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A detection kit for multiplexed amplification of 21 short tandem repeats using a double fluorescent labeling 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 the effects of high fluorescence efficiency, strong amplification specificity, and good species specificity

Active Publication Date: 2019-09-27
江苏苏博生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A detection kit for multiplexed amplification of 21 short tandem repeats using a double fluorescent labeling method
  • A detection kit for multiplexed amplification of 21 short tandem repeats using a double fluorescent labeling method
  • A detection kit for multiplexed amplification of 21 short tandem repeats using a double fluorescent labeling 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] The system amplifies a total of 21 STR loci, including Amelogenin, D3S1358, vWA, D7S820, CSF1PO, D8S1179, D21S11, D16S539, TH01, D13S317, TPOX, D18S51, D5S818, FGA, D2S1338, D19S433, D1S1659, D12S D, D1S1659, D12S Penta E and D6S1043.

[0048] Specific primers were designed for the above 21 loci on the flanks of their repeated sequences. Primers were designed using Oligo7 software, and the Tm value of each primer was close to 60°C. The maximum range of the length of the amplified product is 75-500bp. Each pair of primers is tested and optimized until the amplified peak with sharp peak shape and high peak height is obtained. After the multiplex amplification test, between 56°C and 63°C, there is no non-specific amplification, all have target peaks, and no other interactions or cross-reactions occur. No amplification of DNA from dogs, pigs, cattle, sheep,...

Embodiment 3

[0070] Embodiment 3, compare the consistency of typing of the present invention and other commercial kits

[0071] In order to detect the consistency of typing between the present invention and other commercial kits, this embodiment selects Microreader TM 21 Direct ID System was used as a reference object, and 1316 samples (including 663 irrelevant individuals) were amplified using the present invention and 21 Direct ID kit respectively, and the products were typed by ABI 3100 genetic analyzer capillary electrophoresis, and the type The results were analyzed and compared. 21Direct ID kit is operated according to its instructions, and the steps of the present invention are as follows:

[0072] 3.1. Polymerase chain reaction (PCR) amplification

[0073] 1) Take the buffer, primer mixture, and Taq enzyme, and prepare a mixture according to the table below, shake and mix well, and then dispense into PCR reaction tubes, 25 μL per tube.

[0074] this invention:

[0075]

[00...

Embodiment 4

[0089] Embodiment 4, the sensitivity comparison of the present invention and conventional labeling method amplification system

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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 in particular relates to a 21-short tandem repeat compound amplification detection kit using a double fluorescent labeling method and an application thereof. Background technique [0002] STR genetic markers, also known as microsatellite DNA, widely exist in the genomes of prokaryotic and eukaryotic organisms, and are a kind of repeating sequence consisting of tens to more than one hundred nucleotides consisting of 2 to 6 nucleotides as repeating units . Different numbers of core sequences are arranged in tandem repeats, while the lengths are polymorphic. According to the conserved sequences at both ends, design primers, perform PCR, and then detect polymorphisms of STR genetic markers by polyacrylamide, agarose gel electrophoresis, or capillary electrophoresis. [0003] The individual recognition DNA fluorescence detection kit mainly adopts the most widely used multiplex PCR compound ampl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888
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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