific primer A2 targeting the DIP site, and a universal primer A3 targeting the STR site; the primer A1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.1; the primer A2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.2; the primer A3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.3; The primer set B is used for typing the SDD8 in claim 1 or 2, and is composed of a specific primer B1 and a specific primer B2 targeting the DIP site, and a universal primer B3 targeting the STR site; the primer B1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.4; the primer B2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.5; the primer B3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.6; The primer set C is used for typing the SDD30 in claim 1 or 2, and is composed of a specific primer C1 and a specific primer C2 targeting the DIP site, and a universal primer C3 targeting the STR site; the primer C1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.7; the primer C2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.8; the primer C3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.9; The primer set D is used for typing the SDI7 in claim 1 or 2, and is composed of a specific primer D1 and a specific primer D2 targeting the DIP site, and a universal primer D3 targeting the STR site; the primer D1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.10; the primer D2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.11; the primer D3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.12; The primer set E is used to type the DSI7 in claim 1 or 2, and consists of a specific primer E1 and a specific primer E2 targeting the DIP site, and a universal primer E3 targeting the STR site; the primer E1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.13; the primer E2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.14; and the primer E3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.

15.

4. The primer set according to claim 3, characterized in that: In the primer set A, the universal primer A3 is fluorescently labeled; and / or In the primer set B, the universal primer B3 is fluorescently labeled; and / or In the primer set C, the universal primer C3 is fluorescently labeled; and / or In the primer set D, the universal primer D3 is fluorescently labeled; and / or In the primer set E, the universal primer E3 is fluorescently labeled.

5. A reagent or kit comprising the primer set according to claim 3 or 4.

6. Use of the primer set according to claim 3 or 4 or the reagent or kit according to claim 5 in any of the following: (A1) Mixed DNA detection; (A2) Mixed stain identification.

7. A method for determining which individuals a mixed spot originates from, comprising the following steps: (a1) extracting DNA from the mixed stain to be identified to obtain mixed DNA; sampling each individual in the comparison group to obtain single individual DNA of known origin; the comparison group is composed of each individual from which the mixed stain is suspected to originate; (a2) using the primer set described in claim 3 or 4 or the reagent or kit described in claim 5 to detect the mixed DNA and the single individual DNA of known origin in (a1), respectively, to obtain the typing results of the mixed DNA at the five DIP-STR loci, and the typing results of the single individual DNA of known origin at the five DIP-STR loci; (a3) Determining which individuals in the comparison group the DNA in the mixed stain comes from by comparing the typing results of the mixed DNA obtained in (a2) with the typing results of the single individual DNA whose source is known.

8. The use or the primer set or the reagent or the kit or the method according to any one of claims 1 to 7, characterized in that: The mixed spots are unbalanced mixed spots.

9. The use or primer set or reagent or kit or method according to any one of claims 1 to 7, characterized in that: The mass ratio of the main donor DNA to the secondary donor DNA in the mixed DNA is less than or equal to 1000:1.

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