重组质粒pPAHD1的限制性内切酶图谱分析

本文对我们构建的含有青霉素酰化酶(Acylace)基因的重组质粒pPAHD1进行了限制性内切酶图谱的测绘。使用了限制酶14种,证明BglⅡ,SalⅠ,SmaⅠ和BamHⅠ在重组质粒上各有切点一个;EcoRⅠ有切点两个:HindlⅡ,AvaⅠ各有切点三个;PvuⅠ有切点四个;EcoRⅤ和XmnⅠ各有切点五个。     

丝羽乌骨鸡mtDNA限制酶酶切研究

应用碱变性法制备丝羽乌骨鸡（简称丝羽鸡）肝细胞ｍｔＤＮＡ，测其分子量为１８．５ｋｂ．用限制性内切酶进行酶切分析，结果表明，ＢａｍＨⅠ、ＰｓｔⅠ、ＨｉｎｄⅢ、ＳａｃⅠ在丝羽鸡ｍｔＤＮＡ分子上分别有１、１、１、２个酶切位点．根据单酶和双酶完全酶切片段的分子量，建立了丝羽鸡ｍｔＤＮＡ的限制酶图谱．与由来亨鸡肝细胞ｍｔＤＮＡ为材料所得的研究结果比较，发现丝羽鸡与来亨鸡在ｍｔＤＮＡ种质方面存在着较大差异．     

青鱼线粒体DNA限制性酶切图谱的研究

用10种限制性内切酶对青鱼(Mylopharyngodonpiceus)的肝脏mtDNA进行了分析,其分子质量为9 949×106u,分子大小约为16 60kb.PstⅠ、EcoRⅠ、XhoⅠ、HindⅢ、BamHⅠ、PvuⅡ、SalⅠ、XbaⅠ、BglⅡ、BglⅠ在青鱼的mtDNA分子上分别具有0、3、1、4、4、3、2、2、2、3个切点.根据单酶解及双酶解结果建立了青鱼mtDNA的限制性酶切图谱.     

重要海水养殖鱼斜带髭鲷和花尾胡椒鲷16S rRNA遗传变异研究

应用PCR技术扩增了我国南方重要养殖石鲈科鱼类斜带髭鲷(Hapalogenys nitens)和花尾胡椒鲷(Plectorhinchus cinctus)线粒体DNA(mtDNA)的16S rRNA基因片段,纯化后分别进行EcoRⅠ、MseⅠ、PstⅠ、SacⅠ和HindⅢ等5种限制性内切酶酶切和测序分析.酶切结果为:斜带髭鲷16S rRNA扩增片段被MseⅠ和SacⅠ两种内切酶酶切,花尾胡椒鲷16S rRNA扩增片段只被MseⅠ酶切.两者在MseⅠ和SacⅠ酶切图谱的差异可作为区分两者的遗传标记.16S rRNA序列分析结果表明,斜带髭鲷和花尾胡椒鲷的遗传相似度为86.94 %,遗传差异为13.06 %.进化上,两者分化年代大约为1.98×106年前.     

牛蛙mtDNA的一种提取方法及初步酶切分析

本文中使用牛蛙作为材料提取mtDNA进行RFLP分析.探讨提取牛蛙mtDNA的方法及酶切图谱.结果表明,按本实验操作方法能够提取足量牛蛙的mtDNA,用以酶切分析.采取KpnI.Xho.SacI.PstI.HindⅢ等五种限制性内切酶酶切牛蛙mtDNA,能够粗略求出它的分子量大约在20kb左右.     

三索线蛇与过树容蛇肝线粒体DNA的纯化与限制酶图谱

用Sepharose 4B凝胶柱过滤和NaCl离心法纯化了三索线蛇及过树容蛇肝线粒体DNA(mtDNA),它们的分子长度分别为17.75kb及19.70kb。分别用EcoRⅠ,XbaⅠ,BamHⅠ及BglⅡ等4种限制酶消化这两种mtDNA,结果表明:EcoRⅠ,XbaⅠ,BamHⅠ和BglⅡ在三索线蛇肝mtDNA上分别有1,1,2及3个切点;在过树容蛇肝mtDNA上各有4,1,1和2个切点。根据mtDNA的单酶、双酶和部份酶解片段的分析,建立了三索线蛇及过树容蛇肝mtDNA的限制酶图谱。     

利用线粒体 DNA Cytb 基因 PCR-RFLP 分析方法鉴别青鱼和草鱼

建立了一种以线粒体细胞色素 b（Cyt b）基因为标靶,应用 PCR -RFLP 技术进行草鱼和青鱼种质鉴定的分析方法。设计一对引物 QYCYT -S 和 QYCTY -A 分别对青鱼和草鱼的 Cyt b 基因进行了 PCR 扩增,并选用 BglⅠ和 EcoRⅡ两种限制性内切酶对扩增产物进行酶切分析。结果表明,草鱼和青鱼都可以扩增出1111 bp 的条带,两种酶切检验发现,草鱼扩增产物能被 BglⅠ切成247 bp 和864 bp 两个片段,而青鱼的 PCR 产物能被 EcoRⅡ切成140 bp 和971 bp 两个片段,这表明,mtDNA Cyt b 基因 BglⅠ和EcoRⅡ的酶切位点都可作为鉴定青鱼和草鱼的有效分子标记。利用 PCR -RFLP 分析 mtDNA Cyt b 基因的方法操作简单,是一种快速鉴别草鱼和青鱼的可靠方法。     

丰鲤及其双亲线粒体DNA限制性酶切图谱的研究

采用密度梯度离心及RNase消化法制备并纯化了丰鲤(Fengcarp)、兴国红鲤(Xingguoredcarp)及散鳞镜鲤(Scatterscaledmirrorcarp)的肝脏线粒体DNA,用10种限制性内切酶HindIII、EcoRI、BamHI、XbaI、XhoI、PstI、BglII、SalI、BglI、PvuII进行了分析.丰鲤mtDNA相对分子质量约为9 88×106,大小约为16 49kb;兴国红鲤mtDNA相对分子质量约为9 89×106,大小约为16 50kb;散鳞镜鲤mtDNA相对分子质量约为9 87×106,大小为16 48kb.HindIII、EcoRI、BglI、BamHI、XbaI、XhoI、SalI、BglII、PstI及PvuII在丰鲤、兴国红鲤、散鳞镜鲤线粒体DNA分子上均分别有6、4、3、3、3、1、1、0、1和4个切点.根据单酶切及双酶切结果,构建了丰鲤、兴国红鲤、散鳞镜鲤mtDNA9种酶的限制性酶切图谱,结果表明丰鲤、兴国红鲤及散鳞镜鲤mtDNA间缺乏变异性.     

嗜麦芽假单胞菌质粒pSH1的限制图谱及km^r标记的克隆

对嗜麦芽假单胞菌Ｐ２菌株的质粒ｐＳＨ１进行了限制酶切分析，确定了Ｂｇ１ Ⅱ，ＥｃｏＲ Ⅰ，Ｐｓｔ Ⅰ，Ｘｂａ Ⅰ，ＢａｍＨ Ⅰ，Ｂｇｌ Ⅰ，及Ｐｖｕ Ⅱ共７种限制性内切酶在ｐＳＨ１，质粒上的切割位点，前４种酶均为单一切点，后３种依次为２，７，５个切点。通过双酶切和部分酶切的方法绘制了ｐＳＨ１质粒的限制酶切图谱。将ｐＧＰ１－２质粒上的卡那霉素抗性基因片段插入ｐＳＨ１，获得了重组质粒ｐＳＨ２．ｐＳＨ２     

短小芽孢杆菌质粒pCJ3的限制酶切图谱

从短小芽孢杆菌A3菌株分离的具四环素抗性质粒pCJ3,经电泳法及氯化铯密度梯度法纯化后在琼脂糖凝胶电泳上得到2条带,经电镜观察及限制酶切割证实是一种质粒的两种构型——超线团及开环DNA分子。纯化质粒用13种限制酶进行酶切试验,发现其中有BamH I、Ava I、Xba I切点1个,EcoR I、Pst I、Bgl I、Hinc Ⅱ及Xho I切点2个、Hinf I、Sal I及Bel I切点3个,Hha I切点5个,Hind Ⅲ没有切点。用BamH I、EcoR I、Ava I及Xba I进行双酶解,并根据双酶解后的琼脂糖凝胶电泳带估算出各个片段的分子量,作出质粒pCJ3的4种限制酶的酶切图谱。     

枣实蝇及其他5种检疫性实蝇的PCR-RFLP快速鉴定

枣实蝇(Carpomya vesuviana Costa)是一种新入侵检疫性有害生物。应用PCR-RFLP技术,快速区分鉴定了枣实蝇与我国口岸截获频率较高的5种检疫性实蝇。研究表明:设计出的2对引物对6种供试实蝇线粒体DNA(mtDNA)进行PCR扩增,其扩增片断大小分别为350 bp和450 bp。PCR扩增产物用2种限制性内切酶Dra I和MseI进行酶切,根据不同的酶切位点准确区分了6种供试的实蝇。此方法可用于口岸截获实蝇的快速检疫鉴定。     

Mitochondrial DNA analysis of ancient Sampula population in Xinjiang

The archaeological site of Sampula cemetery was located about 14 km to the southwest of the Luo County in Xinjiang Khotan, China, belonging to the ancient Yutian kingdom. ¹⁴C analysis showed that this cemetery was used from 217 B.C. to 283 A.D. Ancient DNA was analyzed by 364 bp of the mitochondrial DNA hypervariable region I (mtDNA HVR-I), and by six restriction fragment length polymorphism (RFLP) sites of mtDNA coding region. We successfully extracted and sequenced intact stretches of maternally inherited mtDNA from 13 out of 16 ancient Sampula samples. The analysis of mtDNA haplogroup distribution showed that the ancient Sampula was a complex population with both European and Asian characteristics. Median joining network of U3 sub-haplogroup and multi-dimensional scaling analysis all showed that the ancient Sampula had maternal relationship with Ossetian and Iranian.     

Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies

In vitro studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome.     

Mitochondrial DNA analysis of ancient Sampula population in Xinjiang

The archaeological site of Sampula cemetery was located about 14 km to the southwest of the Luo County in Xinjiang Khotan,China,belonging to the ancient Yutian kingdom.~14C analysis showed that this cemetery was used from 217 B.C.to 283 A.D. Ancient DNA was analyzed by 364 bp of the mitochondrial DNA hypervariable region I (mtDNA HVR-I),and by six restriction fragment length polymorphism (RFLP) sites of mtDNA coding region.We successfully extracted and sequenced intact stretches of maternally inher- ited mtDNA from 13 out of 16 ancient Sampula samples.The analysis of mtDNA haplogroup distribution showed that the ancient Sampula was a complex population with both European and Asian characteristics.Median joining network of U3 sub-haplogroup and multi-dimen- sional scaling analysis all showed that the ancient Sampula had maternal relationship with Ossetian and Iranian.     

蓖麻蚕蛹mtDNA的限制性内切酶图谱

采用碱变性法制备蓖麻蚕蛹ｍｔＤＮＡ，经九种限制性内切酶单酶和双酶酶解后，琼脂糖凝胶电泳检测酶切位点数和酶切片段长度。根据片段的大小确定消失片段与新生片段关系，通过对片段重叠、拼接、判断各片段邻近位置，从而构建了９种限制性内切酶、共２４个酶切位点蓖麻蚕蛹ｍｔＤＮＡ酶切图谱。     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

动物线粒体DNA的RFLP在家养动物遗传多样性研究中的应用(综述)

对线粒体ＤＮＡ限制性片段长度多态性（ＲＦＬＰ）在家养动物遗传多样性研究中的应用现状进行了综述,并对其应用前景进行了展望。众多的研究表明,线粒体ＤＮＡ的ＲＦＬＰ可以作为一个可靠的母性遗传标记用于家养动物起源分化及其亲缘关系的研究方面。我国有丰富的能够适应各种不同生态条件的优良畜禽品种遗传资源,因此利用线粒体ＤＮＡ的ＲＦＬＰ研究其遗传多样性对于探讨其起源分化以及今后合理的利用和保存将有十分重要的理论和实践意义。