A forensic dna detection kit with auxiliary gender identification and its identification method

Abstract

The invention aims at solving the technical problem in the prior art that analysis data of the amplified chromosome locus cannot enter the China DNA database, provides a DNA detection kit for auxiliary gender identification for a legal medical expert, or a method for adding auxiliary gender identification to a conventional kit. The key point is that Y special-shaped amplification primer of a fluorescent mark identical to the mark internal standard SIZ mark primer is added on the conventional kit, so as to amplify a segment in SRY zone of the Y chromosome, such as chromosome locus M175 and SRY. Meanwhile, the invention further discloses an SIZ mark primer sequence used for amplifying chromosome locus SRY and M175. Through the improvement, the purpose of auxiliary gender identification can be achieved without adding other components to the kit, or influencing the types of the kit; meanwhile, the amplification segment of Y specificity has the peak at the same fluorescent of the internal standard, peaks of fluorescent marks of other colors are not influenced, the introduction of the data into the public security ministry DNA database is not influenced, thereby conforming to the actual situation of recording for the China public security ministry.

Description

technical field

[0001] The invention belongs to the field of forensic DNA detection, and in particular relates to a method for detecting gender in an internal standard by marking a sex identification locus primer with an internal standard dye. Background technique

[0002] Currently, forensic DNA detection uses multi-color (four-color, five-color, six-color) fluorescent compound amplification of 13-30 STR loci, and simultaneously detects the sex identification locus, the enamel protein gene Ameloginen, as listed in Table 1. Products and patents .

[0003] The forensic detection DNA kit is divided into two parts, one part is called the pre-amplification kit, which includes primers for multiplex amplification (the primers are labeled with a different dye from the labeled internal standard) for PCR amplification; the other part is called the post-amplification kit. The kit, including molecular internal standard and allele standard, is used for capillary electrophoresis detecti...

Examples

Embodiment 1

[0048] Example 1 Add SIZ-marked SRY primers to AGCU EX16 to amplify male and female DNA standards

[0049] 1. Sample source

[0050] 9948, a male DNA standard, was purchased from Promega.

[0051] 9947A, a female DNA standard, was purchased from Promega.

[0052] 2. The AGCU EX16 kit is produced by Wuxi Zhongde Meilian Biotechnology Co., Ltd. Add SIZ-labeled SRY primers to the primer mixture, and configure the final concentrations of SRY to be 0.05 μM, 0.2 μM, 0.8 μM, 1.2 μM, and 2.0 μM respectively. μM.

[0053] The SRY gene is a male sex-determining gene, which refers to a gene segment on the Y chromosome that specifically determines the male sex of a creature. The human SRY gene is located at Yp11.3, contains only one exon, no intron, the transcription unit is about 1.1kb long, and encodes a protein of 204 amino acids. The SRY primer sequence is:

[0054] Upstream primer 5'SIZ-CTGCTATGTTAAGCGTATTCAA-3',

[0055] Downstream primer 5'-GATCTGCGGGAAGCAAACT-3'.

[0056] 3...

Embodiment 2

[0064] Example 2 Add SIZ-marked M175 primers to AGCU EX16 to amplify male and female DNA standards

[0065] 1. Sample source

[0066] 9948, a male DNA standard, was purchased from Promega.

[0067] 9947A, a female DNA standard, was purchased from Promega.

[0068] 2. The AGCU EX16 kit is produced by Wuxi Zhongde Meilian Biotechnology Co., Ltd. Add SIZ-labeled M175 primers to the primer mixture, and configure the final concentrations of M175 to be 0.05 μM, 0.2 μM, 0.8 μM, 1.2 μM, 2.0 μM.

[0069] M175 is a unique indel locus on the Y chromosome.

[0070] The M175 primer sequence is:

[0071] Upstream primer 5'SIZ-CCCAAATCAACTCAACTCCAG-3,

[0072] Downstream primer 5'- TGCAGCATTTTCAGTTAGCC-3'.

[0073] 3. Detection and analysis of amplification and amplification products

[0074] Samples were added and amplified according to the kit instructions.

[0075] Fluorescent detection of the amplified product on a genetic analyzer

[0076] The loading mixture is composed of dei...

Embodiment 3

[0081] Example 3 AGCU EX16 plus SRY primers labeled internal standard fluorescent markers in the Y chromosome to assist in gender identification

[0082] 1. Sample source

[0083] Ameloginen Y has been confirmed to be missing from Wuxi Public Security Bureau's database samples.

[0084] 2. DNA extraction (for the extraction of genomic DNA, refer to "GA / T 383-2002 Forensic Science DNA Laboratory Inspection Specification")

[0085] Genomic DNA was extracted from two whole blood samples by Chelex-100 method:

[0086] Take 0.5-5μl whole blood and place it in a sterilized 1.5ml centrifuge tube, add sdH 2 O 1ml in the tube, shake for a few seconds; place at room temperature for 10 minutes, shake for a few seconds, centrifuge at 12,000rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200μl 5% Chelex -100, shake for a few seconds; keep warm at 56°C for 30 minutes, shake for a few seconds; bath in boiling w...

technical field

[0001] The invention belongs to the field of forensic DNA detection, and in particular relates to a method for detecting gender in an internal standard by marking a sex identification locus primer with an internal standard dye. Background technique

[0002] Currently, forensic DNA detection uses multi-color (four-color, five-color, six-color) fluorescent compound amplification of 13-30 STR loci, and simultaneously detects the sex identification locus, the enamel protein gene Ameloginen, as listed in Table 1. Products and patents .

[0003] The forensic detection DNA kit is divided into two parts, one part is called the pre-amplification kit, which includes primers for multiplex amplification (the primers are labeled with a different dye from the labeled internal standard) for PCR amplification; the other part is called the post-amplification kit. The kit, including molecular internal standard and allele standard, is used for capillary electrophoresis detecti...

Examples

Embodiment 1

[0048] Example 1 Add SIZ-marked SRY primers to AGCU EX16 to amplify male and female DNA standards

[0049] 1. Sample source

[0050] 9948, a male DNA standard, was purchased from Promega.

[0051] 9947A, a female DNA standard, was purchased from Promega.

[0052] 2. The AGCU EX16 kit is produced by Wuxi Zhongde Meilian Biotechnology Co., Ltd. Add SIZ-labeled SRY primers to the primer mixture, and configure the final concentrations of SRY to be 0.05 μM, 0.2 μM, 0.8 μM, 1.2 μM, and 2.0 μM respectively. μM.

[0053] The SRY gene is a male sex-determining gene, which refers to a gene segment on the Y chromosome that specifically determines the male sex of a creature. The human SRY gene is located at Yp11.3, contains only one exon, no intron, the transcription unit is about 1.1kb long, and encodes a protein of 204 amino acids. The SRY primer sequence is:

[0054] Upstream primer 5'SIZ-CTGCTATGTTAAGCGTATTCAA-3',

[0055] Downstream primer 5'-GATCTGCGGGAAGCAAACT-3'.

[0056] 3...

Embodiment 2

[0064] Example 2 Add SIZ-marked M175 primers to AGCU EX16 to amplify male and female DNA standards

[0065] 1. Sample source

[0066] 9948, a male DNA standard, was purchased from Promega.

[0067] 9947A, a female DNA standard, was purchased from Promega.

[0068] 2. The AGCU EX16 kit is produced by Wuxi Zhongde Meilian Biotechnology Co., Ltd. Add SIZ-labeled M175 primers to the primer mixture, and configure the final concentrations of M175 to be 0.05 μM, 0.2 μM, 0.8 μM, 1.2 μM, 2.0 μM.

[0069] M175 is a unique indel locus on the Y chromosome.

[0070] The M175 primer sequence is:

[0071] Upstream primer 5'SIZ-CCCAAATCAACTCAACTCCAG-3,

[0072] Downstream primer 5'- TGCAGCATTTTCAGTTAGCC-3'.

[0073] 3. Detection and analysis of amplification and amplification products

[0074] Samples were added and amplified according to the kit instructions.

[0075] Fluorescent detection of the amplified product on a genetic analyzer

[0076] The loading mixture is composed of dei...

Embodiment 3

[0081] Example 3 AGCU EX16 plus SRY primers labeled internal standard fluorescent markers in the Y chromosome to assist in gender identification

[0082] 1. Sample source

[0083] Ameloginen Y has been confirmed to be missing from Wuxi Public Security Bureau's database samples.

[0084] 2. DNA extraction (for the extraction of genomic DNA, refer to "GA / T 383-2002 Forensic Science DNA Laboratory Inspection Specification")

[0085] Genomic DNA was extracted from two whole blood samples by Chelex-100 method:

[0086] Take 0.5-5μl whole blood and place it in a sterilized 1.5ml centrifuge tube, add sdH 2 O 1ml in the tube, shake for a few seconds; place at room temperature for 10 minutes, shake for a few seconds, centrifuge at 12,000rpm for 3 minutes, discard the supernatant, keep enough supernatant to cover the precipitate, do not stir the precipitate; add 200μl 5% Chelex -100, shake for a few seconds; keep warm at 56°C for 30 minutes, shake for a few seconds; bath in boiling w...

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