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酵母双杂交相关方法的改良及应用 总被引:1,自引:0,他引:1
对酵母双杂交实验过程中较为耗时的阳性克隆鉴定过程进行改进,以期建立一种快速有效的鉴定方法。分别采用液氮冻融法、超声破碎法、渗透压破壁法以及煮沸裂解法裂解酵母细胞,获得质粒作为PCR模板,直接测序鉴定筛选到的相互作用蛋白。以液氮冻融法和超声破碎法裂解细胞获得的质粒为模板进行PCR,得到特异的产物,测序鉴定结果明确,与经典的鉴定方法相比效果相当,但更加经济快捷;而渗透压破壁法和煮沸裂解法则效果不好。说明前两种方法可代替常规方法用于阳性克隆的鉴定,从而加快酵母双杂交实验中大量阳性克隆的筛查工作。 相似文献
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猪β2-AR基因重组质粒的快速筛选 总被引:2,自引:0,他引:2
为建立一种高效快速鉴定重组质粒的实验方法,将猪β2-肾上腺素能受体(β2-AR)基因与pET-32C重组,构建β2-AR基因表达载体pET32CAR,以T7启动子引物和猪β2-肾上腺素能受体基因特异性引物为PCR引物,挑取重组质粒转化单菌落直接进行PCR,产物经电泳发现,5个候选克隆中有3个克隆扩增出了一条约2kb的特异性条带,并且插入方向正确,随机选取其中1个克隆用限制性酶切鉴定以及DNA测序验证PCR筛选的正确性,研究结果表明;在采用PCR方法对重组克隆进行鉴定时,可以直接使用细菌菌落参与反应,而无须提取克隆的DNA,与传统的实验方法相比,筛选的时间缩短至3-4h,大大提高了重组DNA的筛选效率。 相似文献
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利用热激PCR法和菌落直接PCR法进行大肠杆菌和农杆菌的鉴定,筛选含有GFP-linker-C-TAPa或wtGPA1-C-TAPa的pCAMBIA2300EC的阳性克隆,比较热激PCR法和菌落直接PCR法的优劣。热激PCR法阳性检出率是100%,菌落直接PCR法阳性检出率是60%左右。热激PCR法是一种快速可靠的鉴定重组阳性克隆的方法。 相似文献
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PCR简便快速筛选阳性克隆 总被引:4,自引:0,他引:4
目前常用的筛选阳性重组子的方法主要有快提质粒酶切法及杂交筛选法[1] ,但这两种方法均存在不足之处。比如快提质粒法需要提取质粒 ,在筛选大量样品时操作比较繁琐 ;而杂交法存在操作周期长并要使用同位素等不足。由于PCR技术具有操作简便且灵敏度极高的特点[2 ] ,近年出现了一些利用PCR技术筛选阳性克隆的方法[3] ,但这些方法在制备PCR扩增模板时仍然需要提取被筛选样品菌的质粒。我们用溶菌酶处理被筛选样品菌细菌悬液 ,并对悬液加热 ,以使质粒分散于菌细胞之外。然后直接以该悬液为模板 ,使用PCR对重组质粒上的特定序列进行… 相似文献
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Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis
It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis. 相似文献
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RICHARD E. NORRIS 《Botanical journal of the Linnean Society. Linnean Society of London》1991,106(1):1-40
Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera. 相似文献
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JOAN VALUÈS MONTSERRAT TORRELL NÚRIA GARCIA JACAS 《Botanical journal of the Linnean Society. Linnean Society of London》2001,137(4):399-407
Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted. 相似文献
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肝癌中HBV和HCV基因和抗原的分布及意义 总被引:1,自引:0,他引:1
采用原位分子杂交方法检测HCV
RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV
RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV
RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV
X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV
C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细 相似文献
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H.-R. GREGORIUS 《Biological journal of the Linnean Society. Linnean Society of London》1984,23(2-3):157-165
For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment. 相似文献
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment. 相似文献