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DNA fragment related to pig polydactyly

A technology of fragments and peptides, which is applied in the field of mutation and genetic engineering, can solve the problems of no difference found and correlation analysis, etc., and achieve the effect of accurate detection

Inactive Publication Date: 2008-04-09
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no relatively complete pig Lmbr1 gene sequence, and no difference in Lmbr1 gene sequence between normal pigs and polydactyl pigs has been found. To explore the feasibility of Lmbr1 gene as molecular marker-assisted selection for carcass traits in pigs

Method used

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  • DNA fragment related to pig polydactyly
  • DNA fragment related to pig polydactyly
  • DNA fragment related to pig polydactyly

Examples

Experimental program
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Effect test

example 1

[0024] Example 1 Obtaining of DNA fragments of the present invention

[0025] 1 Collection of samples

[0026] Immediately after slaughtering pigs, collect heart, liver, spleen, lung, kidney, forelimb muscles and other tissues, cut into small pieces, put them in cryopreservation tubes, store them in liquid nitrogen immediately, and place them in a -80°C ultra-low temperature freezer after returning to the laboratory save in .

[0027] 2 Searching for porcine homologous EST and designing primers

[0028] Taking the coding sequence of the human / mouse polydactyly candidate gene C7orf2 / Lmbr1 gene as the search object, BLAST was performed on the NCBI website to obtain a pig homologous EST (CV878120, 724bp), and the 6bp-636bp part of the sequence is similar to the human C7orf2 The homology of the 497bp-1148bp part of the gene sequence ((accession number: NM_022458, 2781bp in total, 155bp-1627bp part is CDS)) is 88%. Based on this EST sequence, use Primer5.0 software to design pri...

example 2

[0149] Example 2 Molecular detection of pig developmental traits

[0150] (1) extraction of pig blood total RNA;

[0151] Collect 5 ml of jugular venous blood from individual pigs to be tested, and anticoagulate with heparin. After separating leukocytes, use the total RNA extraction reagent (D312) of Dalian Bao Bioengineering Co., Ltd. to total RNA according to the instructions.

[0152] (2) RT-PCR amplification;

[0153] Use SMARTTM RACE cDNA Amplification kit, reverse transcription reaction: total RNA 1-3ul, 3'-CDSprimer 1ul, add deionized water to 5ul, 70°C for 2min, put on ice for 2min, add 5×First-Strand Buffer 2ul, DTT (20mM) 1ul, dNTP (10mM) 1ul, BD PowerScript Reverse Transcriptase 1ul, 90min at 42°C, diluted with 100ul Tricine-EDTA Buffer, bathed in water at 72°C for 7min, to obtain 3'-RACE-Ready cDNA.

[0154] The first round of PCR was performed using UPM (provided in the kit) and GSP2 (5'-GAC TTG CCG CTC ATT GAC AAC GAC-3'). See the kit instructions for the PCR...

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Abstract

The invention provides a DNA fragment relating to a swine polydactyly. The DNA fragment is one of the following polynucleotide: (1) the polynucleotide with the nucleotide sequence of SEQ NO.1 and SEQ NO.2; (2) the polypeptide polynucleotide with the encoding amino acid sequence of SEQ NO.2 and SEQ NO.4. The DNA fragment of the invention has a close relation with the development of swine limbs. The missense mutation of the former four codons of an Exon 16 directly results the polydactuly of swine toes. Therefore, the invention provides a method for detecting the development phenotype of the swine. The method adopts the following premiers to increase reverse transcription products of the general RNA of the swine: 5'-CGC CGT GGC TTG TAA CAT TCT CTG C-3', 5'-AAG CAG TGG TAT CAA CGC AGA GT-3'. The product of the normal swine is 197bp, the product of the pilydactyly swine is 636bp.

Description

technical field [0001] The invention relates to the fields of mutation and genetic engineering, in particular to a gene related to abnormal development of animal limbs. Background technique [0002] Polydactyly (toe) is a kind of limb deformity caused by wrong signals during the formation of animal limbs. It sometimes occurs in humans, mice, chickens, and pigs, and the phenotypes of different species are similar. At present, a large number of studies have been carried out on polydactyly (toes) of humans, mice, and chickens, and some progress has been made. Chromosomal segment of (toe) gene (human: 7q36, 2q31 and 7p13; mouse: homologous region on chromosome 5 to human 7q36; chicken: 2p45), clones such as Shh (sonic hedgehog), Lmbr1 (lamb region 1) / C7orf2(chromosome 7 open reading frame 2), Lmbr2, Hox, Gli, EN2(ENGRAILED2), FGF, FgF receptors, RAR(retinoic acid receptors), WNT(wg and int), BMP(bone morphogenetic protein) Wait for a large number of candidate genes, and find m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/47C12Q1/68
Inventor 王金勇白小青赵献之
Owner CHONGQING ACAD OF ANIMAL SCI
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