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Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat)

A standard substance and human technology, applied in the biological field, can solve the problems of large cell storage capacity and high requirements for cell culture conditions, and achieve the effect of simple preparation process, low personnel level requirements, and easy storage

Active Publication Date: 2011-11-23
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 2. Large amount of cell preservation
[0013] 3. High requirements for cell culture conditions

Method used

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  • Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat)
  • Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat)
  • Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Primer Design

[0108] Select the appropriate STR loci. According to the main use of ABI company's AmpFISTRSinofiler in our country TM , Promega's PowerPlex16 and the DNATyper15 developed by the Physical Evidence Identification Center of the Ministry of Public Security of my country TM For the STR locus used in the commercial kit, the inventors selected 19 STR loci in Table 1 (CSF1PO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA) and sex marker site Amelogenin were used to prepare standard substances for STR detection.

[0109] Primers for the above-mentioned 19 STR loci and the sex marker locus Amelogenin were designed. The above-mentioned 19 STR loci and the full sequence of the sex marker locus Amelogenin were searched from GenBank and related literature, using the found sequence as a template to design several pairs of primers through Primer Premier softwa...

Embodiment 2

[0116] Next, human genomes K562, 9947A and 9948 were used as templates to amplify the above-mentioned 19 STR loci and sex marker site Amelogenin. The PCR reaction system and conditions are shown in Table 3 and Table 4 as follows:

[0117] Table 3. PCR amplification using 50 μL reaction system

[0118]

[0119] Table 4. PCR Thermal Cycle Parameters

[0120]

[0121] The results of agarose electrophoresis of the amplified product are shown in Figure 2-7 .

Embodiment 3

[0123] According to the different restriction sites of the designed primers, the plasmid pUC18 and the purified PCR product were double-digested with corresponding enzymes, and the enzymes used for different PCR products are listed in Table 5.

[0124] Table 5. Enzymes used for double digestion of PCR products

[0125]

[0126] Plasmid pUC18 was double-derived by Pst I / EcoR I, Hind III / Pst I, Xba I / EcoR I, Xba I / PstI, Pst 1 / BamH I, Xba I / Hind III, EcoR I / Hind III, Xba I / BamH I After enzyme digestion, the electrophoresis results are shown in Figure 8 .

[0127] The pUC18 digested with the same enzyme and the PCR product were ligated overnight with T4 ligase, transformed into Escherichia coli JM109, and the transformants were picked for identification. First, pick 4 colonies on each plate to identify transformants by colony PCR and extraction of plasmids, the identification results are shown in Figure 9-12 . The bacterial fluid identified as a recombinant was sent for s...

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Abstract

The invention discloses a preparation method of a reference material (RM) for detecting a human DNA (deoxyribonucleic acid) STR (short tandem repeat), and the method is used for preparing the RM for detecting the human DNA STR by utilizing a molecular cloning technique. The preparation method is mainly characterized by comprising the following steps: recombining artificially synthesized or PCR amplified fragments containing STR gene loca with plasmids, transferring the recombinant plasmids into cells, screening out clones containing correct insert fragments by sequencing, and performing mass propagation; and extracting the recombinant plasmids, performing enzyme digestion, purifying and mixing to obtain a large quantity of high-quality RMs for detecting the DNA STR. The preparation methodhas the advantages of simple preparation process and low cost; and the RM for detecting the DNA STR prepared by the method is good in genetic stability and easy for storage.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a preparation method of a standard substance for human DNA STR detection, which is used to solve the problems of DNA detection in the fields of forensic science, anthropology, genetics, and diseases, such as individual identification, paternity identification, and genetic diagnosis. Accuracy and standardization issues. Background technique [0002] Reference material is a measurement standard with accurate value. According to the "International General Metrology Basic Terminology" and "ISO Guide 30", the reference material (RM) has the following definition: one or more materials with sufficient uniformity and A material or substance whose property values ​​are well established for calibrating measuring devices, evaluating measuring methods, or assigning values ​​to materials. The transfer and traceability of the quantity value is realized through the standard substance, so t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/63C12N15/66C12R1/19
Inventor 毕万里何召明叶健赵兴春
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY