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  • General

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  • Gene 克隆 - 高级

    Dolly the sheep was the first mammal to be successfully cloned from an adult somatic cell.

    What does it mean to clone?
    ::克隆意味着什么?

    A genetically exact copy. A clone can be a gene , a , an , or a plant. And these clones are produced all the time. Dolly the sheep was the first mammal to be successfully cloned from an adult somatic cell using a technique known as somatic cell nuclear transfer (SCNT). This groundbreaking achievement was announced in 1997 by scientists at the Roslin Institute in Scotland. Theoretically, a clone could also be a human. But would that be a smart thing to do? Is it ethical? Is it even legal?
    ::基因精确的复制件。 克隆可以是基因, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个 基因, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个, 一个

    Gene Cloning
    ::Gene 克隆

    You probably have heard of . A clone is a genetically exact copy. It can be a clone of a gene, a cell or an organism . Even a human. However, whereas of humans has many ethical issues associated with it and is illegal throughout most of the world, the cloning of genes has been ongoing for well over 30 or 40 years, with cloning of occurring more recently. Gene cloning , also known as molecular cloning, refers to the process of isolating a sequence of interest for the purpose of making multiple copies of it. The identical copies are clones. In 1973, Stanley Cohen and Herbert Boyer developed techniques to make , a form of artificial . 
    ::您可能听说过。 克隆是一个基因、 细胞或有机体的复制品。 它可以是基因、 细胞或生物的复制品。 甚至是一个人类。 然而, 人类有许多与此相关的伦理问题, 在世界大部分地区都是非法的, 基因克隆已经持续了30或40多年, 克隆最近才发生。 基因克隆,又称为分子克隆, 是指为制作多种复制品而分离利益序列的过程。 相同的复制品是克隆。 1973年, Stanley Cohen 和 Herbert Boyer 开发了制造技术, 一种人造技术。

    Recombinant DNA is engineered through the combination of two or more strands, combining DNA sequences that would not normally occur together. In other words, selected DNA (or the DNA of "interest") is inserted into an existing organismal genome , such as a bacterial plasmid DNA, or some other sort of vector . The recombinant DNA can then be inserted into another cell, such as a bacterial cell, for amplification and possibly production of the resulting . This process is called transformation , the genetic alteration of a cell resulting from the uptake, incorporation, and expression of foreign genetic material . Recombinant technology was made possible by the discovery of restriction endonucleases.
    ::重组DNA是通过两个或两个以上线条的组合,结合通常不会同时发生的DNA序列来制造的,换句话说,选定的DNA(或“利益”的DNA)被插入现有的生物基因组中,如细菌颗粒式DNA,或某种其他媒介。再组合DNA可以被插入另一个细胞,如细菌细胞,以便放大并可能生产由此产生的DNA。这一过程称为变异,由于吸收、融合和表达外国遗传材料而导致细胞的基因改变。通过发现限制内分泌,重组技术成为可能。

    Restriction Digestion and Ligation
    ::限制消化和排量

    Restriction or restriction endonucleases are prokaryotic enzymes that recognize and cut DNA at specific sequences called restriction sites. It is believed that they evolved as a defense mechanism against foreign DNA, such as viral DNA. Over 3,000 restriction enzymes have been identified. Some of the more common restriction enzymes are shown in the table below, where up and down arrows show the sites of cleavage . Restriction enzymes are named based on the prokaryotic organism they are isolated from. For example, those isolated from Escherichia coli would begin with Eco. As the Table  shows, digestion with the restriction enzymes will result in overlapping or blunt ends. EcoRI produces overlapping "sticky" ends: the cleaves between the G and A on both strands. On the other hand, SmaII restriction enzyme cleavage produces "blunt" ends. The enzyme cleaves between the G and C on both strands.
    ::限制或限制内分泌酶是指在所谓的限制地点的特定序列中识别和切除DNA的蛋白质酶,据信它们演变为防御外国DNA的防御机制,如病毒DNA。已经确定了3,000多个限制酶。下面的表格中列出了一些更常见的限制性酶,向上或向下箭头显示了裂痕的位置。限制性酶的命名基于它们与它隔离的蛋白质有机体。例如,从Eco开始,与Escherichia coli分离的酶将首先从Eco开始。表显示,与限制性酶的消化将导致重叠或钝端。EcoRI产生重叠的“粘性”端:G和A在两根两端的螺丝上的螺旋。另一方面,SmaII限制性酶酶在两端的螺丝上产生“泡”端。G和C在两端的酶间断裂。

    Common Restriction Endonucleases
    Enzyme Source Recognition Sequence Restriction Digest
    EcoRI Escherichia coli 
    5'GAATTC3'CTTAAG


    5'---G↓AATTC---3'3'---CTTAA↑G---5'

    BamHI Bacillus amyloliquefaciens 
    5'GGATCC3'CCTAGG


    5'---G↓GATCC---3'3'---CCTAG↑G---5'

    TaqI Thermus aquaticus 
    5'TCGA3'AGCT


    5'---T↓CGA---3'3'---AGC↑T---5'

    HinfI Haemophilus influenzae 
    5'GANTCA3'CTNAGT


    5'---G↓ANTC---3'3'---CTNA↑G---5'

    Sau3A Staphylococcus aureus 
    5'GATC3'CTAG


    5'---↓GATC---3'3'---CTAG↑---5'

    PvuII Proteus vulgaris 
    5'CAGCTG3'GTCGAC


    5'---CAG↓CTG---3'*3'---GTC↑GAC---5'

    SmaI Serratia marcescens 
    5'CCCGGG3'GGGCCC


    5'---CCC↓GGG---3'*3'---GGG↑CCC---5'

    HaeIII Haemophilus aegyptius 
    5'GGCC3'CCGG


    5'---GG↓CC---3'*3'---CC↑GG---5'

    Key: N = C or G or T or A
    ::键:N=C或G或T或A

    • = blunt ends
      ::=钝端

    Cloning of a segment of DNA of interest can easily be carried out with restriction digestion, followed by ligation and transformation or transfection. In the classical restriction enzyme digestion and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps:
    ::在传统的限制酶消化和隔离克隆协议中,任何DNA碎片的克隆基本上包括四个步骤:

    1. isolation of the DNA of interest (or target DNA),
      ::将感兴趣的DNA(或目标DNA)隔离;
    2. ligation,
      ::排泄物,
    3. transfection (or transformation), and
      ::转口(或变形),以及
    4. a screening/selection procedure.
      ::a 筛选/甄选程序。

    Isolation of DNA
    ::DNA分离

    Initially, the DNA fragment to be cloned must be isolated. This DNA of interest may be a gene, part of a gene, a promoter , or another segment of DNA, and is frequently isolated by the polymerase chain reaction ( ) or restriction enzyme digestion. As discussed above, a restriction enzyme is an enzyme that cuts double-stranded DNA at a specific sequence. The enzyme makes two incisions, one through each strand of the double helix , without damaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or blunt ends. The 1978 in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases. The first practical use of their work was the manipulation of E. coli to produce human insulin for diabetics.
    ::最初, 需要克隆的DNA碎片必须分离。 这种值得注意的DNA可能是基因、 基因的一部分、 促销者 或DNA的另一部分, 并且经常被聚合酶链反应egg 或限制酶消化隔离。 如上所述, 限制酶是一种酶, 在特定序列中切除双层DNA。 酶进行两次切开, 一次切开双螺旋, 一次切开双螺旋, 但不破坏氮基。 这既可以产生重叠端( 也称为粘结端 ) , 也可以产生钝端。 1978年, 药品授予Daniel Nathans 和 Hamilton Smith , 用于发现限制内分泌。 其作品的第一个实际用途是操纵 E. Coli , 用于生产人类糖尿病的胰岛素。

    Ligation
    ::利差

    Once the DNA of interest is isolated, a ligation procedure is necessary to insert the amplified fragment into a vector to produce the recombinant DNA molecule. Restriction fragments (or a fragment and a plasmid/vector) can be spliced together, provided their sticky ends are complementary. Blunt end ligation is also possible.
    ::一旦将感兴趣的DNA分离出来,就必须采取捆绑程序,将放大的碎片插入矢量,以产生重组的DNA分子。 限制性碎片(或一个碎片和一个粒子/粒子)可以组合在一起,只要其粘结端是相辅相成的。 模糊的末端绑绑也是可能的。

    The plasmid or vector (which is usually circular) is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. If the isolated DNA of interest and the plasmid or vector are digested with the same restriction enzyme, their sticky ends will be complementary. The two DNAs are then incubated with DNA ligase , an enzyme that can attach together strands of DNA with double-strand breaks. This produces a recombinant DNA molecule. The figure   depicts a plasmid with two additional segments of DNA ligated into the plasmid, producing the recombinant DNA molecule. 
    ::粒子或矢量(通常是环形的)用限制酶消化,打开矢量,以便插入目标DNA。如果孤立的脱氧核糖核酸和颗粒或矢量用同样的限制酶消化,它们的粘结端将是相辅相成的。然后,两种DNA用脱氧核糖核酸(ligase)孵化,一种酶可以将脱氧核糖核酸的线条与双弦断裂结合在一起。这产生一个重组的DNA分子。图中描绘了一个颗粒,将另外两个DNA捆绑在颗粒中,产生重组的DNA分子。

    lesson content
    (left) This image shows a line drawing of a plasmid. The plasmid is drawn as two concentric circles that are very close together, representing the two strands of DNA, with two large segments and one small segment depicted. The two large segments (blue and green) indicate antibiotic resistances usually used in a screening procedure, and the small segment (red) indicates an origin of replication, used in DNA replication. The resulting DNA is a recombinant DNA molecule. (right) Sticky ends produced by restriction enzyme digestion can be joined with the enzyme DNA ligase.

    Transfection and Selection
    ::切除和选择

    Following ligation, the recombinant DNA is placed into a host cell, usually bacterial, in a process called transfection or transformation. Finally, the transfected are cultured. Many of these cultures may not contain a plasmid with the target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNA of interest must be selected. Many plasmids/ include selectable markers - usually some sort of antibiotic ( Figure  ). When cultures are grown in the presence of an antibiotic, only transfected with the vector containing to that antibiotic should grow. However, these selection procedures do not guarantee that the DNA of insert is present in the . Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing (DNA sequence analysis).
    ::解剖后,将重组的DNA放入宿主细胞,通常是细菌,其过程称为转基因或变异。最后,对转基因人进行了培养。许多这些文化可能并不包含目标DNA的微粒,因为转基因过程通常不100%成功,因此必须选择具有相关DNA的适当培养物。许多粒子/包括可选择的标记――通常包括某种抗生素(图 ) 。当文化在出现抗生素的情况下生长时,只有与含有抗生素的病媒发生切换,但只有与含有抗生素的病媒发生切换。然而,这些选择程序并不能保证插入的DNA存在于(DNA序列分析)中。需要进一步分析由此形成的宿主群,以确认克隆成功。这可以通过一个过程或限制的碎片分析方法实现,两者都需要用凝胶电光和/或DNA测序(DNA序列分析)。

    DNA sequence analysis, , or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge. 
    ::限制的碎片分析是分离的颗粒/颗粒DNA的消化。如果分离的DNA含有目标DNA,则该碎片将通过限制的酶消化而切割。

    Gel Electrophoresis
    ::Gel 电发

    Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. Notice in Figure  that the "gels" are rectangular in shape. The gels are made of a gelatin-like material of either agarose or polyacrylamide. An electric field, with a positive charge applied at one end of the gel, and a negative charge at the other end, forces the fragments to migrate through the gel. DNA molecules migrate from negative to positive charges due to the net negative charge of the phosphate groups in the DNA backbone . Longer molecules migrate more slowly through the gel matrix. After the separation is completed, DNA fragments of different lengths can be visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The resulting stained gel shows bands corresponding to DNA molecules of different lengths, which also correspond to different molecular weights. Band size is usually determined by comparison to DNA ladders containing DNA fragments of known length. Gel electrophoresis can also be used to separate molecules and .
    ::Gel 电极是一种分析技术,用于按大小和电荷区分DNA碎片。图中注意,“凝胶”是长方形的。凝胶是用长方糖或聚丙烯酸的凝胶状材料制成的。电场,在凝胶的一端施用正电荷,在另一端施用负电荷,迫使这些碎片通过凝胶迁移。DNA分子由于DNA脊椎中磷酸盐组的净负电荷而从负向正电荷转移。较长的分子通过凝胶质矩阵迁移较慢。分离完成后,不同长度的DNA碎片可以使用具体针对DNA的荧光染色(例如溴)进行可视化。由此产生的带状凝胶显示与不同长度的DNA分子相对应的带,也与不同的分子重量相对应。通常通过与含有已知长度的DNA碎片的DNA梯子进行比较来确定波段大小。Gel 电阻还可用于分离分子和分离。

    Agarose gel electrophoresis of DNA
    Agarose gel electrophoresis of DNA

     

    Summary
    ::摘要

    • Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it.
      ::基因克隆,又称分子克隆,是指为制作多份DNA副本而分离一个值得注意的DNA序列的过程。
    • Classic gene cloning involves the following steps:
      • Restriction enzyme digestion and ligation
        ::限制酶消化和除虫
      • Isolation of DNA
        ::DNA分离
      • Ligation
        ::利差
      • Transfection and Selection
        ::切除和选择
      • Gel electrophoresis
        ::热电极

      ::典型基因克隆涉及以下步骤:

    Review
    ::回顾

    1. What are restriction endonucleases?
      ::什么是限制终止?
    2. How are gene cloning and recombinant DNA related?
      ::基因克隆和重新组合的DNA如何相关?
    3. Describe the process of gene cloning.
      ::描述基因克隆的过程。
    4. How does gel electrophoresis analyze DNA?
      ::凝胶电光如何分析DNA?