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1. 发明授权

US5928908A Method for introducing unidirectional nested deletions 失效
标题翻译：引入单向嵌套缺失的方法
公开(公告)号：US5928908A
公开(公告)日：1999-07-27
申请号：US966958
申请日：1997-11-10
申请人： John J. Dunn , Mark A. Quesada , Matthew Randesi
发明人： John J. Dunn , Mark A. Quesada , Matthew Randesi
IPC分类号： C12N15/10 , C12N15/64
CPC分类号： C12N15/102
摘要： Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.
摘要翻译：公开了在克隆的DNA片段中引入单向缺失的方法。更具体地，该方法包括提供包含插入克隆载体中的目标DNA片段的重组DNA构建体，该克隆载体具有与感兴趣的DNA片段的插入位点相邻的f1内切核酸酶识别序列。然后将重组DNA构建体与由噬菌体f1的基因II编码的蛋白质pII接触，从而产生单链切口。然后将切口的DNA与大肠杆菌外切核酸酶III接触，从而将单链缺口扩大为单链间隙。然后将单链间隙DNA与单链特异性内切核酸酶接触，从而产生含有与单链间隙大小相对应的双链缺失的线性化DNA分子。然后以适合于连接的条件，用这种方式处理的DNA与DNA连接酶一起温育。还公开了用于生产单链DNA探针的方法。在该实施方案中，如上所述制备的单链间隙DNA在DNA标记的核苷酸的存在下与DNA聚合酶接触以填充间隙。然后通过用限制性内切酶消化DNA直接切割该DNA，该限制性内切酶在感兴趣的DNA片段之外。然后将该消化产物变性以产生标记的单链核酸探针。

2. 发明授权

US06248569B1 Method for introducing unidirectional nested deletions 有权
标题翻译：引入单向嵌套缺失的方法
公开(公告)号：US06248569B1
公开(公告)日：2001-06-19
申请号：US09342353
申请日：1999-06-29
申请人： John J. Dunn , Mark A. Quesada , Matthew Randesi
发明人： John J. Dunn , Mark A. Quesada , Matthew Randesi
IPC分类号： C12N1566
CPC分类号： C12N15/102
摘要： Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment in the context of a cloning vector which contains an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment. Also disclosed is a method for producing single-stranded DNA probes utilizing the same cloning vector. An optimal vector, PZIP is described. Methods for introducing unidirectional deletions into a terminal location of a cloned DNA sequence which is inserted into the vector of the present invention are also disclosed. These methods are useful for introducing deletions into either or both ends of a cloned DNA insert, for high throughput sequencing of any DNA of interest.
摘要翻译：公开了在克隆载体的上下文中引入克隆DNA片段中的单向缺失的方法，该克隆载体含有与DNA片段的插入位点相邻的f1内切核酸酶识别序列。还公开了利用相同克隆载体产生单链DNA探针的方法。描述了最佳向量PZIP。还公开了将插入到本发明的载体中的克隆DNA序列的末端位置引入单向缺失的方法。这些方法可用于将缺失引入到克隆的DNA插入物的任一端或两端，用于任何感兴趣的DNA的高通量测序。

3. 发明授权

US5968786A Method for producing labeled single-stranded nucleic acid probes 失效
标题翻译：生产标记单链核酸探针的方法
公开(公告)号：US5968786A
公开(公告)日：1999-10-19
申请号：US215817
申请日：1998-12-18
申请人： John J. Dunn , Mark A. Quesada , Matthew Randesi
发明人： John J. Dunn , Mark A. Quesada , Matthew Randesi
IPC分类号： C12N15/10 , C12P19/34
CPC分类号： C12N15/102
摘要： Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.
摘要翻译：公开了在克隆的DNA片段中引入单向缺失的方法。更具体地，该方法包括提供包含插入克隆载体中的目标DNA片段的重组DNA构建体，该克隆载体具有与感兴趣的DNA片段的插入位点相邻的f1内切核酸酶识别序列。然后将重组DNA构建体与由噬菌体f1的基因II编码的蛋白质pII接触，从而产生单链切口。然后将切口的DNA与大肠杆菌外切核酸酶III接触，从而将单链缺口扩大为单链间隙。然后将单链间隙DNA与单链特异性内切核酸酶接触，从而产生含有与单链间隙大小相对应的双链缺失的线性化DNA分子。然后以适合于连接的条件，用这种方式处理的DNA与DNA连接酶一起温育。还公开了用于生产单链DNA探针的方法。在该实施方案中，如上所述制备的单链间隙DNA在DNA标记的核苷酸的存在下与DNA聚合酶接触以填充间隙。然后通过用限制性内切酶消化DNA直接切割该DNA，该限制性内切酶在感兴趣的DNA片段之外。然后将该消化产物变性以产生标记的单链核酸探针。

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