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Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method

A technology of short tandem repeats and detection kits, applied in the field of molecular genetics, can solve the problems of difficult sensitivity and achieve high fluorescence efficiency, fast amplification speed, and good species specificity

Active Publication Date: 2017-05-24
江苏苏博生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, ROX dye labeling is more than FAM labeling, the same amount of product, the signal intensity is only one-fourth to one-eighth, under such a large difference in fluorescence efficiency, better balance between the various fluorescent channels and a better High sensitivity becomes difficult

Method used

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  • Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method
  • Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method
  • Multiple amplification detection kit for 21 short tandem repeat sequences using novel bifluorescence marking method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Primer design and determination of the position of the fluorescent label on the primer sequence

[0047] A total of 21 STR loci were amplified by the system, including Amelogenin, D3S1358, vWA, D7S820, CSF1PO, D8S1179, D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433, D1S1656, D12S391, D12S391 Penta E and D6S1043.

[0048] Specific primers were designed on the flanks of the repetitive sequences of the above 21 loci. The primer design uses Oligo7 software, and the Tm value of each primer is close to 60°C. The maximum range of the length of the amplified product is 75-500bp, and the amplification test and optimization of each pair of primers are performed until an amplified peak with sharp peak shape and high peak height is obtained. After the composite amplification test, between 56°C and 63°C, no non-specific amplification is generated, there are target peaks, and no other interactions or cross-reactions occur. There is no amplificati...

Embodiment 3

[0068] Example 3. Comparison of typing consistency between the present invention and other commercial kits

[0069] In order to test the typing consistency between the present invention and other commercial kits, Microreader was selected in this example TM 21 Direct ID System was used as the reference object. The present invention and 21 Direct ID kit were used to amplify 1316 samples (663 unrelated individuals), and the products were typed by capillary electrophoresis using ABI 3100 genetic analyzer. The results are analyzed and compared. 21 The Direct ID kit is operated in accordance with its instructions. The steps of the present invention are as follows:

[0070] 3.1. Polymerase chain reaction (PCR) amplification

[0071] 1) Take the buffer, primer mixture, and Taq enzyme, prepare the mixture according to the following table, shake and mix, and distribute it into PCR reaction tubes, 25μL per tube.

[0072] this invention:

[0073] Component Dosage (μL) 2.5×PCR Mix10 5×Prim...

Embodiment 4

[0086] Example 4. Comparison of the sensitivity of the amplification system of the present invention and conventional labeling methods

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Abstract

The invention discloses a multiple amplification detection kit for 21 short tandem repeat sequences using a novel bifluorescence marking method. The kit comprises a compound primer group comprising 21 pairs of specific primers, 21 gene bases for compound amplification, a reaction mixed liquid and a warm start Taq enzyme, wherein the first basic group at 5' end of a labeled primer is marked by a conventional dye, and some basic groups at 5' and 3' ends of the primer are marked by a short excitation wavelength fluorescent dye, so that fluorescent energy transfer is achieved. The amplification result disclosed by the invention is more stable and balanced, and meanwhile, the kit is higher in sensitivity, can be widely applied to individual recognition, DNA database building and paternity test of Chinese population, and is suitable for various detection materials.

Description

Technical field [0001] The invention belongs to the field of molecular genetics, and specifically relates to a 21 short tandem repeat sequence compound amplification detection kit adopting a novel double fluorescent labeling method and its application. Background technique [0002] STR genetic markers, also known as microsatellite DNA, are widely present in the genomes of prokaryotes and eukaryotes. They are a type of repeating sequences of dozens to more than one hundred nucleotides composed of 2-6 nucleotides as repeating units. . Different numbers of core sequences are arranged in tandem repeats, and their lengths are polymorphic. According to the conservative sequences at both ends, primers are designed, PCR is performed, and then polymorphisms of STR genetic markers are detected by polyacrylamide, agarose gel electrophoresis or capillary electrophoresis. [0003] Individual identification DNA fluorescence detection kits mainly use the most widely used multiplex PCR complex a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 葛斌文陈拓
Owner 江苏苏博生物医学科技有限公司
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