• Create Time: 2022-06-24
  • Update Time: 2022-06-27

2.1 Introduction

我们的生命科学研究离不开工具的辅佐，这一章主要介绍了一些相关的分子生物学研究方法，其中包含了非常重要的两类：

  • 限制性内切酶（restriction endonucleases）

  • 克隆载体（cloning vectors）

随着发展，我们已经进入到了可以在全基因组层面研究功能和表达了。所以这章将介绍一些起源工具及一些最近的工具。

2.2 Nucleases

  • Nucleases能水解磷酸二酯键中的酯

  • Phosphatases只能水解磷酸单酯键中的酯

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Phosphatase也是很重要的，尽管它只能水解末端的磷酸酯键，这样它就能去掉末端的磷酸盐，这样的作用可以用于连接多条链（ligating or connecting chains）。也可以用来使用32P来做标记。

Nucleases可以分成两类：

  1. 外切酶（exonuclease）：只能从链的末尾开始水解

  2. 内切酶（endonuclease）：可以从链的中间开始水解

不同的nucleases的targets也不同，例如有针对DNA的也有针对RNA的，有针对单链的也有针对双链的，还有针对于DNA/RNA hybrid的（例如：RNaseH，在lib prep中最常用到的，至少对我来说）。而且不同的nuclease可能还有序列的偏好，例如pancreatic RNase A，就偏好于在pyrimidine（C，T）后切。这种有很强的序列特异性的，我们就叫做限制性内切酶（restriction enzyme）。

限制性内切酶也有三种分类：

  1. Type I：剪切的位点与识别的位点相差1000bp

  2. Type II：序列的识别与剪切位点相同，换句话说就是剪切识别位点。这类特异性序列是回文的（inversely palindromic）

  3. Type III：剪切的位点与识别的位点相差20-30bp

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剪切完后的末端可能形成两种，一种是staggered，一种是blunt。来源不同的限制性内切酶如果识别的位点相同，但是也可能产生不同的剪切结果，主要是产生的末端可能有差异，这样的限制性内切酶集合叫做isoschizomers。

When a DNA molecule is cut with a suitable restriction enzyme, it is cleaved into distinct, negatively charged fragments.

2.3 Cloning

recombinant DNA**: ***a DNA molecule from two (or more) different *sources

这是一些题外话，我的TA还是有关于cloning的（虽说我的部分是做生信方面），但是我都没有好好的了解过，这次读「Gene XII」才算是让我有些明白了原理，感觉非常的好。

对于Cloning来说最重要的两个组成部分就是1. insert和2. vector

  • insert： 就是我们想要进行复制的DNA

  • vector：作为载体

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这里介绍了一个比较常见的Vector（blue/while selection vector），这个vector中包含着一些很重要的基因（部分），如上图中的 ori ，主要是控制vector的自我复制（这样的vector进入宿主细胞后就能自我复制）。还有如上图的amp，这个基因的作用就是对于抗生素ampicillin的拮抗，这样就能挑选出哪些host cells是包含vector的。另外一个非常重要的组成部分就是lacZ，这个基因序列包含两个作用，第一个是提供了multiple cloning sites（MCS），这样不同的endonuclease都可以剪切这一段；第二个作用就是区分包含insert的vector（也有可能不是包含insert）和不包含的。

这个lacZ基因就是blue/white selection的关键，因为，如果lacZ基因是完整的，没有被插入打断，那么他会进行一些反应，将media中的ß-gal切成有蓝色颜色，而那些有insert的，因为lacZ已经不完整了，所以并没有这项功能，media中的ß-gal仍旧是白色的。以此，来区分那些是有有效vector，哪些是没有有效vector的（或者不包含vector）。

其实可以用一个决策树来判断：

  • 在ampicillin培养基中

    • 能活：包含vector

      • 白色：vector中有insert或者其他序列（vector被打断过）

      • 蓝色：vector是完整的，没有insert

    • 不能活：不包含vector

所以最终我们会选择活下来，并且呈白色的colony。

Transformation:

  • washing the bacteria in a high salt wash of calcium chloride：使得host cell的表面破洞

  • electroporation: 电流刺激，使得host cell的表面破洞

因为存在假阳性，所以并不能将白色的colony当作我们所要的，还是需要做测序，来确保结果是我们想要的东西。

2.4 Cloning Vectors Can Be Specialized for Different Purposes

New words

单词读音词性释义例句
precise/priˈsaɪs/To make precise or definite; to define precisely or exactly; to particularize.Two essential items in the molecular biologists’ toolkit are restriction endonucleases, which allow DNA to be cut into precise pieces, and cloning vectors, such as plasmids or phages used to “carry” inserted foreign DNA fragments for the purpose of producing more material or a protein product.
nuclease/ˈn(j)ukliˌeɪz/An enzyme that catalyses the hydrolysis of a phosphodiester bond of a nucleic acid, cleaving the nucleic acid into smaller units.Nucleases hydrolyze an ester bond within a phosphodiester bond.
ester/ˈɛstər/A derivative of an acid in which one or more acidic hydrogen atoms are replaced by an alkyl, aryl, or similar group;  Nucleases hydrolyze an ester bond within a phosphodiester bond.
Archaea/ɑrˈkiə/A taxon comprising the archaebacteria; (also  archaea) organisms of this taxon (collectively or individually), archaebacteria.These are endonucleases from eubacteria and Archaea that recognize a specific DNA sequence.
cleavage/ˈklivɪdʒ/The action of cleaving or splitting crystals and certain rocks along their lines of natural fissure; the state of being so cleft.Type II enzymes are distinguished because the recognition site and cleavage site are the same.
palindromic/ˌpælənˈdrɑmɪk/Designating or consisting of a nucleic acid sequence that is a palindromeThe sites are typically inversely palindromic, that is, reading the same forward and backward on complementary strands.
stagger/ˈstæɡər/To cause to waver, throw into confusionThe first and more common is a staggered cut, which leaves single-stranded overhangs, or “sticky ends”.
palindrome/ˈpælənˌdroʊm/n. and adjA word or a sequence of words that reads, letter for letter, the same backwards as forwards.Types I and III enzymes differ from type II enzymes in that the recognition site and cleavage site are different and are usually not palindromes.
irrespective/ɪrɪˈspɛktɪv/adj. and adv.Not respectful, disrespectfulA restriction map can be obtained for any sequence of DNA, irrespective of whether we have any knowledge of its function.
era/ˈɪərə/n.The portion of historical time to which an event is to be assigned; the approximate dateNow that we have entered the era of routine whole-genome sequencing, the function and expression of entire genomes have become commonplace.
multiplicity/ˌməltəˈplɪsᵻdi/n.A large number or variety ofNucleases have a multiplicity of specificities.
pancreatic/ˌpæŋkriˈædɪk/adj.Of or relating to the pancreasNucleases might have a sequence preference, such as pancreatic RNase A, which preferentially cuts after a pyrimidine.
ampicillin/æmpɪˈsɪlɪn/n.
electroporation/iˌlɛktroʊpɔˈreɪʃ(ə)n/n.The application of a high-voltage pulse of electricity to cells or protoplasts in order to cause reversible breakdown of the cell membranes, so creating temporary pores through which foreign material may be introduced; the use of this technique as a means of introducing genetic material into cells.There are two common methods of transformation: washing the bacteria in a high salt wash of calcium chloride, or electroporation, in which an electric current is applied.
agar/ˈɑˌɡɑr/n.A gelatinous substance obtained from certain red seaweeds and used in biological culture media and as a thickener in foods;The transformed bacterial cells are plated on an agar plate containing both the antibiotic ampicillin and an artificial ß-gal inducer called isopropylthiogalactoside (IPTG).
recombinant/riˈkɑmbənənt/adj. and n.Of genetic material: assembled by genetic recombination or genetic engineering. Of an organism, cell, or protein: being or containing the expressed product of such genetic material.Cloning DNA typically involves recombinant DNA.
vehicle/ˈviək(ə)l/n.any substance which serves as a base or carrier medium for other substances added to it.There are two different DNAs needed: a vector, or cloning vehicle, and an insert, or the molecule to be cloned.
specifically/spəˈsɪfək(ə)li/adv.In respect of specific or inherent qualitiesOver the years, vectors have been specifically engineered for safety, selection ability, and high growth rate.
necessitate/nəˈsɛsəˌteɪt/adj.The size of the insert is important; too large (over ~10 kilobases) an insert will not be efficiently cloned in a plasmid vector, which will necessitate using an alternative virus-based vector.
propagate/ˈprɑpəˌɡeɪt/v.To produce (offspring); to cause (a plant, animal, etc.) to reproduce or multiply; to cause (a race, species, etc.) to continue in being by procreation. Also: to produce (a new individual) by natural processes from
 a parent stock, seed, etc.Shuttle vectors can be propagated in more than one type of host cell.
desirable/dɪˈzʌɪərəb(ə)l/adj. and n.Worthy to be desired; to be wished for. In early use often standing for the qualities which cause a thing to be desired: Pleasant, delectable, choice, excellent, goodly.It is often desirable to clone larger inserts, though, and sometimes the goal is not just to amplify the DNA but also to express cloned genes in cells, investigate properties of a promoter, or create various fusion proteins (defined shortly).
capacity/kəˈpæsədi/n.Ability to receive or contain; holding power. *Obsolete *(in general sense).The HAC is the newest addition of the line of vectors and it offers the advantage of having virtually unlimited capacity.
quantitate/ˈkwɑn(t)əˌteɪt/v.To measure the quantity or extent of; to perform quantitative analysis upon; to express in numerical terms; to quantify.The type of reporter gene that is most appropriate depends on whether we are interested in quantitating the efficiency of the promoter (and , for example, determining the effects of mutations in it or the activities of transcription factors that bind to it) or determining its tissue-specific pattern of expression.
assay/ˈæˌseɪ/v.To put to the testFigure2.7 summarizes a common system for assaying promoter activity.
luciferase/l(j)uːˈsɪfəreɪs/n.Any enzyme which catalyses a reaction by which a specific luciferin produces light.A cloning vector is created that has a eukaryotic promoter linked to the coding region of luciferase, a gene that encodes the enzyme responsible for bioluminescence in the firefly.
bioluminescence/ˌbaɪəˌluməˈnɛs(ə)ns/n.The emission of light by living organisms such as fireflies, glow-worms, and many deep-sea creatures.the same as above.
fluorescent/ˌflʊˈrɛs(ə)nt/adj.Of light or other radiation: arising from or of the nature of fluorescence; (of a substance or object) possessing the property of fluorescence; relating to or resulting from fluorescence. Also in extended use: vividly colourful.One of the most popular reporters that can be used to visualize patterns of gene expression is green fluorescent protein (GFR), which is obtained from jellyfish.
fluoresce/flʊˈrɛs/v.To emit light or other radiation by fluorescence; to be or become fluorescent.In addition to the original GFP, numerous variants that fluoresce in different colors, such as yellow (YFP), cyan (CFP), and blue (BFP), have been developed.
permeabilize/ˈpərmiəbəˌlaɪz/v.To make permeable.Bacteria and simple eukaryotes like yeast can be transformed easily, using chemical treatments that permeabilize the cell membranes (as discussed in the section Cloning earlier in this chapter).
puncture/ˈpəŋ(k)(t)ʃər/v.To make a puncture in; to pierce with a sharp point; to prick; to perforate. Also: to make a puncture in the tyre of (a bicycle or its rider). Also figurative.Micro-injection uses a very fine needle to puncture the cell membrane.
impediment/ɪmˈpɛdɪmənt/n.The fact of impeding or condition of being impeded; hindrance, obstruction; *concrete *something that impedes, hinders, or obstructs; a hindrance, an obstruction.The thick cell walls of plants are an impediment to many transfer methods;
propel/prəˈpɛl/v.To drive away or out; to eject; to expel.A gene gun shoots very small particles into the cell by propelling them through the wall at high velocity.