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Kit used for paternity test of giant pandas

A paternity test and kit technology, applied in the biological field, can solve the problems of inability to diagnose, no preparation of allelic typing standards, large scale, etc., and achieve the effect of rectifying blood relationship, accurate paternity test and genetic monitoring, and improving pedigree

Inactive Publication Date: 2011-01-19
CHENGDU RES BASE OF GIANT PANDA BREEDING
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these kits do not prepare corresponding allelic typing standards, but use traditional ROX-350 or ROX-500 for genotyping
Due to the large scale of these standards, the typing products of different batches and different systems (such as instrument differences, personnel replacement) are very prone to systematic errors
Therefore, these kits are still in trial phase and cannot really be used for diagnosis
Moreover, in terms of wild animals, there is currently no report on the development of microsatellite paternity testing kits

Method used

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  • Kit used for paternity test of giant pandas
  • Kit used for paternity test of giant pandas
  • Kit used for paternity test of giant pandas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Amplification of alleles at individual loci

[0049] The materials include: ① DNA samples of giant pandas: 110 DNA samples of captive giant pandas. Extract DNA from giant panda tissues and blood according to conventional methods, adjust the concentration to 0.5g / L, and store at -20°C;

[0050] ② Primers shown in Table 3.

[0051] The steps are: select the corresponding DNA samples, and then perform PCR amplification with primers without fluorescent labels. PCR was carried out on GeneAmp9700 PCR machine. The total reaction system is 50L: 50ng template DNA, 5L10×PCR buffer, 1.5mM MgCl 2 , 200MdNTP, 1M primers and 1.5UTaqDNA polymerase (Fermentas). The reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 40 s, annealing temperature for 50 s, extension at 72°C for 55 s, a total of 35 cycles; final cycle of extension at 72°C for 10 min.

[0052] The PCR product was subjected to 2% agarose gel electrophoresis, stained with eth...

Embodiment 2

[0055] Construction of allelic plasmid and identification of its sequence

[0056] Materials include: ① strains and plasmids:

[0057] Escherichia coli JM109 was purchased from Huamei Biological Co., Ltd. The genotype is: recAsupE44endA1hsdR17gyrA96relA1thiΔ(lac-proAB)F'[traD36proAB + lac q lacZΔM15];

[0058] ② The cloning vector pGEM-T, produced by Promega, was purchased from Shanghai Jinmai Biotechnology Co., Ltd.

[0059] The steps are: connect the PCR product to the pGEM-T vector, and the connection reaction conditions are: 22°C, 12 hours. The ligation product was transformed into Escherichia coli (JM109) competent cells, spread on LB plates (Luria-Bertani Medium) containing 50 μg / mL ampicillin, 200 mg / mL IPTG and 20 mg / mL X-Gal, cultivated overnight, and screened positive clones by blue-white spots.

[0060] Positive clones were streaked and plasmids were extracted. Two-way sequencing was performed after electrophoresis detection, and the sequencing primers were T7...

Embodiment 3

[0062] Preparation of microsatellite allele standards

[0063] Materials include: ①TaqGold enzyme, produced by Applied Biosystems, USA, purchased from Chengdu Gold Thread Biotechnology Co., Ltd.;

[0064] ②GS-350StandardSize (ROX), produced by Applied Biosystems, USA, purchased from Chengdu Golden Thread Biotechnology Co., Ltd.;

[0065] ③ Add ROX fluorescently labeled primer b at the 5' end;

[0066] ④ POP4 gel. Produced by Applied Biosystems, Inc., purchased from Chengdu Golden Thread Biotechnology Co., Ltd.

[0067] The corresponding plasmid was amplified by PCR using primer b with ROX fluorescent label at the 5' end. The total reaction system is 50L: 50ng template DNA, 5L10×PCR buffer, 1.5mM MgCl 2 , 200MdNTP, 1M primers and 1.5UTaqDNA polymerase (Fermentas). The reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 40 s, annealing temperature for 50 s, extension at 72°C for 55 s, a total of 35 cycles; final cycle of extensi...

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Abstract

The invention discloses a kit used for a paternity test of giant pandas. 68 allelic genes of 11 STR gene loci, obtained by microsatellite study, of the giant pandas are taken as typing standard substances, and parameters of annealing temperature and magnesium ion concentration in a PCR process of the STR gene loci are optimized, wherein the typing standard substances correspond to the allelic genes of the gene loci one by one, so that the microsatellite typing can be performed accurately. The kit of the invention can perform the paternity test and genetic monitoring accurately on species groups of captive giant pandas, complete genealogy, reorganize blood relationship and provide very useful molecular markers for the study in the aspects such as the molecular evolution of the giant pandas, the population migration and the behavioral ecology of wild giant pandas and the like in future, and has very significant theoretical and practical significance on the formulation and implementation of the conservation and breeding plans of rare species.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene identification kit, in particular to a kit for paternity identification of giant pandas. Background technique [0002] The small size of the giant panda (Ailuropodamelanoleuca) population, redistribution, and the danger of unnatural selection may lead to the loss of genetic variation. reproductive issues. The application of genetic markers to the genetic relationship identification, genetic evaluation and monitoring of giant pandas has become an important topic in the research of giant panda conservation biology. [0003] Microsatellite (microsatellite) is considered to be the most effective new genetic marker for determining the relationship between humans and animals and for population genetics research. The repeat unit is only composed of 2-7bp, also known as Short Tandem Repeat (STR) . At present, the microsatellite markers used for paternity identification of giant panda...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 沈富军张志和侯蓉杨建东张亮张文平
Owner CHENGDU RES BASE OF GIANT PANDA BREEDING
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