A method for recovering original template DNA from STR kit amplification products containing dUTP

Abstract

The application discloses a method for recovering original template DNA from STR kit containing dUTP amplification product, comprising the following steps: S1, using the STR kit containing dUTP to amplify the detection sample as the original template, obtaining the amplification product containing dUTP, electrophoretic detection, and obtaining the first typing information; S2, adding UNG enzyme in the remaining amplification product for digestion treatment; S3, inactivating the product after digestion by high temperature, using a purification kit for purification recovery, removing the excess primers, dNTP and digestion fragments, and retaining the original template DNA. The application uses the STR kit containing dUTP amplification product, the amplification product can be digested by UNG enzyme without affecting the original template, and the original template is recovered by purification, so that the original template can be used for STR kit detection again, and more typing information can be provided by the combined use of multiple kits.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for recovering original template DNA from STR kit amplification products containing dUTP. Background Technology

[0002] Short tandem repeats (STRs) are typically DNA repetitive sequences consisting of 1 to 6 base units in the genome. Due to the high variability in the number of core repeats among individuals and their abundance in the genome, STR loci exhibit excellent genetic polymorphism. They are currently the second-generation genetic markers after restriction fragment length polymorphism (RFLP) and are widely used. Compared to previous techniques such as RFLP, STRs, with their small locus size and ability to multiplex under similar amplification conditions, demonstrate significant advantages in the rapid and efficient detection of trace and degraded samples.

[0003] Currently, crime scenes in common cases are often complex, and collected samples are frequently affected by adverse factors such as soil microbial decomposition, high temperatures, and chemical corrosion, leading to DNA degradation or extremely low DNA content. Forensic evidence is often obtained in trace or ultra-trace quantities. However, forensic DNA testing has specific requirements regarding the amount of DNA used, and for some special cases, forensic experts need to use different STR kits in combination. For example, DNA testing is an established part of sexual assault investigations and prosecutions. The main purpose of DNA evidence is to identify suspects or prove sexual contact. However, due to the extremely unbalanced ratio of female and male DNA in typical stains, conventional autosomal analysis often fails to detect the DNA of sexual assault perpetrators. In traces from typical sexual assault cases, additional Y-STR analysis can increase the information content of reported autosomal STR analysis results by 21%. Compared to autosomal STR analysis, Y-STR analysis is more likely to detect multiple male contributors, and it is particularly valuable in complex mixtures that cannot be interpreted by autosomal analysis.

[0004] A conventional STR kit contains template DNA, amplification products, residual primers, and dNTPs after amplification. If these components could be removed, and the template DNA purified and recovered, the recovered DNA could theoretically be used for detection with a new STR kit. However, currently available STR kits use an amplification system constructed with thymine dTTP. Amplification products from systems containing thymine dTTP cannot be specifically eliminated, making it impossible to target and recover the original template within the system. Therefore, combined detection using multiple kits is not feasible with limited sample sizes.

[0005] Chinese patent CN107828758A discloses recombinant uracil-DNA glycosidase, its encoding gene, preparation method, and application. Chinese patent CN106701716B discloses a heat-labile UNG enzyme and its application. Both patents add the UNG enzyme to the PCR reaction system solely to prevent product contamination and do not utilize the UNG enzyme for the recovery of the original template from the product.

[0006] Therefore, a method for digesting amplification products with UNG enzyme and recovering the original template DNA from amplification products of STR kits containing dUTP is urgently needed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for recovering original template DNA from amplification products of STR kits containing dUTP. The amplification products are amplified using STR kits containing dUTP, and the amplification products can be digested by UNG enzyme without affecting the original template. The original template is then purified and recovered, and can be reused for STR kit detection. The combined use of multiple kits can provide more genotyping information.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] This invention provides a method for recovering original template DNA from STR amplification products containing dUTP, comprising the following steps:

[0010] S1. Using the test sample as the original template, amplify it using an STR kit containing dUTP to obtain the amplification product containing dUTP, detect it by electrophoresis, and obtain the first genotyping information.

[0011] S2. Add UNG enzyme to the remaining amplification products for digestion.

[0012] S3. The digested product is inactivated by high temperature and purified and recovered using a purification kit to remove excess primers, dNTPs and digested fragments, while retaining the original template DNA.

[0013] Preferably, the amount of UNG enzyme in step S2 is 0.1-1U.

[0014] Preferably, the digestion process in step S2 is performed on a PCR instrument and incubated at 50°C for 1-3 hours.

[0015] Preferably, the high-temperature inactivation temperature in step S3 is 95°C, and the high-temperature inactivation time is 5-10 min.

[0016] Preferably, the purification step in step S3 is as follows:

[0017] S31. Place the deionized product into a centrifuge tube, add lysis buffer and proteinase K;

[0018] S32, Place in a metal bath;

[0019] S33. Continue adding magnetic beads and binding liquid to the centrifuge tube, shake to mix, place on a magnetic rack, and remove the waste liquid.

[0020] S34. Add rinsing solution I, shake to mix well, place on a magnetic rack, and remove the waste liquid.

[0021] S35. Add rinsing solution II, shake to mix well, place on a magnetic rack, and remove the waste liquid.

[0022] S36. Open the centrifuge tube lid and let the magnetic beads dry;

[0023] S37. Add the eluent and place it in a metal bath.

[0024] Preferably, in step S31, the amount of lysis buffer is 400-600 µL, the amount of proteinase K is 10-20 µL, in step S33, the amount of magnetic beads is 20-30 µL, the amount of binding buffer is 200-300 µL, and in step S37, the amount of elution buffer is 20-50 µL.

[0025] Preferably, in step S32, the temperature of the metal bath is 56°C and the time is 30 min; in step S37, the temperature of the metal bath is 60°C and the time is 10 min.

[0026] Preferably, the rinsing solution in step S34 contains 50% polyethylene glycol, and the rinsing solution II in step S35 contains 80% ethanol and 3M sodium chloride.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention utilizes the presence of dUTP in newly synthesized amplicones. The UNG enzyme effectively catalyzes the hydrolysis of dUTP and N-glycosidic bonds in the phosphate backbone of single-stranded or double-stranded DNA, allowing the DNA strand lacking a base to undergo further hydrolysis and breakage under alkaline conditions and high temperatures, while simultaneously preserving the native thymine-containing DNA. Therefore, in forensic STR typing, when the sample nucleic acid is sufficient for only one test and repeated sampling is not possible, dUTP is used instead of dTTP. After obtaining typing information in the first round of PCR, the product is incubated with the UNG enzyme at a specific temperature for a certain period, which degrades any PCR product containing dUTP, eliminating residual contamination and preserving the original DNA template. Subsequent high-temperature incubation of the reaction tube effectively inactivates the UNG enzyme. The product is then purified and recovered, and the recovered template DNA is used for typing detection in other STR kits to obtain more typing information. Attached Figure Description

[0029] Figure 1 This is the detection pattern of mixed male and female DNA using the NHID® 25A Case Kit (Anti-Contamination Version) in Example 1 of this invention;

[0030] Figure 2 This is the Captain Y detection spectrum for mixed male and female DNA product recovery using the NHID® 25A Case Kit (Anti-Contamination Version) in Example 1 of this invention;

[0031] Figure 3 This is the peak height diagram of the control group in Example 2 of the present invention;

[0032] Figure 4 This is the peak height diagram of template recovery detection in Embodiment 2 of the present invention;

[0033] Figure 5 This is an electrophoresis image of the template recovered by elution in the PCR system of Example 3 of this invention;

[0034] Figure 6 This is an electrophoresis image of the template recovered from beaded amplification detection in Embodiment 3 of the present invention;

[0035] Figure 7 This is the electrophoresis image of the control group in Example 4 of the present invention;

[0036] Figure 8 This is an electrophoresis image of the magnetic bead recovery template detection in Embodiment 4 of the present invention;

[0037] Figure 9 This is an electrophoresis image of the silicon bead recovery template detection in Embodiment 4 of the present invention;

[0038] Figure 10 Electrophoresis diagram of template recovery detection in the silica membrane centrifuge column of embodiment 4 of the present invention. Detailed Implementation

[0039] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0040] Example 1: After amplification of a mixed sample with more females than males, the template was recovered for Y chromosome detection.

[0041] The NHID® 25A Case Kit (anti-contamination version) from Suzhou Xinhai Biotechnology Co., Ltd. (hereinafter referred to as Suzhou Xinhai) was used to amplify and detect mixed DNA (9948 DNA (1 ng / μL) and 9947 DNA (1 ng / μL), with a male to female DNA mixing ratio of 1:4). The amplification products were digested with UNG enzyme, followed by enzyme inactivation and purification. The recovered original template was then analyzed using the NHID® Captain Y-STR kit produced by Suzhou Xinhai. The specific implementation method is as follows:

[0042] 1. Sample preparation.

[0043] The mixed DNA (9948 DNA and 9947 DNA) was amplified and detected using a kit containing dUTP to obtain the amplification products.

[0044] Prepare the reaction mixture (25 μL) according to the table below:

[0045]

[0046] Amplification was performed using an ABI 9700 PCR instrument (Golden Base) under the following conditions:

[0047]

[0048] 2. Electrophoresis detection.

[0049] Formamide (VWR) and internal standard (Suzhou Xinhai) were mixed at a ratio of 10:0.2, and 9 μL was dispensed into each well. Then, 1 μL of the amplification product was added. Electrophoresis was performed using a 3500 xL DX genetic analyzer with the program (1.2 kV, 24 s). One allele genotyping control was electrophoresed for each amplification.

[0050] 3. Experimental results and analysis.

[0051] After electrophoresis, the data were analyzed using GeneMapper ID-X software (see...). Figure 1 According to the mixed sample detection results, it can be seen that both male and female samples exist in the mixed sample.

[0052] 4. Digestion and recycling.

[0053] (1) Add UNG enzyme (0.1-1U) to the remaining amplification product, add TE buffer to make up to 50μL, place on a PCR instrument for digestion, digestion program is 50℃, 2h; then inactivate at 95℃ for 5min;

[0054] (2) Place the deionized product into a centrifuge tube, add 400 μL of lysis buffer and 10 μL of proteinase K;

[0055] (3) Place in a metal bath at 56°C for 30 minutes;

[0056] (4) Continue to add 20 μL of magnetic beads and 250 μL of binding solution to the centrifuge tube, shake to mix, place on the magnetic rack, and remove the waste liquid;

[0057] (5) Add 500 μL of rinsing solution I, shake to mix, place on a magnetic rack, and remove the waste liquid;

[0058] (6) Add 500 μL of rinsing solution II, shake to mix, place on a magnetic rack, and remove the waste liquid;

[0059] (7) Open the centrifuge tube lid and let the magnetic beads dry;

[0060] (8) Add 20 μL of elution buffer, place in a metal bath, 60 °C, for 10 min.

[0061] 5. NHID® Captain Y-STR kit detection and recovery template.

[0062] Prepare the reaction mixture (25 μL) according to the table below:

[0063]

[0064] Amplification was performed using an ABI 9700 PCR instrument (Golden Base) under the following conditions:

[0065]

[0066] 6. Electrophoresis detection.

[0067] Formamide and internal standard were mixed at a ratio of 10:0.2, and 9 μL was dispensed into each well. Then, 1 μL of the amplification product was added. Electrophoresis was performed using a 3500 xL DX genetic analyzer with the following program: 1.2 kV, 24 s. One allele genotyping control was analyzed by electrophoresis for each amplification.

[0068] 7. Experimental Results and Analysis.

[0069] After electrophoresis, the data were analyzed using GeneMapper ID-X software (see...). Figure 2The NHID® Captain Y-STR assay detects Y chromosome loci that are male-specific, indicating that mixed genotyping information has been obtained from the original template and the original mixed template male samples.

[0070] Example 2: Template recovery rate.

[0071] The NHID® 25A Case Kit (anti-contamination version) amplifies 0.25 ng of 9948 DNA (25 µL system). After amplification, the DNA is digested with UNG enzyme, inactivated at 95 °C, purified and recovered. 20 µL of elution buffer is added to the magnetic beads for elution. The recovered template is detected by the NHID® CaptainY-STR kit.

[0072] A control group was set up (the original template was not amplified, purified, or recovered using the NHID® 25A Case Kit (Anti-Contamination Version)). 0.25 ng of 9948 DNA was diluted to 20 µL with elution buffer and directly amplified and detected using the NHID® Captain Y-STR Kit.

[0073] The template recovery and control groups were each performed in four replicates. Recovery rate was calculated by comparing the average peak heights of the recovered template and the control group. The amplification system for the NHID® Captain Y-STR kit was as follows:

[0074]

[0075] according to Figure 3 and Figure 4 The average peak heights of the recovered template and the control group detected by the NHID® Captain Y-STR kit were obtained (see table below). The recovery rate could be calculated as: Recovery rate = Peak height of recovered template / Peak height of control group * 100%. The results showed that the template recovery rate could reach over 90%.

[0076]

[0077] Example 3: PCR amplification system elution and PCR system bead amplification.

[0078] The NHID® 25A Case Kit (Anti-contamination Version) amplifies 0.25 ng of 9948 DNA (25 µL system). After amplification, the DNA is digested with UNG enzyme, inactivated at 95 °C, purified, and recovered. The magnetic beads are then eluted with the 25 µL NHID® Captain Y-STR kit PCR system. After elution, the PCR system is magnetically aspirated into a PCR tube for amplification. Alternatively, the magnetic beads can be mixed and suspended with the 25 µL PCR system, and the PCR system and magnetic beads can be directly transferred into a PCR tube for amplification. The PCR reaction conditions are as described in Example 1.

[0079] In conventional recovery processes, elution buffer is typically used for elution. However, the PCR system typically adds a maximum of 17.5 μL of elution buffer. Since there may be liquid loss during elution, the volume of elution buffer needs to be more than 17.5 μL, resulting in some of the recovered nucleic acids not being added to the PCR system. In this embodiment, the PCR system elution and beaded amplification can amplify and detect all the nucleic acids.

[0080] Example 4: Purification of reagent (Xinhai Bio NH9547) was performed using magnetic beads, silica beads, and silica membrane centrifuge columns.

[0081] The NHID® 25A Case Kit (Anti-contamination Version) amplifies 0.25 ng of 9948 DNA (25 µL system). After amplification, the DNA is digested with UNG enzyme and inactivated at 95 °C. The digestion products are lysed with lysis buffer and proteinase K. After adding binding buffer, the DNA is bound to magnetic beads, silica beads, and silica gel membrane centrifuge column for purification and recovery of the original template DNA.

[0082] Elution was performed using 20 µL of elution buffer. A control group (original template was not amplified, purified, or recovered using the NHID® 25A Case Kit (anti-contamination version)) was set up. 0.25 ng of 9948 DNA was diluted to 20 µL with elution buffer and amplified using the NHID® CaptainY-STR kit. Each of the three purification methods was performed in four replicates. The PCR reaction conditions were as described in Example 1. All three methods were able to recover more than 90% of the original template.

[0083]

[0084] This invention uses a PCR system containing dUTP to amplify the template, which can be used for normal sample detection. The amplification product contains dUTP, which can be digested and cleaved by UNG enzyme, while the original template does not contain dUTP and is not affected by UNG enzyme. Thus, the original template can be purified and recovered from the amplification product for use in other kits.

[0085] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for recovering original template DNA from a dUTP-containing STR kit amplification product, characterized by, The method comprises the following steps: S1, using a dUTP-containing STR kit to amplify the detection sample as an original template to obtain a dUTP-containing amplification product, and performing electrophoresis detection to obtain first typing information; S2, adding UNG enzyme to the remaining amplification product to perform digestion treatment; S3, inactivating the digested product at high temperature, purifying and recovering the product using a purification kit, removing excess primers, dNTPs and digestion fragments, and retaining the original template DNA; After purification and recovery, the magnetic beads are eluted, and the PCR system is taken out by magnetic attraction and placed in a PCR tube for amplification, or the magnetic beads are mixed and suspended with the PCR system, and the PCR system and the magnetic beads are taken out together and placed in a PCR tube for amplification; the purification steps are as follows: S31, placing the inactivated product in a centrifuge tube, adding lysis solution and proteinase K; S32, placing in a metal bath; S33, continuing to add magnetic beads and binding solution to the centrifuge tube, mixing and oscillating, placing on a magnetic stand, and discarding the waste liquid; S34, adding rinse solution I, mixing and oscillating, placing on a magnetic stand, and discarding the waste liquid; rinse solution I contains 50% polyethylene glycol; S35, adding rinse solution II, mixing and oscillating, placing on a magnetic stand, and discarding the waste liquid; rinse solution II contains 80% ethanol and 3M sodium chloride; S36, opening the centrifuge tube cover and air-drying the magnetic beads; S37, adding elution solution and placing in a metal bath.

2. The method of recovering original template DNA from a dUTP-containing STR kit amplification product according to claim 1, characterized in that, The amount of UNG enzyme in step S2 is 0.1-1U.

3. The method of recovering original template DNA from a dUTP-containing STR kit amplification product according to claim 1, wherein, The digestion treatment in step S2 is performed on a PCR instrument, and incubation is performed at 50°C for 1-3h.

4. The method of recovering original template DNA from amplification products of a dUTP- containing STR kit according to claim 1, characterized in that, The high-temperature inactivation temperature in step S3 is 95°C, and the high-temperature inactivation time is 5-10min.

5. The method of recovering original template DNA from amplification products of a dUTP- containing STR kit according to claim 1, characterized in that, The amount of lysis solution in step S31 is 400-600µL, the amount of proteinase K is 10-20µL, the amount of magnetic beads in step S33 is 20-30µL, the amount of binding solution is 200-300µL, and the amount of elution solution in step S37 is 20-50µL.

6. The method of recovering original template DNA from amplification products of a dUTP- containing STR kit according to claim 1, characterized in that, The temperature of the metal bath in step S32 is 56°C, and the time is 30min; the temperature of the metal bath in step S37 is 60°C, and the time is 10min.

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