基因组2-4 DNA研究

4. Studying DNA Learning outcomes When you have read Chapter 4, you should be able to: ​ Give outline descriptions of the events involved in DNA cloning and the polymerase chain reaction (PCR), and state the applications and limitations of these techniques ​ Describe the activities and main applications of the different types of enzyme used in recombinant DNA research ​ Identify the important features of DNA polymerases and distinguish between the various DNA polymerases used in genomics research ​ Describe, with examples, the way that restriction endonucleases cut DNA and explain how the results of a restriction digest are examined ​ Distinguish between blunt- and sticky-end ligation and explain how the efficiency of blunt-end ligation can be increased ​ Give details of the key features of plasmid cloning vectors and describe how these vectors are used in cloning experiments, using pBR322 and pUC8 as examples ​ Describe how bacteriophage λ vectors are used to clone DNA ​ Give examples of vectors used to clone long pieces of DNA, and evaluate the strengths and weaknesses of each type ​ Summarize how DNA is cloned in yeast, animals and plants ​ Describe how a PCR is performed, paying particular attention to the importance of the primers and the temperatures used during the thermal cycling the toolkit of techniques used by molecular biologists to study DNA molecules was assembled during the 1970s and 1980s. Before then, the only way in which individual genes could be studied was by classical genetics, using the procedures that we will examine in Chapter 5. Classical genetics is a powerful approach to gene analysis and many of the fundamental discoveries in molecular biology were made in this way. The operon theory proposed by Jacob and Monod in 1961 (Section 9.3.1), which describes how the expression of some bacterial genes is regulated, was perhaps the most heroic achievement of this era of genetics. But the classical approach is limited because it does not involve the direct examination of genes, information on gene structure and activity being inferred from the biological characteristics of the organism being studied. By the late 1960s these indirect methods had become insufficient for answering the more detailed questions that molecular biologists had begun to ask about the expression pathways of individual genes. These questions could only be addressed by examining directly the segments of DNA containing the genes of interest. This was not possible using the current technology, so a new set of techniques had to be invented. The development of these new techniques was stimulated by breakthroughs in biochemical research which, in the early 1970s, provided molecular biologists with enzymes that could be used to manipulate DNA molecules in the test tube. These enzymes occur naturally in living cells and are involved in processes such as DNA replication, repair and recombination (see Chapters 13 and 14). In order to determine the functions of these enzymes, many of them were purified and the reactions that they catalyze studied in the test tube. Molecular biologists then adopted the pure enzymes as tools for manipulating DNA molecules in pre-determined ways, using them to make copies of DNA molecules, to cut DNA molecules into shorter fragments, and to join them together again in combinations that do not exist in nature ( Figure 4.1 ). These manipulations, which are described in Section 4.1, form the basis of recombinant DNA technology, in which new or ‘recombinant' DNA molecules are constructed in the test tube from pieces of naturally occurring chromosomes and plasmids. Recombinant DNA methodology led to the development of DNA or gene cloning, in which short DNA fragments, possibly containing a single gene, are inserted into a plasmid or virus chromosome and then replicated in a bacterial or eukaryotic host ( Figure 4.2 ). We will examine exactly how gene cloning is performed, and the reasons why this technique resulted in a revolution in molecular biology, in Section 4.2. Gene cloning was well established by the end of the 1970s. The next major technical breakthrough came some 5 years later when the polymerase chain reaction (PCR) was invented ( HYPERLINK "javascript:PopUpMenu2_Set(Menu_id384460);" Mullis, 1990). PCR is not a complicated technique - all that it achieves is the repeated copying of a short segment of a DNA molecule ( Figure 4.3 ) - but it has become immensely important in many areas of biological research, not least the study of genomes. PCR is covered in detail in Section 4.3. 4.1. Enzymes for DNA Manipulation Recombinant DNA technology was one of the main factors that contributed to the rapid advance in knowledge concerning gene expression that occurred during the 1970s and 1980s. The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the test tube. This, in turn, depends on the availability of purified enzymes whose activities are known and can be controlled, and which can therefore be used to make specified changes to the DNA molecules that are being manipulated. The enzymes available to the molecular biologist fall into four broad categories: ​ DNA polymerases (Section 4.1.1), which are enzymes that synthesize new polynucleotides complementary to an existing DNA or RNA template ( Figure 4.4A ); ​ Nucleases (Section 4.1.2), which degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next ( Figure 4.4B ); ​ Ligases (Section 4.1.3), which join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends of two different molecules, or at the two ends of a single molecule ( Figure 4.4C ); ​ End-modification enzymes (Section 4.1.4), which make changes to the ends of DNA molecules, adding an important dimension to the design of ligation experiments, and providing one means of labeling DNA molecules with radioactive and other markers (Technical Note 4.1). 4.1.1. DNA polymerases Many of the techniques used to study DNA depend on the synthesis of copies of all or part of existing DNA or RNA molecules. This is an essential requirement for PCR (Section 4.3), DNA sequencing (Section 6.1), DNA labeling (Technical Note 4.1) and many other procedures that are central to molecular biology research. An enzyme that synthesizes DNA is called a DNA polymerase and one that copies an existing DNA or RNA molecule is called a template-dependent DNA polymerase. The mode of action of a template-dependent DNA polymerase A template-dependent DNA polymerase makes a new DNA polynucleotide whose sequence is dictated, via the base-pairing rules, by the sequence of nucleotides in the DNA molecule that is being copied ( Figure 4.5 ). The mode of action is very similar to that of a template-dependent RNA polymerase (Section 3.2.2), the new polynucleotide being synthesized in the 5′→3′ direction: DNA polymerases that make DNA in the other direction are unknown in nature. One important difference between template-dependent DNA synthesis and the equivalent process for synthesis of RNA is that a DNA polymerase is unable to use an entirely single-stranded molecule as the template. In order to initiate DNA synthesis there must be a short double-stranded region to provide a 3′ end onto which the enzyme will add new nucleotides ( Figure 4.6A ). The way in which this requirement is met in living cells when the genome is replicated is described in Chapter 13. In the test tube, a DNA copying reaction is initiated by attaching to the template a short synthetic oligonucleotide, usually about 20 nucleotides in length, which acts as a primer for DNA synthesis. At first glance, the need for a primer might appear to be an undesired complication in the use of DNA polymerases in recombinant DNA technology, but nothing could be further from the truth. Because annealing of the primer to the template depends on complementary base-pairing, the position within the template molecule at which DNA copying is initiated can be specified by synthesizing a primer with the appropriate nucleotide sequence ( Figure 4.6B ). A short specific segment of a much longer template molecule can therefore be copied, which is much more valuable than the random copying that would occur if DNA synthesis did not need to be primed. You will fully appreciate the importance of priming when we deal with PCR in Section 4.3. A second general feature of template-dependent DNA polymerases is that many of these enzymes are multifunctional, being able to degrade DNA molecules as well as synthesize them. This is a reflection of the way in which DNA polymerases act in the cell during genome replication (Section 13.2.2). As well as their 5′→3′ DNA synthesis capability, DNA polymerases can also have one or both of the following exonuclease activities ( Figure 4.7 ): ​ A 3′→5′ exonuclease activity enables the enzyme to remove nucleotides from the 3′ end of the strand that it has just synthesized. This is called the proofreading activity because it allows the polymerase to correct errors by removing a nucleotide that has been inserted incorrectly. ​ A 5′→3′ exonuclease activity is less common, but is possessed by some DNA polymerases whose natural function in genome replication requires that they must be able to remove at least part of a polynucleotide that is already attached to the template strand that the polymerase is copying. The types of DNA polymerases used in research Several of the template-dependent DNA polymerases that are used in molecular biology research ( Table 4.1 ) are versions of the Escherichia coli DNA polymerase I enzyme, which plays a central role in replication of this bacterium's genome (Section 13.2.2). This enzyme, sometimes called the Kornberg polymerase, after its discoverer Arthur Kornberg (Kornberg, 1960), has both the 3′→5′ and 5′→3′ exonuclease activities, which limits it usefulness in DNA manipulation. Its main application is in DNA labeling, as described in Technical Note 4.1. Of the two exonuclease activities, it is the 5′→3′ version that causes most problems when a DNA polymerase is used to manipulate molecules in the test tube. This is because an enzyme that possesses this activity is able to remove nucleotides from the 5′ ends of polynucleotides that have just been synthesized ( Figure 4.8 ). It is unlikely that the polynucleotides will be completely degraded, because the polymerase function is usually much more active than the exonuclease, but some techniques will not work if the 5′ ends of the new polynucleotides are shortened in any way. In particular, DNA sequencing is based on synthesis of new polynucleotides, all of which share exactly the same 5′ end, marked by the primer used to initiate the sequencing reactions. If any nibbling of the 5′ ends occurs, then it is impossible to determine the correct DNA sequence. When DNA sequencing was first introduced in the late 1970s, it made use of a modified version of the Kornberg enzyme called the Klenow polymerase. The Klenow polymerase was initially prepared by cutting the natural E. coli DNA polymerase I enzyme into two segments with a protease. One of these segments retained the polymerase and 3′→5′ exonuclease activities, but lacked the 5′→3′ exonuclease of the untreated enzyme. The enzyme is still often called the Klenow fragment in memory of this old method of preparation, but nowadays it is almost always prepared from E. coli cells whose polymerase gene has been engineered so that the resulting enzyme has the desired properties. But in fact the Klenow polymerase is now rarely used in sequencing and has its major application in DNA labeling (see Technical Note 4.1). This is because an enzyme called Sequenase (see Table 4.1 ), which has superior properties as far a sequencing is concerned, was developed in the 1980s. We will return to the features of Sequenase, and why they make the enzyme ideal for sequencing, in Box 6.1 The E. coli DNA polymerase I enzyme has an optimum reaction temperature of 37 °C, this being the usual temperature of the natural environment of the bacterium, inside the intestines of mammals such as humans. Test-tube reactions with either the Kornberg or Klenow polymerases, and with Sequenase, are therefore incubated at 37 °C, and terminated by raising the temperature to 75 °C or above, which causes the protein to unfold or denature, destroying its activity. This regimen is perfectly adequate for most molecular biology techniques but, for reasons that will become clear in Section 4.3, PCR requires a thermostable DNA polymerase - one that is able to function at temperatures much higher than 37 °C. Suitable enzymes can be obtained from bacteria such as Thermus aquaticus, which live in hot springs at temperatures up to 95 °C, and whose DNA polymerase I enzyme has an optimum working temperature of 72 °C. The biochemical basis of protein thermostability is not fully understood, but probably centers on structural features that reduce the amount of protein unfolding that occurs at elevated temperatures. One additional type of DNA polymerase is important in molecular biology research. This is reverse transcriptase, which is an RNA-dependent DNA polymerase and so makes DNA copies of RNA rather than DNA templates. Reverse transcriptases are involved in the replication cycles of retroviruses (Section 2.4.2), including the human immunodeficiency viruses that cause AIDS, these having RNA genomes that are copied into DNA after infection of the host. In the test tube, a reverse transcriptase can be used to make DNA copies of mRNA molecules. These copies are called complementary DNAs (cDNAs). Their synthesis is important in some types of gene cloning and in techniques used to map the regions of a genome that specify particular mRNAs (Section 7.1.2). 4.1.2. Nucleases A range of nucleases have found applications in recombinant DNA technology ( Table 4.2 ). Some nucleases have a broad range of activities but most are either exonucleases, removing nucleotides from the ends of DNA and/or RNA molecules, or endonucleases, making cuts at internal phosphodiester bonds. Some nucleases are specific for DNA and some for RNA; some work only on double-stranded DNA and others only on single-stranded DNA, and some are not fussy what they work on. We will encounter various examples of nucleases in later chapters when we deal with the techniques in which they used. Only one type of nuclease will be considered in detail here: the restriction endonucleases, which play a central role in all aspects of recombinant DNA technology. Restriction endonucleases enable DNA molecules to be cut at defined positions A restriction endonuclease is an enzyme that binds to a DNA molecule at a specific sequence and makes a double-stranded cut at or near that sequence. Because of the sequence specificity, the positions of cuts within a DNA molecule can be predicted, assuming that the DNA sequence is known, enabling defined segments to be excised from a larger molecule. This ability underlies gene cloning and all other aspects of recombinant DNA technology in which DNA fragments of known sequence are required. There are three types of restriction endonuclease. With Types I and III there is no strict control over the position of the cut relative to the specific sequence in the DNA molecule that is recognized by the enzyme. These enzymes are therefore less useful because the sequences of the resulting fragments are not precisely known. Type II enzymes do not suffer from this disadvantage because the cut is always at the same place, either within the recognition sequence or very close to it ( Figure 4.9 ). For example, the Type II enzyme called EcoRI (isolated from E. coli) cuts DNA only at the hexanucleotide 5′-GAATTC-3′. Digestion of DNA with a Type II enzyme therefore gives a reproducible set of fragments whose sequences are predictable if the sequence of the target DNA molecule is known. Over 2500 Type II enzymes have been isolated and more than 300 are available for use in the laboratory (Brown, 1998). Many enzymes have hexanucleotide target sites, but others recognize shorter or longer sequences ( Table 4.3 ). There are also examples of enzymes with degenerate recognition sequences, meaning that they cut DNA at any of a family of related sites. HinfI (from Haemophilus influenzae), for example, recognizes 5′-GANTC-3′, where ‘N' is any nucleotide, and so cuts at 5′-GAATC-3′, 5′-GATTC-3′, 5′-GAGTC-3′ and 5′-GACTC-3′. Most enzymes cut within the recognition sequence, but a few, such a BsrBI, cut at a specified position outside of this sequence. Restriction enzymes cut DNA in two different ways. Many make a simple double-stranded cut, giving a blunt or flush end; others cut the two DNA strands at different positions, usually two or four nucleotides apart, so that the resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky or cohesive ends because base-pairing between them can stick the DNA molecule back together again ( Figure 4.10A ). Some sticky-end cutters give 5′ overhangs (e.g. Sau3AI, HinfI) whereas others leave 3′ overhangs (e.g. PstI) ( Figure 4.10B ). One feature that is particularly important in recombinant DNA technology is that some pairs of restriction enzymes have different recognition sequences but give the same sticky ends, examples being Sau3AI and BamHI, which both give a 5′-GATC-3′ sticky end even though Sau3AI has a 4-bp recognition sequence and BamHI recognizes a 6-bp sequence ( Figure 4.10C ). Examining the results of a restriction digest After treatment with a restriction endonuclease, the resulting DNA fragments can be examined by agarose gel electrophoresis (see Technical Note 2.1) to determine their sizes. Depending on the concentration of agarose in the gel, fragments between 100 bp and 50 kb can be separated into sharp bands after electrophoresis ( Figure 4.11 ). Fragments less than 150 bp can be separated in a 4% or 5% agarose gel, making it possible to distinguish bands representing molecules that differ in size by just a single nucleotide. With larger fragments, however, it is not always possible to separate molecules of similar size, even in gels of lower agarose concentration. If the starting DNA is long, and so gives rise to many fragments after digestion with a restriction enzyme, then the gel may simply show a smear of DNA because there are fragments of every possible length that all merge together. This is the usual result when genomic DNA is restricted. If the sequence of the starting DNA is known then the sequences, and hence the sizes, of the fragments resulting from treatment with a particular restriction enzyme can be predicted. The band for a desired fragment (for example, one containing a gene) can then be identified, cut out of the gel, and the DNA purified. Even if its size is unknown, a fragment containing a gene or another segment of DNA of interest can be identified by the technique called Southern hybridization, providing that some of the sequence within the fragment is known or can be predicted. The first step is to transfer the restriction fragments from the agarose gel to a nitrocellulose or nylon membrane. This is done by placing the membrane on the gel and allowing buffer to soak through, taking the DNA from the gel to the membrane, where it becomes bound ( Figure 4.12A ). This process results in the DNA bands becoming immobilized in the same relative positions on the surface of the membrane. The next step is to prepare a hybridization probe, which is a labeled DNA molecule whose sequence is complementary to the target DNA that we wish to detect. The probe could, for example, be a synthetic oligonucleotide whose sequence matches part of an interesting gene. Because the probe and target DNAs are complementary, they can base-pair or hybridize, the position of the hybridized probe on the membrane being identified by detecting the s