A accurate and simple Anthropoides virgo sex appraisal method

An identification method, Demoiselle technology, applied in the field of molecular biology, can solve the problems of few applications, and achieve the effects of reducing damage, strong repeatability, and convenient communication

Inactive Publication Date: 2007-12-12
YANGZHOU UNIV
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Problems solved by technology

[0003] At present, as a simple sex identification method, there are few studies on the sex identification of birds, especially demoiselle cranes, using EE0.6. The CHD gene, Chromobox-heli-case protein gene (Chromobox-heli-case -DNA binding gene), the application of these two pairs of primer combinations for EE0.6 gene is also rare

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  • A accurate and simple Anthropoides virgo sex appraisal method

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Embodiment Construction

[0013] 1. Using improved phenol-chloroform extraction method to extract Demoiselle Crane genomic DNA:

[0014] Take 30 μL of anticoagulant blood in a 1.5 mL centrifuge tube, add 300 μL of lysate (2M urea, 100 mM Tris-HCl, 100 mM EDTA, 1% SDS, pH 8.0) to lyse for one day, add 6 μL of proteinase K (PrK, the concentration is 5 μg / μL) and 2 μL RNase (concentration: 2.5 μg / μL), in a 55°C water bath for 10 to 15 hours.

[0015] Add equal volumes of saturated phenol, phenol-chloroform-isoamyl alcohol (25:24:1) mixture and chloroform-isoamyl alcohol (24:1) mixture to extract once respectively, keep the supernatant, add ice-free DNA was precipitated with ethanol, washed with 70% ethanol, dried, dissolved in TE, and stored at 4°C for later use.

[0016] 2. Using the obtained genomic DNA as a template, carry out PCR reaction:

[0017] Use 10μL system: take 0.5 U Taq DNA polymerase, 1.5mM MgCl 2 , 200μM dNTP, 4pmol primers EE1 and EE2 or EE3 and EE4, 100ng template DNA, add double dis...

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Abstract

The invention relates to an accurate and simple method for determining sex for Anthropoides virgo, belonging to molecular biology technology field. It comprises following steps: augmenting the special squence on W and Z chromosome on Anthropoides virgo genetic DNA by using two units of primer combination; gel electrophoresizing with 1.5% agarose, photographic recording with Gene Genius gelatin imager, determining sex according to band type: two bands are female, while one band is male. The invention is characterized in that it determines sex according to band type, needs no extra equipment, but normal operation of PCR and agar gel electrophoresis, which is simple with strong repeatability.

Description

technical field [0001] The invention relates to an accurate and simple method for gender identification of demoiselle cranes, belonging to the technical field of molecular biology. Background technique [0002] The sex chromosome-linked EE0.6 (0.6kb-EcoRI-fragment) gene is cloned from the chicken W chromosome and is a single sequence that is conserved in ratite and non-ratite birds that have been tested so far , You can select appropriate primers to specifically amplify the homologous copies on the Z and W chromosomes to identify gender. Since females are of ZW type and males are of ZZ type, fragments of different lengths can be obtained in females during electrophoresis detection of PCR amplification products, while only fragments of the same length can be obtained in males. Itoh et al. (2001) used EE1 and EE4 primer combinations, EE2 and EE3 primer combinations to amplify the fragments on the EE0.6 gene to identify the sex of several birds such as red-crowned cranes, whit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈国宏张红霞包文斌吴信生徐琪胡飞孙志明束婧婷程金花陈同海王金美刘鑫吴云良陈清谢飞
Owner YANGZHOU UNIV
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