Chicken mtDNA D-loop region complete-sequence amplifying and sequencing method and special-use primer

A technology for sequencing primers and full sequences, applied in the field of molecular biology, to achieve the effects of cost saving, harm reduction and high speed

Active Publication Date: 2015-11-11
JIANGSU INST OF POULTRY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some people at home and abroad have used the hypervariable region of the D-loop region to study the genetic diversity and kinship evolution of chickens, the complete sequence contains more abundant genetic information. At present, the complete sequence of the D-loop region has not been used to analyze the local poultry breeds in my country. To carry out systematic research, it is necessary to establish a method for full-sequence amplification and sequencing of the mtDNAD-loop region

Method used

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  • Chicken mtDNA D-loop region complete-sequence amplifying and sequencing method and special-use primer
  • Chicken mtDNA D-loop region complete-sequence amplifying and sequencing method and special-use primer
  • Chicken mtDNA D-loop region complete-sequence amplifying and sequencing method and special-use primer

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Embodiment 1

[0067] Embodiment 1 Tibetan Chicken Phylogenetic Evolution and Genetic Diversity Research

[0068] 1.1 DNA sample preparation

[0069] The feathers just grown after neck moulting of 30 Tibetan chickens were collected, half male and half female, randomly sampled. Use scissors to cut off the down feathers on the upper part of the feathers, cut them into pieces on clean PE gloves, put them into a 2mL centrifuge tube, add 1000 μL of lysate and 40 μL of proteinase K to each sample, shake well, and put them in a shaker for digestion in a water bath at 59 °C. After fully digested, add 700-800uL saturated phenol, shake well for 10min and centrifuge at 10000rpm for 5min. Draw the supernatant into a 2mL centrifuge tube, add 1000uL saturated phenol, shake well for 5min and centrifuge at 10000rpm for 5min. Draw the supernatant into a 2mL centrifuge tube, add 1000uL of chloroform, shake well for 5min, and centrifuge at 10000rpm for 5min. Aspirate the supernatant and put it into a 2mL ce...

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Abstract

The invention discloses a chicken mtDNA D-loop region complete-sequence amplifying and sequencing method and a special-use primer and belongs to the field of chicken mitochondrial genome researching. The primer for chicken mtDNA D-loop region complete-sequence amplifying and sequencing is represented as the Seq ID No.1, the Seq ID No.2 and the Seq ID No.3. The amplifying and sequencing method includes the steps of (1) extracting DNA from chicken feather and detecting the quality of the DNA; (2) designing a pair of PCR primer (represented as the formula of the Seq ID No.1 and the Seq ID No.2) in upstream and downstream conserved domains of the chicken mtDNA D-loop region, performing PCR reaction to amplify a fragment containing the Cytb gene and performing agarose electrophoresis detection; (3) designing a sequencing primer (represented as the formula of the Seq ID No.3), and sequencing a PCR product; and (4) splicing the sequence. The detection method is quick in speed, low in cost and easy to master. An obtained chicken mtDNA D-loop region complete sequence can be applied in chicken genetic resource variety evaluation, maternal line origin, system evolution analysis and germplasm resources identification and protection and the like fields.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a chicken mtDNAD-loop full sequence amplification and sequencing method. Background technique [0002] The diversity of animal genetic resources can provide materials for breed improvement and can adapt well to changes in the environment. my country has abundant local breeds of poultry resources, and it is of great significance to study and evaluate the genetic diversity of local breeds of poultry in my country, and formulate reasonable utilization and protection measures for the sustainable development of animal husbandry in my country. The methods for studying animal genetic diversity mainly include microsatellites and mitochondrial DNA, etc. Both methods have their own advantages and disadvantages, and mitochondrial DNA is easier to operate. Most of the breeds of livestock (poultry) are affected by the hybrid improvement of foreign males (poultry), and the number of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 高玉时唐修君贾晓旭陆俊贤陈大伟葛庆联顾荣刘茵茵
Owner JIANGSU INST OF POULTRY SCI
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