贵州小香羊FSH-β基因部分序列的RFLP多态性

为寻找与小香羊繁殖力相关的分子标记提供基础数据和科学依据,以贵州小香羊为试验材料,颈静脉采血后,提取血液全基因组DNA,根据NCBI Gene Bank山羊FSH-β基因序列(S64745.1)设计引物,采用限制性内切酶片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)方法对FSH-β基因内含子1与内含子2的序列进行多态性检测。结果表明:在两段特异性扩增产物中存在限制性酶切位点,不存在RFLP多态性。     

Direct cloning and sequence analysis of enzymatically amplified genomic sequences

A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.     

Human beta-globin gene sequences injected into mouse eggs, retained in adults, and transmitted to progeny

Foreign gene sequences were retained in two adult mice (out of 62 analyzed) from fertilized eggs injected with a recombinant plasmid containing the human beta-globin genomic region and the herpes simplex viral thymidine kinase gene. The intact human and viral genes were found in DNA of one of the animals and, in the other, at least part of the human globin gene was present. The latter individual transmitted these sequences to its progeny in a Mendelian ration. Thus, human DNA may be incorporated into the germ line of mice for in vivo studies of regulation of gene expression in development, genetic diseases, and malignancy.     

Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes

Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.     

一种新的获取海洋贝类基因组DNA的取样方法

[目的]探讨一种新的获取海洋贝类基因组DNA的取样方法,为开展珍稀贝类的分子生物学研究提供参考。[方法]以文蛤、栉江瑶、翡翠贻贝、香港巨牡蛎、毛蚶为供试材料,以闭壳（合）肌全基因组DNA作为参考,采用常规酚-氯仿法抽提贝类贝腔液基因组DNA,使用生物光度计法、琼脂糖凝胶电泳、目的片段扩增测序验证其质量,并建立系统发育树检测其来源的真实可靠性。[结果]采用常规酚/氯仿基因组DNA提取方法获得的贝腔液DNA质量优于闭壳（合）肌DNA,蛋白质、多酚色素污染较少,完全满足目的片段扩增测序;系统发育进化树则证实了贝腔液并非外源污染。[结论]从贝腔液中获取基因组DNA是完全可行的,并可将其用于目的基因片段的扩增。     

一种新的获取海洋贝类基因组DNA的取样方法（摘要）

[目的]探讨一种新的获取海洋贝类基因组DNA的取样方法,为开展珍稀贝类的分子生物学研究提供参考。[方法]以文蛤、栉江瑶、翡翠贻贝、香港巨牡蛎、毛蚶为供试材料,以闭壳（合）肌全基因组DNA作为参考,采用常规酚-氯仿法抽提贝类贝腔液基因组DNA,使用生物光度计法、琼脂糖凝胶电泳、目的片段扩增测序验证其质量,并建立系统发育树检测其来源的真实可靠性。[结果]采用常规酚/氯仿基因组DNA提取方法获得的贝腔液DNA质量优于闭壳（合）肌DNA,蛋白质、多酚色素污染较少,完全满足目的片段扩增测序;系统发育进化树则证实了贝腔液并非外源污染。[结论]从贝腔液中获取基因组DNA是完全可行的,并可将其用于目的基因片段的扩增。     

灰盖鬼伞基因组中微卫星序列的组成

本研究利用已公布的灰盖鬼伞基因组测序结果,对该真菌基因组中的微卫星(microsatellite)或简单重复序列(simplesequence repeats,SSRs)进行了系统分析。结果表明,在已公布的36.2 Mb的基因组序列中,共有7 859个SSR序列(长度大于15bp,匹配值大于80%)。SSR的碱基总数达143 kb,约占整个基因组碱基数的0.40%,平均4.61 kb中就有1个大于15 bp的SSR序列。其中数量最多的是3碱基SSR,数量达到3 033个,其次为6碱基重复序列(2 121个)、5碱基重复序列(1 820个),这3种SSR总数达6 974个,占SSR总数的84.9%,单碱基重复序列数量最少,仅有285个。与子囊菌中的稻瘟病菌和粗糙脉孢菌相比,灰盖鬼伞菌基因组中每百万碱基中的SSR数量和密度都较小。这些研究结果可为该担子菌基因组的特征描述、注释及分子标记的筛选提供基础信息。     

Correction of murine beta-thalassemia by gene transfer into the germ line

A murine beta-thalassemia was corrected by the transfer of cloned beta-globin genes into the mouse germ line. The cloned mouse beta maj-globin gene or the cloned human beta-globin gene was introduced into mice deficient in beta-globin synthesis because of a deletion of the beta maj-globin gene. Both introduced genes produced functional beta-globin chains, leading to a reduction in one case, and elimination in another case, of the anemia and associated abnormalities of the red blood cells.     

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.