本文選題：聚合酶鏈反應(yīng) + 遺傳監(jiān)測(cè)��； 參考：《鄭州大學(xué)》2007年碩士論文

【摘要】： 近交系小鼠(Inbred strain mice)是指經(jīng)連續(xù)20代以上的全同胞兄妹或親子交配培育而成，品系內(nèi)所有個(gè)體都可追溯到起源于第20代或以后代數(shù)的一對(duì)共同祖先的遺傳群。采用近交系的動(dòng)物可以提高實(shí)驗(yàn)結(jié)果的準(zhǔn)確性、可比性和可重復(fù)性，減少實(shí)驗(yàn)動(dòng)物數(shù)量和實(shí)驗(yàn)重復(fù)次數(shù)。近交系小鼠的顯著特征是基因純合性及遺傳穩(wěn)定性，但在培育、保種及繁殖生產(chǎn)過程中，存在著發(fā)生遺傳污染或遺傳變異的可能，所以有必要對(duì)近交系小鼠進(jìn)行嚴(yán)格、長期、定時(shí)的遺傳監(jiān)測(cè)。一些傳統(tǒng)的方法如：皮膚移植、生化標(biāo)記等通過檢測(cè)其表型變化來推測(cè)其基因變化的方法有明顯的局限性。 微衛(wèi)星DNA是核苷酸短串聯(lián)重復(fù)序列(short tandem repeats)，具有分布廣、數(shù)目多、穩(wěn)定、易于擴(kuò)增等特點(diǎn)，采用PCR擴(kuò)增微衛(wèi)星DNA可直接檢測(cè)DNA水平的變化，能夠全面地反映出基因組的遺傳概貌及變異情況，是近交系動(dòng)物理想的遺傳監(jiān)測(cè)標(biāo)記。本研究采用10條染色體上的20個(gè)微衛(wèi)星DNA位點(diǎn)對(duì)近交系小鼠的DNA多態(tài)性進(jìn)行研究：提取小鼠基因組DNA并利用微衛(wèi)星引物PCR擴(kuò)增，然后通過變性聚丙烯酰胺凝膠電泳及銀染顯色等技術(shù)顯示微衛(wèi)星DNA擴(kuò)增產(chǎn)物。最后采用遺傳學(xué)數(shù)據(jù)分析方法進(jìn)行分析，以期篩選出具有顯著多態(tài)性的微衛(wèi)星位點(diǎn)，用于近交系小鼠的遺傳監(jiān)測(cè)。 方法：選用七品系近交系小鼠(DBA/2、CBA、C57、C3H/HeJ、BALB/C、FVB/NJ、YYHL小鼠)及一個(gè)封閉群小鼠(昆明小鼠)各五只。用酚—氯仿抽提法提取小鼠的基因組DNA，用PCR擴(kuò)增以下位點(diǎn)：D4 mit9、D5mit31、D6mit15、D6mit16、D3mit36、D7mit12、D7mit29、D8mit11、D8mit22、D8mit24、D8mit30、D9mit21、 D10mit29、D11mit35、D11mit36、D12mit8、D12mit29、D12mit34、D12mit35、D13mit18，將PCR產(chǎn)物與等位基因分型標(biāo)準(zhǔn)物同步高壓4-6％變性聚丙烯酰胺凝膠電泳，電泳后經(jīng)過固定、染色、顯色觀察帶型。根據(jù)DNA在變性聚丙烯酰胺凝膠上泳動(dòng)的距離，通過微衛(wèi)星基因座擴(kuò)增條帶與對(duì)照等位基因分型標(biāo)準(zhǔn)物的比較，進(jìn)行結(jié)果判讀并統(tǒng)計(jì)八品系小鼠在這些位點(diǎn)上表現(xiàn)出多態(tài)性條帶的數(shù)目。擴(kuò)增結(jié)果按Lynth氏法計(jì)算相似系數(shù)并分析其相似性。 結(jié)果： 1．近交系小鼠純合性的分析：不同近交系品系及同品系不同個(gè)體小鼠之間的擴(kuò)增產(chǎn)物中，同一微衛(wèi)星位點(diǎn)呈現(xiàn)一清晰條帶，表明河南省及全國目前有關(guān)這些近交系小鼠及其群體沒有發(fā)生遺傳污染或遺傳變異，符合近交系的要求。 2．不同品系近交系小鼠之間微衛(wèi)星多態(tài)性比較的結(jié)果： (1)：不同品系近交系小鼠之間微衛(wèi)星多態(tài)性比較：在20個(gè)微衛(wèi)星位點(diǎn)中有16個(gè)位點(diǎn)：D4Mit9、D5Mit31、D6Mit15、D6Mit16、D6Mit36、D7Mit12、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit8、D12Mit29、D12Mit34、D12Mit35、D13Mit18表現(xiàn)出多態(tài)性，，其中13個(gè)位點(diǎn)：D4Mit9、D5Mit31、D6Mit15、D6Mit36、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit29、D12Mit34、D12Mit35、D13Mit18表現(xiàn)出顯著多態(tài)性，其余4個(gè)位點(diǎn)：D7mit29、D8mit11、D8mit22、D10mit29擴(kuò)增產(chǎn)物電泳距離一致，表現(xiàn)為單態(tài)性，即不具有多態(tài)性。 (2)：不同品系小鼠之間的相似系數(shù)：近交系小鼠中CBA與DBA/2，F(xiàn)VB/NJ與C3H/HeJ的相似系數(shù)分別為0.650，反映出二者親緣關(guān)系較近；其次是FVB/NJ與DBA/2和CBA的相似系數(shù)為0.600；親緣關(guān)系最遠(yuǎn)的是CBA與BALB/C，其相似系數(shù)分別為0.350。而封閉群昆明小鼠與BALB/C的相似系數(shù)為0.700，可見二者親緣關(guān)系最近；昆明小鼠與C3H/HeJ的相似系數(shù)為0.450，二者親緣關(guān)系最遠(yuǎn)。昆明小鼠與YYHL小鼠、DBA、C57的相似系數(shù)中等，為0.550。 3．同一品系內(nèi)不同個(gè)體之間微衛(wèi)星多態(tài)性分析：在20個(gè)位點(diǎn)上同一品系內(nèi)的小鼠在同一位點(diǎn)擴(kuò)增的產(chǎn)物條帶動(dòng)距離相同，表現(xiàn)為單態(tài)性：各位點(diǎn)呈現(xiàn)一條清晰圖帶，且電泳距離一致。 結(jié)論： 1．經(jīng)過20個(gè)位點(diǎn)檢測(cè)，7個(gè)近交系小鼠符合近交系要求，說明河南省及周邊 地區(qū)有關(guān)這些近交系小鼠及其群體沒有發(fā)生遺傳污染或遺傳變異，符合近交系的要求。 2．可以運(yùn)用PCR穩(wěn)定、特異、有效地?cái)U(kuò)增8個(gè)品系小鼠的微衛(wèi)星DNA，微衛(wèi)星DNA技術(shù)的建立可以對(duì)近交系小鼠的品系鑒別、個(gè)體識(shí)別、遺傳背景監(jiān)測(cè)等提供一個(gè)更好的方法和手段。 3．在不同近交系小鼠之間，篩選出的16個(gè)微衛(wèi)星位點(diǎn)：D4Mit9、D5Mit31、D6Mit15、D6Mit16、D6Mit36、D7Mit12、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit8、D12Mit29、D12Mit34、D12Mit35、D13Mit18具有多態(tài)性，其中13個(gè)位點(diǎn)：D4Mit9、D5Mit31、D6Mit15、D6Mit36、D8Mit24、D8Mit30、D9Mit21、D11Mit35、D11Mit36、D12Mit29、D12Mit34、D12Mit35、D13Mit18表現(xiàn)出顯著多態(tài)性，可利用這些位點(diǎn)快速、經(jīng)濟(jì)地對(duì)有關(guān)近交系小鼠進(jìn)行遺傳監(jiān)測(cè)。 4．各品系小鼠之間的相似系數(shù)，反映了其祖代之間的親緣關(guān)系。
[Abstract]:The inbred mouse (Inbred strain mice) refers to the mating and breeding of all sibling siblings or parents of more than 20 generations. All of the individuals in the strain can be traced to a pair of common ancestor that originated in the twentieth generation or later algebra. The significant characteristics of mice in the inbred line are gene homozygosity and genetic stability, but there is a possibility of genetic and genetic variation in breeding, breeding and reproduction. Therefore, it is necessary to carry out strict, long-term and regular genetic monitoring of inbred mice. Some traditional methods are necessary. Method: skin transplantation, biochemical markers and other methods of detecting their gene changes by detecting their phenotypic changes have obvious limitations.
Microsatellite DNA is the nucleotide short tandem repeat (short tandem repeats), which has the characteristics of wide distribution, large number, stable and easy amplification. The PCR amplification of microsatellite DNA can directly detect the changes of DNA level, and can fully reflect the genetic profile and variation of the genome. This is an ideal genetic monitoring marker for the inbred animal. The study used 20 microsatellite DNA loci on 10 chromosomes to study the DNA polymorphism of inbred mice. The genomic DNA of mice was extracted and the microsatellite primer PCR was used to amplify the microsatellite primers. Then the microsatellite DNA expansion was displayed by denatured polyacrylamide gel electrophoresis and silver staining and other techniques. Finally, the genetic data analysis was used. Analysis was performed to identify microsatellite loci with significant polymorphisms for genetic monitoring in inbred mice.
Methods: five mice (DBA/2, CBA, C57, C3H/HeJ, BALB/C, FVB/NJ, YYHL mice) and a closed group of mice (Kunming mice) were selected. The genomic DNA of mice was extracted with phenol chloroform extraction method, and the following loci were amplified by PCR. 30, D9mit21,
D10mit29, D11mit35, D11mit36, D12mit8, D12mit29, D12mit34, D12mit35, D13mit18, synchronize high pressure 4-6% denatured polyacrylamide gel electrophoresis with PCR products and allele typing standards. After electrophoresis, the electrophoresis is fixed, stained and coloured to observe the band pattern. According to the distance of DNA on denatured polyacrylamide gel, the microsatellite loci are expanded. The number of polymorphic bands in the eight strain mice showed the number of polymorphic bands. The results of the amplification were calculated by Lynth's method and the similarity was analyzed.
Result錛

本文編號(hào)：1774908