CN116377078A - Six-color fluorescent Indel kit for paternity test supplementary use and application thereof - Google Patents
Six-color fluorescent Indel kit for paternity test supplementary use and application thereof Download PDFInfo
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Abstract
The invention discloses a six-color fluorescent Indel kit for paternity test supplement and application thereof, wherein the kit comprises specific amplification primers of 28 Indel loci and 3 autosomal STR loci; these 28 Indel loci are preferably Indel locus sites, the upstream and downstream DNA sequences flanking these locus sites being free of the presence of other Indel locus sites or the presence of other Indel locus sites without affecting the typing of the original locus site; the 3 STR loci can provide paternity test and data of other STR detection kits for comparison, so that errors of detection results caused by sample mistake can be prevented. The kit can obtain more locus information in a trace degradation DNA sample, and is compatible with database information of STR loci of the existing mainstream kit.
Description
Technical Field
The invention relates to the technical field of forensic genetics detection, in particular to a six-color fluorescent marker composite amplification detection system, and particularly relates to a six-color fluorescent Indel kit for paternity test supplement and application.
Background
The Short Tandem Repeat (STR) locus, which is the second generation genetic marker following the restriction fragment length polymorphism, has been widely used for forensic study and identification since the eighth nineties of the twentieth century, and is one of the most widely used genetic markers at present. Compared with other genetic markers, the STR marker has the advantages of small STR locus fragment, easy amplification, more suitability for trace and degradation detection materials, capability of carrying out composite amplification on a plurality of STR loci simultaneously, rapidness, high efficiency, accuracy, sensitivity, large information quantity and the like, and is widely applied to individual identification and parent identification in forensic material evidence.
The main flow kit applied to STR detection in the current forensic detection field mostly adopts a five-color or six-color fluorescent compound amplification technology, the maximum amplified fragments can reach 600bp, and the detected STR sites can reach 20-30. The kit can obtain more STR locus information when normal samples are amplified, but the mutation probability of STR loci reaches about one thousandth, the situation that the loci need to be detected is often encountered in paternity test, the conventional locus adding kit or STR kit has larger accumulated mutation probability along with the increase of the number of the STR loci to be detected, and if new mutation occurs in the STR loci to be detected, the situation that the genetic mutation is non-autogenous is difficult to distinguish.
Disclosure of Invention
In order to avoid the occurrence of the situation, the applicant developed an Indel locus kit as a test kit, wherein the Indel locus is characterized in that mutation probability is one part per million, the mutation probability is one thousand times more stable than that of an STR locus, and the situation of wrong judgment can be avoided by the Indel locus kit. The invention aims to provide a six-color fluorescent Indel kit for paternity test supplement and application thereof, which can obtain more locus information in a trace degradation DNA sample and is compatible with database information of STR loci of the existing mainstream kit so as to overcome the defects of the prior art.
The technical scheme for solving the problems is as follows: a six color fluorescent Indel kit for paternity test complementation, the kit comprising specific amplification primers for 28 Indel loci and 3 autosomal STR loci;
the 28 Indel locus sites were: rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, rs2307963, rs34287950, rs35119276, rs11571577, rs145261968, rs3842225, rs3834129, rs31274670; the 3 autosomal STR loci are: D1S1656, D7S820, D2S1338.
The further technical scheme is as follows: the sequence of the specific amplification primers is as follows:
rs6144644SEQ ID NO:1～2；rs5786049SEQ ID NO:3～4；rs34862123SEQ ID NO:5～6；rs77422519SEQ ID NO:7～8；rs34705786SEQ ID NO:9～10；rs146582211SEQ ID NO:11～12；rs3842715SEQ ID NO:13～14；rs1611048SEQ ID NO:15～16；rs67443324SEQ ID NO:17～18；rs10558392SEQ ID NO:19～20；rs3832592SEQ ID NO:21～22；rs34525141SEQ ID NO:23～24；rs34202831SEQ ID NO:25～26；rs34647099SEQ ID NO:27～28；rs34421865SEQ ID NO:29～30；rs3217112SEQ ID NO:31～32；rs5787309SEQ ID NO:33～34；rs5789990SEQ ID NO:35～36；rs3830737SEQ ID NO:37～38；rs2307561SEQ ID NO:39～40；rs2307963SEQ ID NO:41～42；rs34287950SEQ ID NO:43～44；rs35119276SEQ ID NO:45～46；rs11571577SEQ ID NO:47～48；rs145261968SEQ ID NO:49～50；rs3842225SEQ ID NO:51～52；rs3834129SEQ ID NO:53～54；rs31274670SEQ ID NO:55～56；D1S1656 SEQ ID NO:57～58；D7S820 SEQ ID NO:59～60；D2S1338 SEQ ID NO:61～62。
the further technical scheme is as follows: the concentrations of the specific amplification primers for the 28 Indel loci and 3 autosomal STR loci in the amplification system are shown in table 1.
The further technical scheme is as follows: specific amplification primers were divided into 5 groups: group 1 rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, D1S1656; group 2 rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, D7S820; group 3 rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, D2S1338; group 4 rs2307963, rs34287950, rs35119276, rs11571577, rs145261968; group 5 rs3842225, rs3834129, rs31274670; at least one of the primers is labeled with a fluorescent dye at its 5' end.
The further technical scheme is as follows: the fluorescent dye is any one of 6-FAM, HEX, TAMRA, 6-FAM-ROX and 6-FAM-Alexa594, the fluorescent dyes adopted by each group of primers are different, and the internal standard is orange fluorescent 6-FAM-Cy5; wherein 6-FAM-ROX, 6-FAM-Alexa594, 6-FAM-Cy5 are energy transfer labels.
The further technical scheme is as follows: the fluorescent dyes adopted in the primers are respectively as follows: group 1, 6-FAM, group 2 HEX, group 3 TAMRA, group 4, 6-FAM-ROX, group 5, 6-FAM-Alexa594.
The further technical scheme is as follows: the kit also comprises PCR mixed liquor, an allelic typing standard substance, a DNA standard substance and sdH 2 O and fluorescent molecular weight internal standard; the PCR mixture comprises the following components: mgCl 2 3.0mM, tris-HCl50mM, KCl 100mM,dNTPs 8.0mM,BSA 2g/L, hot start Taq enzyme 1.5U/. Mu.L.
The further technical scheme is as follows: the length of the amplicon fragments of the PCR products is less than 250bp; the amplified fragments of 28 Indel loci are all smaller than 170bp.
The other related technical proposal is as follows: the application of the six-color fluorescent Indel kit for paternity test supplement in forensic identification, paternity test or DNA pedigree construction.
The further technical scheme is as follows: samples to which the kit is applied include aged samples, degraded samples.
By adopting the technical scheme, the six-color fluorescent Indel kit for parent-child paternity test supplement and application have the following beneficial effects compared with the prior art:
(1) The kit is a six-color fluorescent composite amplification kit, and can accommodate more locus sites than the six-color fluorescent kit within the range of the same fragment length.
(2) The 28 Indel locus loci rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, rs2307963, rs34287950, rs35119276, rs11571577, rs145261968, rs3842225, rs3834129, rs31274670 are preferred Indel locus loci, and the upstream and downstream DNA sequences flanking these loci are free of the presence of other Indel locus loci or of the presence of other Indel locus loci without affecting the typing of the original locus, see fig. 4 to 6.
(3) The kit comprises 3 STR loci, and the 3 STR loci can provide paternity test and compare with data of other STR detection kits, so that errors of detection results caused by sample mistake can be prevented.
(4) The 28 Indel locus amplified fragments of the kit are smaller than 170bp, and the kit has a very good detection effect on trace-degraded detection materials, and can obtain enough locus typing information even under the condition that DNA detection material samples are extremely degraded.
(5) The kit comprises a kit of 28 Indel loci, wherein the 28 Indel loci can provide enough conservation data and can provide more accurate data judgment when mutation conditions are encountered in paternity test.
(6) The primer in the kit has the advantages of strong specificity, high sensitivity and accurate typing result, and can completely meet the requirements of actual case inspection, DNA database construction and paternity test.
The technical characteristics of the six-color fluorescent Indel kit for parent-child paternity test supplement and application of the six-color fluorescent Indel kit are further described below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a graph showing the amplification results of a conventional sample of the kit of the present invention;
FIG. 2 is a graph showing the amplification result of a degraded sample of the kit of the present invention;
FIG. 3 is a graph of amplification results of an imported hexachrome kit degradation sample;
FIG. 4 is a screenshot of the rs6144644 gene locus DNA sequence;
FIG. 5 is a screenshot of the rs5786049 gene locus DNA sequence;
FIG. 6 is a screenshot of the rs34862123 gene locus DNA sequence.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1:
a six color fluorescent Indel kit for paternity test complementation, the kit comprising specific amplification primers for 28 Indel loci and 3 autosomal STR loci;
the 28 Indel locus sites were: rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, rs2307963, rs34287950, rs35119276, rs11571577, rs145261968, rs3842225, rs3834129, rs31274670; the 3 autosomal STR loci are D1S1656, D7S820, D2S1338.
The sequence of the specific amplification primers is as follows:
rs6144644SEQ ID NO:1～2；rs5786049SEQ ID NO:3～4；rs34862123SEQ ID NO:5～6；rs77422519SEQ ID NO:7～8；rs34705786SEQ ID NO:9～10；rs146582211SEQ ID NO:11～12；rs3842715SEQ ID NO:13～14；rs1611048SEQ ID NO:15～16；rs67443324SEQ ID NO:17～18；rs10558392SEQ ID NO:19～20；rs3832592SEQ ID NO:21～22；rs34525141SEQ ID NO:23～24；rs34202831SEQ ID NO:25～26；rs34647099SEQ ID NO:27～28；rs34421865SEQ ID NO:29～30；rs3217112SEQ ID NO:31～32；rs5787309SEQ ID NO:33～34；rs5789990SEQ ID NO:35～36；rs3830737SEQ ID NO:37～38；rs2307561SEQ ID NO:39～40；rs2307963SEQ ID NO:41～42；rs34287950SEQ ID NO:43～44；rs35119276SEQ ID NO:45～46；rs11571577SEQ ID NO:47～48；rs145261968SEQ ID NO:49～50；rs3842225SEQ ID NO:51～52；rs3834129SEQ ID NO:53～54；rs31274670SEQ ID NO:55～56；D1S1656 SEQ ID NO:57～58；D7S820 SEQ ID NO:59～60；D2S1338 SEQ ID NO:61～62。
the specific amplification primer sequences and the concentrations thereof corresponding to 31 loci (namely 28 Indel loci and 3 autosomal STR loci) included in the kit are shown in Table 1:
table 1: concentration meter of specific amplification primer
Specific amplification primers were divided into 5 groups: group 1 rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, D1S1656; group 2 rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, D7S820; group 3 rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, D2S1338; group 4 rs2307963, rs34287950, rs35119276, rs11571577, rs145261968; group 5 rs3842225, rs3834129, rs31274670; at least one of the primers is labeled with a fluorescent dye at its 5' end.
The fluorescent dye is any one of 6-FAM, HEX, TAMRA, 6-FAM-ROX and 6-FAM-Alexa594, the fluorescent dyes adopted by each group of primers are different, and the internal standard is orange fluorescent 6-FAM-Cy5; wherein 6-FAM-ROX, 6-FAM-Alexa594, 6-FAM-Cy5 are energy transfer labels (see patent CN 106566827B).
In this example, the fluorescent dyes used in the primers were: group 1, 6-FAM, group 2 HEX, group 3 TAMRA, group 4, 6-FAM-ROX, group 5, 6-FAM-Alexa594.
The kit also comprises a PCR mixed solution (PCR Master Mix), an allelic typing standard AllelicLadder, DNA standard and sdH 2 O and fluorescent molecular weight internal standard; the composition of the PCR Master Mix is: mgCl 2 3.0mM, tris-HCl50mM,KCl 100mM,dNTPs 8.0mM,BSA 2g/L, hot start Taq enzyme 1.5U/. Mu.L.
The length of the PCR product amplicon fragments of the primers is smaller than 250bp. The amplified fragments of 28 Indel loci are all smaller than 170bp.
The six-color fluorescent Indel kit for paternity test supplement can be applied to forensic identification, paternity test or DNA pedigree construction.
Example 2: flow for detecting actual sample by using kit of the invention in actual application
The six-color fluorescent Indel kit for paternity test supplement use described in the embodiment 1 is adopted in the embodiment, and the application steps in forensic identification, paternity test or DNA pedigree construction are as follows: genomic DNA was collected and amplified by PCR, and the amplified product was analyzed.
1. Collection of genomic DNA
Sample sources used in forensic identification, paternity identification or DNA pedigree construction include human genomic DNA extracted by using a Chelex method, a magnetic bead extraction method or an organic extraction method; or human blood or oral cells collected by any one carrier of filter paper, FTA card, cotton swab and gauze without extraction. Sources of test materials include human blood, blood marks, semen, saliva, body fluids, hair, muscle, or tissue organs.
2. PCR amplification
A. Preparation of amplification System according to the Components of Table 2
Table 2: PCR amplification system
| Component (A) | Standard system | Commonly used system |
| PCR Master Mix | 5μL | 2μL |
| Genomic DNA | 0.033-3ng | 0.025-2.5ng |
| Specific primer mixture | 5μL | 2μL |
| Deionized sterilizing water | Make up 25. Mu.L | Make up 10 mu L |
；
B. Expansion thermal cycle
(1) Placing the PCR amplification tube on a thermal cycler;
(2) Selecting the procedure recommended in table 3 for amplification;
(3) The amplified sample should be preserved in dark;
table 3: amplification program of thermal cycler
C. Fluorescence detection of amplified products on genetic analyzer
The sample mixture was composed of deionized formamide and the molecular weight internal standard SD-520 in the system [ (0.5. Mu.L SD-520 (self-produced))X (sample number) + (10. Mu.L deionized formamide) ]. 10.0. Mu.L of the loading mixture was mixed with 1. Mu.L of amplification product or an allelic typing standard AlllicLadder (manufactured by Corp.) to avoid the generation of air bubbles. Denaturation at 95℃for 3 min, ice bath for 3 min, and electrophoresis detection using a genetic analyzer were performed as soon as possible.
3. Typing analysis
The collected data were analyzed by a fragment analysis software analysis genetic analyzer.
Example 3: effect of the kit of the present invention in the test of degraded samples
The detection flow of the degradation detection material by using the kit of the invention is as follows:
1. genomic DNA extraction of degradation assay
The extraction of the genome DNA of various detection materials is carried out by referring to GA/T383-2014 court science DNA laboratory test Specification, and the samples are extracted by a magnetic bead method.
2. The operation steps are as follows:
2.1 formulation of amplification systems according to the components of Table 4:
table 4: PCR amplification system
| Component (A) | Standard system |
| PCR Master Mix | 5μL |
| Genomic DNA | 0.033-3ng |
| Specific primer mixture | 5μL |
| Deionized sterilizing water | Make up 25. Mu.L |
；
2.2 amplification procedure is table 3:
table 3: amplification program of thermal cycler
2.3 fluorescence detection of amplified products on genetic Analyzer
The sample mixture was composed of deionized formamide and the molecular weight internal standard SD-520 in the system [ (0.5. Mu.L SD-520 (self-produced))X (sample number) + (10. Mu.L deionized formamide) ]. 10.0. Mu.L of the loading mixture was mixed with 1. Mu.L of amplification product or an allelic typing standard AlllicLadder (manufactured by Corp.) to avoid the generation of air bubbles. Denaturation at 95℃for 3 min, ice bath for 3 min, and electrophoresis detection using a genetic analyzer were performed as soon as possible.
2.4 typing analysis
The collected data were analyzed by a fragment analysis software analysis genetic analyzer.
3. Conclusion(s)
The kit of the invention is used for amplifying a certain degradation sample, the amplification result is shown in figure 2, and the amplification is carried outThe phenomenon of amplification peak loss is not seen in the amplification detection result; verifeler using applied biosystems TM The Plus kit amplifies a certain degradation sample, the amplification result is shown in figure 3, and the amplification peak of the STR locus of a large fragment larger than 260bp is lost. Compared with the results of the detection, the detection result of the amplified and degraded sample of the kit is obviously superior to that of the traditional STR six-color kit in detection rate, and more effective gene locus information can be obtained.
Claims (10)
1. A six-color fluorescence Indel kit for paternity test supplement use is characterized in that: the kit comprises specific amplification primers of 28 Indel loci and 3 autosomal STR loci;
the 28 Indel locus sites were: rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, rs2307963, rs34287950, rs35119276, rs11571577, rs145261968, rs3842225, rs3834129, rs31274670; the 3 autosomal STR loci are: D1S1656, D7S820, D2S1338.
2. A six color fluorescent Indel kit for paternity test complementation according to claim 1, wherein: the sequence of the specific amplification primers is as follows:
rs6144644 SEQIDNO:1～2；rs5786049 SEQIDNO:3～4；rs34862123 SEQIDNO:5～6；rs77422519 SEQIDNO:7～8；rs34705786 SEQIDNO:9～10；rs146582211 SEQIDNO:11～12；rs3842715 SEQIDNO:13～14； rs1611048 SEQIDNO: 15～16；rs67443324 SEQIDNO: 17～18；rs10558392 SEQIDNO: 19～20；rs3832592 SEQIDNO: 21～22；rs34525141 SEQIDNO: 23～24； rs34202831 SEQIDNO: 25～26；rs34647099 SEQIDNO: 27～28；rs34421865 SEQIDNO: 29～30；rs3217112 SEQIDNO: 31～32；rs5787309 SEQIDNO: 33～34；rs5789990 SEQIDNO: 35～36；rs3830737 SEQIDNO: 37～38；rs2307561 SEQIDNO: 39～40； rs2307963 SEQIDNO: 41～42；rs34287950 SEQIDNO: 43～44；rs35119276 SEQIDNO: 45～46；rs11571577 SEQIDNO: 47～48；rs145261968 SEQIDNO: 49～50；rs3842225 SEQIDNO: 51～52；rs3834129 SEQIDNO: 53～54；rs31274670 SEQIDNO: 55～56；D1S1656 SEQIDNO: 57～58；D7S820 SEQIDNO: 59～60；D2S1338 SEQIDNO: 61～62。
3. a six color fluorescent Indel kit for paternity test complementation according to claim 1, wherein: the concentrations of the specific amplification primers for the 28 Indel loci and 3 autosomal STR loci in the amplification system are shown in table 1.
4. A six color fluorescent Indel kit for paternity test complementation according to claim 1, wherein: specific amplification primers were divided into 5 groups: group 1 rs6144644, rs5786049, rs34862123, rs77422519, rs34705786, rs146582211, rs3842715, D1S1656; group 2 rs1611048, rs67443324, rs10558392, rs3832592, rs34525141, D7S820; group 3 rs34202831, rs34647099, rs34421865, rs3217112, rs5787309, rs5789990, rs3830737, rs2307561, D2S1338; group 4 rs2307963, rs34287950, rs35119276, rs11571577, rs145261968; group 5 rs3842225, rs3834129, rs31274670; at least one of the primers is labeled with a fluorescent dye at its 5' end.
5. A six color fluorescent Indel kit for paternity test complementation according to claim 4, wherein: the fluorescent dye is as follows: any one of 6-FAM, HEX, TAMRA, 6-FAM-ROX and 6-FAM-Alexa594, wherein fluorescent dyes adopted by each group of primers are different, and an orange fluorescent 6-FAM-Cy5 is adopted as an internal standard; wherein 6-FAM-ROX, 6-FAM-Alexa594, 6-FAM-Cy5 are energy transfer labels.
6. A six color fluorescent Indel kit for paternity test complementation according to claim 5, wherein: the fluorescent dyes adopted in the primers are respectively as follows: group 1, 6-FAM, group 2 HEX, group 3 TAMRA, group 4, 6-FAM-ROX, group 5, 6-FAM-Alexa594.
7. A six color fluorescent Indel kit for paternity test complementation according to any one of claims 1-6, wherein: the kit also comprises PCR mixed liquor, an allelic typing standard substance, a DNA standard substance and sdH 2 O and fluorescent molecular weight internal standard; the PCR mixture comprises the following components: mgCl 2 3.0mM, tris-HCl50mM,KCl 100mM,dNTPs 8.0mM,BSA 2g/L, hot start Taq enzyme 1.5U/. Mu.L.
8. A six color fluorescent Indel kit for paternity test complementation according to claim 7, wherein: the length of the amplicon fragments of the PCR products is less than 250bp; the amplified fragments of 28 Indel loci are all smaller than 170bp.
9. Use of a six-color fluorescent Indel kit for paternity test supplementation according to any one of claims 1-8 in forensic identification, paternity test or DNA pedigree construction.
10. Use of a six color fluorescent Indel kit for paternity test complementation according to claim 9, characterized in that: samples to which the kit is applied include aged samples, degraded samples.
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