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A fluorescent-labeled 32-plex InDels compound amplification system and its application

A composite amplification system and fluorescent labeling technology, applied in the field of composite amplification system and fluorescently labeled 32-plexInDels composite amplification system, can solve problems such as low recognition power

Active Publication Date: 2020-07-21
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that compared with European populations, Investigator The kit’s ability to identify the Chinese population is low, and there is no InDel commercialized kit developed for Chinese people in China

Method used

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  • A fluorescent-labeled 32-plex InDels compound amplification system and its application
  • A fluorescent-labeled 32-plex InDels compound amplification system and its application
  • A fluorescent-labeled 32-plex InDels compound amplification system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of 32-plex InDels multiplex amplification system

[0025] 1. DNA sample

[0026] Oral swabs or blood samples were collected from 204 unrelated individuals of Han nationality in southern China, including 78 females and 126 males. Genomic DNA was extracted by Chelex-100 method. DNA products were quantified using a Nanodrop 2000 spectrophotometer (ThermoScientific, USA). This study was approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

[0027] 2. Selection of InDel markers and primer design

[0028] InDel markers were selected from 1000 Genomes Project data (the 1000Genomes Project) or published articles. All candidate InDel markers meet the following criteria: (1) indel markers are located on autosomes; (2) in the Chinese Han population, the minimum allele frequency (MAF) of each marker must be ≥ 0.4; (3) The insertion / deletion length of each InDel marker is 3-6bp, and a few of th...

Embodiment 2

[0044] Example 2 Sensitivity Research and Degradation DNA Research

[0045] 1. Sensitivity

[0046] To determine the sensitivity of the 32-plex InDels Multiplex Amplification System, experiments were performed using control DNA 9947A. The control standard DNA 9947A was serially diluted to 500pg, 250pg, 125pg, 62.5pg, 31.25pg and 15.625pg, and PCR and capillary electrophoresis were performed according to the conditions in Example 1.

[0047] Typing map results such as figure 2 Shown (wherein the 500pg concentration DNA typing map is not shown). When the DNA amount is greater than or equal to 31.25pg, the complete typing result can be detected; while the DNA amount is 15.625pg, the peak height of a few sites is lower than the detection threshold. Although 31.25 pg of DNA is sufficient for detection, we recommend using DNA template amounts between 0.5-1 ng for PCR in forensic practice.

[0048] 2. Degrade DNA

[0049] In order to verify the ability of this method to detect ...

Embodiment 3

[0051] Example 3 Species Specificity Test

[0052] Multiplex amplification systems used in forensic applications also need to be specific for Homo genus. We used the Chelex-100 method to extract the DNA of five common animals at crime scenes, including rats, rabbits, pigs, cows, and chickens, for amplification and electrophoresis, and verified the 32-plexInDels multiplex amplification system.

[0053] The results showed that no amplified products were detected in rats, rabbits and chickens, while an insertion at 87 bp of a locus (rs3042783) was detected in DNA from cattle and pigs, see Figure 5 . After primer blast, it was confirmed that a pair of primers at rs3042783 could indeed amplify the DNA fragments of these two species. Overall, however, none of these animals were able to obtain complete DNA profiles using this multiplex amplification system, demonstrating that the 32-plex InDels multiplex amplification system can provide the species-specificity required for human D...

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Abstract

The invention belongs to the technical field of biological detection and particularly relates to a fluorescent-labeled 32-plex InDels composite amplification system and application thereof. The constructed composite amplification system comprises 32 InDel sites and one sex site, the InDel marker with the minor allele frequency (MAF) equal to or greater than 0.4 in the Chinese Han population is selected, and amplified fragments range from 70 bp to 160bp. The composite amplification system is adopted for carrying out genetic typing on 204 unrelated individuals of the Chinese Han population, the cumulative personal recognition probability is 0.999999999999966, and the cumulative non-parent exclusion probability is 0.9982. Studies shown that the constructed composite amplification system has high recognition ability for the Chinese Han population and meanwhile can correctly type DNA templates as low as 31.25 pg and have significant advantages in degrading amplified types of DNA.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a fluorescent-labeled 32-plex InDels composite amplification system and an application thereof. The composite amplification system is a composite amplification system containing 32 insertion-deletion sites. Background technique [0002] In the field of forensic genetics, the problem of personal identification and paternity testing is mainly solved by genotyping DNA genetic markers. Short tandem repeats (STRs) have become the most commonly used genetic markers due to their high polymorphism and known allele frequency data in different national populations. However, the amplified fragments of the STR amplification system are long, the resolution of highly degraded DNA samples is low, and there are defects of high mutation rate. [0003] Single nucleotide polymorphism (SNP) has the characteristics of short amplicon, low mutation rate and high density of hum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 黄代新黄钰洁刘聪肖超易少华
Owner HUAZHONG UNIV OF SCI & TECH
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