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1.
We constructed a series of cosmid vectors that carry the two cohesive end sites (cos) of lambda phage, arrayed in tandem, which enabled us to clone fragments of genomic DNA of up to 50 kb without a vector background. An equimolar mixture of the left and right vector arms of equal length was prepared from the vector DNA, simply by treating the DNA sequentially with three enzymes, restriction enzyme PvuII, alkaline phosphatase, and restriction enzyme BamHI (or BglII), without purification by agarose gel electrophoresis. After phenol extraction and ethanol precipitation, the equimolar mixture of the vector arms, which carried a single cos oriented from left to right, was directly ligated with insert DNA without further manipulation. We established conditions for cosmid cloning, using two kinds of DNA fragment of 40-50 kb, prepared from mouse L cell genomic DNA, as insert DNAs, namely, three cloned BamHI fragments and Sau3AI fragments, size-selected on a sucrose density gradient. The most important parameters affecting the cloning efficiency were the quality of the insert DNA and the molar ratio of the insert and vector arms. We achieved cloning efficiencies of 3.6 X 10(6)-1.3 X 10(7) colony forming units (cfu)/micrograms of insert DNA and 1.7 X 10(5)-1.0 X 10(6) cfu/micrograms of insert DNA, using the cloned BamHI fragments and the Sau3AI fragments, respectively. We examined more than 5000 clones and found that they all contained insert DNA.  相似文献   

2.
柴建华 《遗传学报》1990,17(2):136-142
cosmld克隆的线性化用λcos末端酶来完成,线性的cosmid或λDNA经部份限制性内切酶酶解后,分别与已标记的cos顺序探针杂交(探针为分别与λ的左端或右端的cos顺序互补的12核苷酸单链片段),杂交后的部份酶解片段经电泳分离和自显影后,酶切点位置可直接在X-底片上读出。在本实验室条件下,可一次完成二个克隆包括5—6种限制性内切酶的图谱分析,分析和作图可通过计算机或手工进行。  相似文献   

3.
We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.  相似文献   

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5.
Summary We used a mouse-human somatic cell hybrid to construct a chromosome 21-enriched library in phage vector EMBL4. In all, 35 phage clones containing human inserts were identified by differential screening with total human and mouse DNA. Whole recombinant phages were regionally mapped on chromosome 21 by Southern blot analysis using competitive hybridisation conditions to block repetitive sequences. Ten phage clones mapped proximal to a translocation breakpoint in band 21q21.2, while 25 mapped distal to this point. Three of the phage clones identify restriction fragment length polymorphisms. Polymorphic chromosome 21 markers may be useful in the genetic analysis of Alzheimer's dementia and Down syndrome.  相似文献   

6.
7.
Amyloplasts were isolated from a heterotrophic culture cell line of a woody plant, sycamore (Acer pseudoplatanus), and their DNA was purified. Conventional procedures for making a physical map were not easily applicable to the amyloplast DNA, since the yield of DNA was too low and the presence of repeated sequences interfered with the analysis. Therefore, the pieces of amyloplast DNA starting with a few micrograms of DNA were cloned in the lambda Fix vector, which is a derivative of lambda EMBL vectors improved for efficient cloning and gene walking. Cloned DNA fragments were randomly picked, mapped for restriction endonuclease sites by a refined procedure, and combined by overlapping their physical maps. The DNA library was also subjected to screening by gene walking using promoters recognized by T3 and T7 RNA polymerases in the vector to fill the gaps between sequences determined by overlapping the physical maps. In this way, we constructed the entire DNA library and the complete physical map of the amyloplast DNA. The sycamore amyloplast genome was composed of 141.7-kbp nucleotides with the same gene arrangement as that of tobacco chloroplasts.  相似文献   

8.
Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.  相似文献   

9.
We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.  相似文献   

10.
Analysis of cosmids using linearization by phage lambda terminase   总被引:29,自引:0,他引:29  
A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage λ terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested' with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage λ (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.  相似文献   

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