Cloning and Application of Sex-Specific DNA Sequences in Taiwan Yellow Cattle,Pigs and Pigeons

Autor:	Yan-Ming Horng, 洪炎明
Rok vydání:	2004
Druh dokumentu:	學位論文 ; thesis
Popis:	92 Random primers were used for random amplified polymorphic DNA fingerprinting to sexing Taiwan yellow cattle, pigs, and pigeon. The OPAG-06 primer produced a sex-specific fragment in all males but no females for Taiwan yellow cattle. Moreover, the OPAV-18 primer produced a novel roughly 1.1 kb DNA fragment found only in tested males of Yorkshire, Landrace, and Duroc pigs. The OPC-20 primer produced an approximately 800 bp sex-specific band in all pigeon females. These sex-specific DNA fragments of Taiwan yellow cattle, Yorkshire pig, and pigeon were isolated and constructed into pCRII-TOPO vector for nucleotide sequencing. Three novel 1529 bp, 1098 bp, and 732 bp sex-specific DNA sequences were obtained from Taiwan yellow cattle, Yorkshire pig, and pigeon, respectively. Two primers of YCaOPAG06F and YCaOPAG06R were designed based on the cloned male-specific sequence of Taiwan yellow cattle. The genomic DNA samples of Taiwan yellow cattle were amplified using these two primers by PCR. The sex-specific fragment in the gel appeared in all Taiwan yellow cattle males but no females. The pair of primers then were used to sex Taiwan yellow cattle, Holstein cattle, Angus cattle, Hereford cattle, and Taiwan water buffalo. The sex-specific band also appeared in Taiwan yellow cattle, Holstein cattle, Angus cattle, Hereford cattle males. The condition demonstrated that the pair of primers could be used to sex the four breeds of cattle. The sex-specific band was absent in Taiwan water buffalo, showing that the primer bind sites was absent in Taiwan water buffalo Two primers (PigSexOPAV18F and PigSexOPAV18R) were designed based on the cloned male-specific sequence to amplify the male-specific band using PCR for pig sexing. Sex-specific bands in the PCR gel products were found in males but not in females when Yorkshire, Landrace, and Duroc pigs genomic DNA samples were amplified using these two primers by PCR. The PCR products in the gel transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment, and clear hybridization signal existed in all of the samples from the male pigs, but not in those from the female pigs. Male and female genomic DNA samples from the pigs were spotted onto nylon membranes and hybridized using the male-specific probe. The probe only hybridized strongly to males. High degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Landrace, and Duroc pigs. The sex of these three breeds of pigs could be easily and effectively determined using these two primers. The random primer OPAV-18 also was used to amplify genomic DNA from Taiwan wild pigs, Small-ear pigs, Taoyuan pigs, and Meishan pigs by PCR for studies on the male-specific sequence. Experimental results demonstrated that OPAV-18 cannot amplify the sex-specific band in these Asian pig breeds. Moreover, the pair of primers (PigSexOPAV18F&R) can amplify a band in these four species of male genome DNA only, but no such band exists in female pigs. The band on gel was transferred to nylon membranes, then hybridized with 32P-labeled the male-specific DNA probe. The male pigs displayed a strong signal while the female pigs did not. Male and female genomic DNA samples from these Asian pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. These four male-specific fragments were sequencing. Notably, the sequence homologies among these four species of pig were extremely high. A pair of primer (DoveOPC20F&R) was designed based on the cloned female-specific sequence to amplify the female-specific band by PCR for pigeons sexing. Sex-specific bands in the gel of PCR products were represent in females but not males. The PCR products in the gel were transferred to nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. There was a clear hybridization signal in samples from all of the female pigeons, but not from those of male pigeons. Male and female genomic DNA samples from pigeons were spotted onto nylon membranes and hybridized with the female-specific probe. The probe hybridized strongly to females only. In squab, it is difficult to determine the gender of individuals based on morphology. The problem can be solved using these two novel primers by PCR. In conclusion, The sex-specific sequences of animals obtained from these trails in this study were all novel sequences. Three pairs of primers were designed according to the cloned sex-specific sequences for sexing. YCaOPAG06F&R primers could be used for sex determination in Taiwan yellow cattle, Holstein cattle, Angus cattle and Hereford cattle. PigSexOPAV18F&R primers could be employed for the sexing of Yorkshire pigs, Landrace pigs, Duroc pigs, Taiwan wild pigs, Small-ear pigs, Taoyuan pigs and Meishan pigs. The sex determination of pigeons could be performed easily with DoveOPC20F&R primers by PCR.
Databáze:	Networked Digital Library of Theses & Dissertations
Externí odkaz:	http://ndltd.ncl.edu.tw/handle/36477697708167867112 Zobrazit plný text záznamu