Fluorescent labeling complex amplification kit for insertion-deletion (InDel) polymorphism detection, and application of kit

A technology of polymorphism detection and multiple amplification, which is applied in the field of biological detection to achieve accurate typing results, high sensitivity, and good amplification effects

Active Publication Date: 2018-05-22
JIANGSU SUPERBIO LIFE SCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, domestic research on InDel is les

Method used

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  • Fluorescent labeling complex amplification kit for insertion-deletion (InDel) polymorphism detection, and application of kit
  • Fluorescent labeling complex amplification kit for insertion-deletion (InDel) polymorphism detection, and application of kit
  • Fluorescent labeling complex amplification kit for insertion-deletion (InDel) polymorphism detection, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Design of primers, optimization of concentration and establishment of reaction system

[0055] 1. Design and screening of primers

[0056] 1.1 Serial download

[0057] Template sequence download, downloaded the gene sequence of the InDel site from NCBI genebank, the specific sequence information is shown in the following table:

[0058] Table 2 Sequence information of 34 InDel sites

[0059]

[0060]

[0061] The InDel labels in Table 2 correspond to those in Table 1.

[0062] 1.2 Design and screening of primers

[0063] After determining the gene sequences of the above 34 InDel loci and sex loci, follow the general principles of primer design and use Oligo7.0 software to design primers. In order to optimize the concentration of subsequent primers, the Tm values ​​of the primers designed here are all within Between 58 and 62°C, the length of the amplified product ranges from 95 to 199 bp. Perform amplification test for each pair of primers to obtai...

Embodiment 2

[0083] Embodiment 2: kit sensitivity test

[0084] Apply kit of the present invention to detect the sensitivity of amplified DNA template, prepare the Chinese Han male genomic DNA (1ng, 500pg, 250pg, 125pg, 62.5pg and 31.25pg) of different concentrations respectively, according to table 4 in embodiment 1 The amplification system and the amplification program in Table 5 were used for amplification detection. The test results show that the sensitivity of the kit of the present invention can reach 125pg, and the balance of peak heights between colors is good. attached figure 2 It is the electropherogram of the amplification result of 125pg sample.

Embodiment 3

[0085] Embodiment 3: the application of kit (paternity test)

[0086] The kit of the present invention has detected and genotyped 456 unrelated individuals from different ethnic groups across the country in the inventor's laboratory. The cumulative personal identification rate reached 0.99999985, and the cumulative non-parent exclusion rate was 0.9989. The results showed that the The typing results of the kit are accurate, repeatable, and sensitive, which can meet the needs of actual cases.

[0087] A practical case in which the test kit of the present invention is used for paternity testing is listed below.

[0088] The samples were provided by the Suqian Ziyuan Judicial Appraisal Institute.

[0089] 1. Collection of blood spots in paternity test cases: samples for paternity test are provided by Suqian Ziyuan Judicial Test Center.

[0090] 2. Genomic DNA extraction of various test materials: refer to "GA / T 383-2002 Forensic Science DNA Laboratory Test Specifications" to car...

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Abstract

The invention discloses a fluorescent labeling complex amplification kit for insertion-deletion (InDel) polymorphism detection, and application of the kit. The kit comprises thirty-four InDel polymorphic sites and a sex locus (amelogenin). The fluorescent labeling complex amplification kit can be used for detecting the thirty-four InDel polymorphic sites and the one sex locus (amelogenin) at the same time; the kit can be used for detecting and genotyping 456 unrelated individuals from different ethnic groups from all over the country, has a cumulative personal identification rate reaching 0.99999985, and has a cumulative non-parent exclusion rate of 0.9989; the results prove that the kit is accurate in genotyping results, good in repeatability, high in sensitivity and accurate in genotyping results; therefore, a new method is provided for genetic relationship identification, sibling identification, individual identification, medical diagnosis, and the like.

Description

technical field [0001] The invention relates to a fluorescent label compound amplification kit for detection of insertion-deletion polymorphism and an application thereof, belonging to the field of biological detection. Background technique [0002] The research on DNA molecular marker technology began in the 1880s. It refers to a specific DNA fragment that can reflect a certain difference in the genome of an individual or a population. It is a direct reflection of genetic polymorphism at the DNA level. Most DNA molecular markers are based on The form of electrophoretic bands shows the DNA differences between individuals, and is one of the important technical indicators in the research of taxonomy, breeding, genetics, and the origin and evolution of species. [0003] Forensic genetics realizes paternity testing and personal identification through the detection of DNA genetic markers on biological samples. Insertion-deletion polymorphism (Insertion-Deletion, InDel) is a spec...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2563/107
Inventor 李小方葛斌文陈拓
Owner JIANGSU SUPERBIO LIFE SCI CO LTD
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