Method for recovering original template DNA from STR kit amplification product containing dUTP

Abstract

The invention discloses a method for recovering original template DNA (deoxyribonucleic acid) from an STR (short tandem repeat) kit amplification product containing dUTP (deoxyuridine triphosphate), which comprises the following steps: S1, taking a detection sample as an original template, performing amplification by using the STR kit containing dUTP to obtain an amplification product containing dUTP, and performing electrophoresis detection to obtain first typing information; s2, adding UNG enzyme into an amplification product left after detection, and carrying out digestion treatment; and S3, carrying out high-temperature inactivation on the digested product, carrying out purification and recovery by using a purification kit, removing redundant primers, dNTP and digested fragments, and retaining the original template DNA. An STR kit amplification product containing dUTP is used, the amplification product can be digested by UNG enzyme without affecting an original template, the original template is recycled through purification and then used for STR kit detection again, and more typing information can be provided through combined use of multiple kits.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a method for recovering the original template DNA from the amplification products of an STR kit containing dUTP. Background Art

[0002] Short tandem repeats (STRs) are usually a section of DNA repeat sequences composed of 1 - 6 base units in the genome. Due to the high variability of the number of core sequence repeats among individuals and their abundance in the genome, STR loci have good genetic polymorphism. They are the second-generation genetic markers after restriction fragment length polymorphism and are also a commonly used type of genetic marker. Compared with previous techniques such as restriction fragment length polymorphism, STRs have obvious advantages in quickly and efficiently detecting trace and degraded samples because of their small locus fragments and similar amplification conditions that allow multiplex amplification.

[0003] Currently, in common cases, the situation at the crime scene is often complex, and the collected samples are often affected by adverse factors such as decomposition by soil microorganisms, high-temperature environments, and chemical corrosion, resulting in degradation of the sample DNA or extremely low DNA content in the collected samples. The forensic samples obtained are often trace or ultra-trace. However, when forensic scientists conduct DNA testing, there are certain requirements for the amount of DNA used. And for some special cases, forensic scientists need to jointly use different STR kits to detect samples. 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 the sexual assailant. In the traces of typical sexual assault cases, additional use of Y-STR analysis can increase the high-information content by 21% compared to the results of reported autosomal STR analysis. Compared with autosomal STR analysis, Y-STR analysis is more likely to detect multiple male contributors, and Y-STR analysis is particularly valuable in complex mixtures that cannot be interpreted by autosomal analysis.

[0004] The system after amplification by a conventional STR kit contains template DNA, amplification products, residual primers, and dNTPs. If the amplification products, residual primers, and dNTPs in the amplification system can be removed and the template DNA is purified and recovered, in principle, the recovered DNA can be used for detection with a new STR kit. However, currently, the STR kits on the market use an amplification system constructed with thymine base dTTP. The amplification products of the amplification system constructed with thymine base dTTP cannot be specifically eliminated, and the original template cannot be targeted and recovered in the system. Therefore, it is impossible to perform joint detection using multiple kits with limited samples.

[0005] The Chinese patent with the publication number CN107828758A discloses a recombinant uracil-DNA glycosylase and its encoding gene, preparation method and application. The Chinese patent with the publication number CN106701716B discloses a thermally unstable UNG enzyme and its application. In both cases, adding the UNG enzyme to the PCR reaction system is only used to prevent product contamination, and the UNG enzyme is not applied to the recovery of the original template in the product.

[0006] Therefore, a method for digesting the amplified product with UNG enzyme and recovering the original template DNA from the amplified product of the STR kit containing dUTP is urgently needed to be proposed. Summary of the Invention

[0007] To solve the defects existing in the prior art, the present invention provides a method for recovering the original template DNA from the amplified product of the STR kit containing dUTP. Using the amplified product of the STR kit containing dUTP, the amplified product can be digested by the UNG enzyme without affecting the original template. The original template is recovered by purification and then used again for STR kit detection. Using multiple kits in combination can provide more typing information.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a method for recovering the original template DNA from the amplified product of the STR kit containing dUTP, including the following steps: S1. Using the test sample as the original template, amplify it with the STR kit containing dUTP to obtain the amplified product containing dUTP, and perform electrophoresis detection to obtain the first typing information; S2. Add the UNG enzyme to the remaining amplified product for digestion; S3. Inactivate the digested product at high temperature, and use a purification kit for purification and recovery to remove the excess primers, dNTPs and digestion fragments, and retain the original template DNA.

[0009] Preferably, the amount of the UNG enzyme in the step S2 is 0.1-1U.

[0010] Preferably, the digestion treatment in the step S2 is carried out on a PCR instrument and incubated at 50°C for 1-3h.

[0011] Preferably, the high-temperature inactivation temperature in the step S3 is 95°C, and the high-temperature inactivation time is 5-10min.

[0012] Preferably, the purification steps in the step S3 are as follows: S31. Put the digested product into a centrifuge tube, and add the lysis solution and proteinase K; S32. Place it in a metal bath; S33. Continue to add magnetic beads and binding solution to the centrifuge tube, mix well by oscillation, place it on a magnetic stand, and aspirate and discard the waste liquid; S34. Add Wash Buffer I, mix well by oscillation, place it on a magnetic stand, and aspirate and discard the waste liquid; S35. Add Wash Buffer II, mix well by oscillation, place it on a magnetic stand, and aspirate and discard the waste liquid; S36. Open the lid of the centrifuge tube to air-dry the magnetic beads; S37. Add elution buffer and place it in a metal bath.

[0013] 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 solution is 200 - 300 µL, and in step S37, the amount of elution buffer is 20 - 50 µL.

[0014] 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.

[0015] Preferably, the Wash Buffer in step S34 contains 50% polyethylene glycol, and the Wash Buffer II in step S35 contains 80% ethanol and 3 M sodium chloride.

[0016] Compared with the prior art, the present invention has the following beneficial effects: Since the newly synthesized amplicons in the present invention contain dUTP, UNG enzyme can effectively catalyze the hydrolysis of the N-glycosidic bond between dUTP and the sugar-phosphate backbone in single-stranded or double-stranded DNA, enabling the DNA strand lacking bases to be further hydrolyzed and broken in an alkaline medium and at high temperature, while keeping the natural DNA containing thymine bases intact. Therefore, in forensic STR typing detection, when the sample nucleic acid is only sufficient for one detection and repeated sampling is not possible, dTTP is replaced with dUTP. After obtaining the typing information at the end of the first round of PCR, the product is incubated with UNG enzyme for a certain time under certain temperature conditions to degrade any PCR product containing dUTP, eliminate residual contamination, and retain the original DNA template. Subsequently, incubating the reaction tube at high temperature can effectively inactivate UNG enzyme, and then purify and recover the product. The recovered template DNA is used for typing detection with other STR kits to obtain more typing information. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the detection chromatogram of male-female mixed DNA by the NHID® 25A case kit (anti-contamination version) in Example 1 of the present invention; Figure 2It is the detection map of Captain Y for the recovery of mixed DNA products of male and female in the NHID® 25A case kit (anti-pollution version) in Example 1 of the present invention; Figure 3 It is the peak height detection map of the control group in Example 2 of the present invention; Figure 4 It is the peak height detection map of template recovery in Example 2 of the present invention; Figure 5 It is the electrophoresis map of the recovered template for the elution detection of the PCR system in Example 3 of the present invention; Figure 6 It is the electrophoresis map of the recovered template for the bead amplification detection in Example 3 of the present invention; Figure 7 It is the electrophoresis map of the control group detection in Example 4 of the present invention; Figure 8 It is the electrophoresis map of the magnetic bead recovered template detection in Example 4 of the present invention; Figure 9 It is the electrophoresis map of the silica bead recovered template detection in Example 4 of the present invention; Figure 10 It is the electrophoresis map of the recovered template detected by the silica gel membrane centrifugal column in Example 4 of the present invention. Detailed implementation manners

[0018] 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 only used to illustrate and explain the present invention and are not used to limit the present invention. Example 1: Detect the Y chromosome after recovering the template by amplifying the mixed sample with more females and fewer males.

[0019] The NHID® 25A case kit (anti-pollution version) of Suzhou Xinhai Biotechnology Co., Ltd. (hereinafter referred to as Suzhou Xinhai) was used to amplify and detect the mixed DNA (9948 DNA (1 ng / μL) and 9947 DNA (1 ng / μL), and the mixing ratio of male DNA to female DNA was 1:4). The amplified product was digested with UNG enzyme, followed by enzyme inactivation and purification and recovery. The recovered original template was detected and analyzed by the NHID® Captain Y-STR kit produced by Suzhou Xinhai. The specific implementation methods are as follows: 1. Sample preparation.

[0020] The mixed DNA (9948 DNA and 9947 DNA) was amplified and detected by a kit containing dUTP to obtain the amplified product.

[0021] The reaction system (25 μL) was prepared according to the following table:

[0022] Amplification was carried out using the 9700 PCR instrument Golden Seat (ABI), and the amplification conditions were as follows:

[0023] 2. Electrophoresis detection.

[0024] 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 detection was carried out using a 3500 xL DX genetic analyzer, and the electrophoresis program was (1.2 kV, 24 s). One allelic typing control was required for electrophoresis detection for each amplification.

[0025] 3. Experimental results and analysis.

[0026] After electrophoresis, the data was analyzed using GeneMapper ID-X software (see Figure 1 ). According to the detection results of the mixed samples, it can be seen that both male and female samples are present in the mixed samples.

[0027] 4. Digestion and recovery.

[0028] (1) UNG enzyme (0.1 - 1 U) was added to the remaining amplified product for detection, and TE buffer was added to make up to 50 μL. It was placed on a PCR instrument for digestion, and the digestion program was 50 °C for 2 h; then inactivated at 95 °C for 5 min; (2) The digested product was placed in a centrifuge tube, and 400 μL of lysis buffer and 10 μL of proteinase K were added; (3) Placed in a metal bath at 56 °C for 30 min; (4) Continue to add 20 μL of magnetic beads and 250 μL of binding solution to the centrifuge tube, mix well by shaking, place it on a magnetic rack, and aspirate the waste liquid; (5) Add 500 μL of wash buffer I, mix well by shaking, place it on a magnetic rack, and aspirate the waste liquid; (6) Add 500 μL of wash buffer II, mix well by shaking, place it on a magnetic rack, and aspirate the waste liquid; (7) Open the lid of the centrifuge tube to dry the magnetic beads; (8) Add 20 μL of elution buffer, place it in a metal bath at 60 °C for 10 min.

[0029] 5. Detect the recovered template using the NHID® Captain Y-STR kit.

[0030] Prepare the reaction system (25 μL) according to the following table:

[0031] Amplification was carried out using the 9700 PCR instrument Golden Seat (ABI), and the amplification conditions were as follows:

[0032] 6. Electrophoresis detection.

[0033] Mix formamide and internal standard at a ratio of 10:0.2, dispense 9 μL into each well, and then add 1 μL of the amplification product. Perform electrophoresis detection using a 3500 xL DX genetic analyzer, and the electrophoresis program is (1.2 kV, 24 s). One allelic typing control needs to be electrophoretically detected for each amplification.

[0034] 7. Experimental results and analysis.

[0035] After electrophoresis, the data is analyzed using GeneMapper ID-X software (see Figure 2 ). The NHID® Captain Y-STR detects Y chromosome loci, which are unique to males, indicating that the original template has been recovered and the mixed genotyping information of male samples in the original mixed template has been detected. Example 2: Template recovery rate.

[0036] The NHID® 25A case kit (anti-contamination version) amplifies 0.25 ng of 9948 DNA (25 μL system). After amplification, it is digested with UNG enzyme, inactivated at 95 °C, purified and recovered. 20 μL of eluent is added to elute the magnetic beads, and the recovered template is detected using the NHID® Captain Y-STR kit.

[0037] Set up a control group (the original template is not amplified, purified and recovered using the NHID® 25A case kit (anti-contamination version)). 0.25 ng of 9948 DNA is diluted to 20 μL with eluent and directly amplified and detected using the NHID® Captain Y-STR kit.

[0038] Both the template recovery and the control group are subjected to 4 repeated experiments. The recovery rate is calculated by comparing the average peak heights of the detected recovered template and the control group. For the NHID® Captain Y-STR kit amplification system, the reaction conditions are as follows:

[0039] According to Figure 3 and Figure 4 the average peak heights of the NHID® Captain Y-STR kit detections of the recovered template and the control group can be obtained (see the following table), and the recovery rate can be calculated. Recovery rate = (peak height of detected recovered template / peak height of detected control group) * 100%. The results show that the template recovery rate can reach over 90%.

[0040]

[0041] Example 3: Elution of the PCR amplification system and bead-amplification of the PCR system.

[0042] The NHID® 25A Case Kit (anti-contamination version) was used to amplify 0.25 ng of 9948 DNA (25 μL system). After amplification, it was digested with UNG enzyme, inactivated at 95°C, purified and recovered. The PCR system of the NHID® Captain Y-STR Kit (25 μL) was prepared to elute the magnetic beads. After elution, the PCR system was magnetically removed and transferred to a PCR tube for amplification, or the magnetic beads were mixed and suspended with the PCR system (25 μL), and then the PCR system and the magnetic beads were directly taken out and transferred to a PCR tube for amplification. The PCR reaction conditions were the same as those in Example 1.

[0043] During the conventional recovery process, eluent is generally used for elution. However, the maximum amount of eluent added to the PCR system is 17.5 μL, and there may be liquid loss during the elution process. Therefore, the volume of the eluent should be more than 17.5 μL, resulting in a part of the recovered nucleic acid not being added to the PCR system. In this example, the elution of the PCR system and bead-amplification can amplify and detect all the nucleic acids. Example 4: The purification reagent (NH9547 of Xinhai Biotech) was used for purification with magnetic beads, silica beads and silica gel membrane centrifugal columns.

[0044] The NHID® 25A Case Kit (anti-contamination version) was used to amplify 0.25 ng of 9948 DNA (25 μL system). After amplification, it was digested with UNG enzyme and inactivated at 95°C. The digestion product was lysed with lysis buffer and proteinase K. After adding the binding buffer, it was bound to magnetic beads, silica beads and silica gel membrane centrifugal columns for purification and recovery of the original template DNA.

[0045] Elution was carried out with 20 μL of eluent. A control group was set up (the original template was not amplified, purified and recovered with the NHID® 25A Case Kit (anti-contamination version)). 0.25 ng of 9948 DNA was diluted to 20 μL with eluent and amplified and detected with the NHID® Captain Y-STR Kit. Four replicate experiments were performed for each of the three purification methods. The PCR reaction conditions were the same as those in Example 1. All three methods could recover more than 90% of the original template.

[0046]

[0047] The PCR system with dUTP of the present invention is used to amplify the template, which can be used for normal detection of samples. And the amplification product contains dUTP and can be digested and cut by UNG enzyme, while the original template does not contain dUTP and will not be affected by UNG enzyme. Thus, the original template can be purified and recovered from the amplification product for detection with other kits.

[0048] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not used 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 perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for recovering original template DNA from amplification products of a STR kit containing dUTP, characterized in that: The following steps are involved: S1. Use the test sample as the original template, use the STR kit containing dUTP to amplify, obtain the amplification product containing dUTP, perform electrophoresis detection, and obtain the first typing information; S2, adding UNG enzyme to the remaining amplification product for digestion; S3. The digested product is inactivated by high temperature, purified and recovered using a purification kit, and excess primers, dNTPs and digested fragments are removed, retaining the original template DNA.

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

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

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

5. The method for recovering original template DNA from amplification products of a STR kit containing dUTP according to claim 1, characterized in that: The purification steps in step S3 are as follows: S31, the product after the deionization is placed in a centrifuge tube, and lysis buffer and proteinase K are added; S32, put into metal bath; S33, continue to add magnetic beads and binding solution to the centrifuge tube, shake and mix, place on the magnetic rack, and discard the waste liquid; S34, add rinsing solution I, shake and mix, place on the magnetic rack, and discard the waste liquid; S35, add rinsing solution II, shake and mix, place on the magnetic rack, and discard the waste liquid; S36. Open the centrifuge tube cover and dry the magnetic beads; S37. Add eluent and place in a metal bath.

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

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

8. The method for recovering original template DNA from amplification products of a STR kit containing dUTP according to claim 1, characterized in that: The rinsing solution I in step S34 contains 50% polyethylene glycol, and the rinsing solution II in step S35 contains 80% ethanol and 3 M sodium chloride.

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