DNA Cloning: The Basics:- Cloning Vectors Allow Amplification of Inserted DNA Segments

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast.

Plasmids are circular DNA molecules that replicate separately from the host chromosome. Natu rally occurring bacterial plasmids range in size from 5,000 to 400,000 bp. They can be introduced into bacterial cells by a process called transformation. The cells (generally E. coli) and plasmid DNA are incubated together at 0 C in a calcium chloride solution, then subjected to a shock by rapidly shifting the temperature to 37 to 43 C. For reasons not well understood, some of the cells treated in this way take up the plasmid DNA. Some species of bacteria are naturally competent for DNA uptake and do not require the calcium chloride treatment. In an alternative method, cells incubated with the plasmid DNA are subjected to a high-voltage pulse. This approach, called electroporation, transiently renders the bacterial membrane permeable to large molecules.

Regardless of the approach, few cells actually take up the plasmid DNA, so a method is needed to select those that do. The usual strategy is to use a plasmid that includes a gene that the host cell requires for growth under specific conditions, such as a gene that confers resistance to an antibiotic. Only cells transformed by the recombinant plasmid can grow in the presence of that antibiotic, making any cell that contains the plasmid “selectable” under those growth conditions. Such a gene is called a selectable marker. Investigators have developed many different plasmid vectors suitable for cloning by modifying naturally occurring plasmids. The E. coli plasmid pBR322 offers a good example of the features useful in a cloning vector (Fig. 9–4):

1. pBR322 has an origin of replication, ori, a sequence where replication is initiated by cellular enzymes (Chapter 25). This sequence is required to propagate the plasmid and maintain it at a level of 10 to 20 copies per cell.

 2. The plasmid contains two genes that confer resistance to different antibiotics (tet^R, amp^R), allowing the identification of cells that contain the intact plasmid or a recombinant version of the plasmid (Fig. 9–5).

3. Several unique recognition sequences in pBR322 (PstI, EcoRI, BamHI, SalI, PvuII) are targets for different restriction endonucleases, providing sites where the plasmid can later be cut to insert foreign DNA.

4. The small size of the plasmid (4,361 bp) facilitates its entry into cells and the biochemical manipulation of the DNA.

Transformation of typical bacterial cells with purified DNA (never a very efficient process) becomes less successful as plasmid size increases, and it is difficult to clone DNA segments longer than about 15,000 bp when plasmids are used as the vector.

Bacteriophages λ has a very efficient mechanism for delivering its 48,502 bp of DNA into a bacterium, and it can be used as a vector to clone somewhat larger DNA segments (Fig. 9–6). Two key features contribute to its utility:

1. About one-third of the genome is nonessential and can be replaced with foreign DNA.

2. DNA is packaged into infectious phage particles only if it is between 40,000 and 53,000 bp long, a constraint that can be used to ensure packaging of recombinant DNA only.

FIGURE 9–5 Use of pBR322 to clone and identify foreign DNA in E. coli.

FIGURE 9–6 Bacteriophage λ cloning vectors. Recombinant DNA methods are used to modify the bacteriophage genome, removing the genes not needed for phage production and replacing them with “filler” DNA to make the phage DNA large enough for packaging into phage particles. As shown here, the filler is replaced with foreign DNA in cloning experiments. Recombinants are packaged into viable phage particles in vitro only if they include an appropriately sized foreign DNA fragment as well as both of the essential λ DNA end fragments.

Researchers have developed bacteriophage vectors that can be readily cleaved into three pieces, two of which contain essential genes but which together are only about 30,000 bp long. The third piece, “filler” DNA, is discarded when the vector is to be used for cloning, and additional DNA is inserted between the two essential segments to generate ligated DNA molecules long enough to produce viable phage particles. In effect, the packaging mechanism selects for recombinant viral DNAs.

Bacteriophage λ vectors permit the cloning of DNA fragments of up to 23,000 bp. Once the bacteriophage λ fragments are ligated to foreign DNA fragments of suit able size, the resulting recombinant DNAs can be pack aged into phage particles by adding them to crude bacterial cell extracts that contain all the proteins needed to assemble a complete phage. This is called in vitro packaging (Fig. 9–6). All viable phage particles will contain a foreign DNA fragment. The subsequent trans mission of the recombinant DNA into E. coli cells is highly efficient.

Bacterial Artificial Chromosomes (BACs) Bacterial artificial chromosomes are simply plasmids designed for the cloning of very long segments (typically 100,000 to 300,000 bp) of DNA (Fig. 9–7). They generally include selectable markers such as resistance to the antibiotic chloramphenicol (Cm^R), as well as a very stable origin of replication (ori) that maintains the plasmid at one or two copies per cell. DNA fragments of several hundred thousand base pairs are cloned into the BAC vector. The large circular DNAs are then introduced into host bacteria by electroporation. These procedures use host bacteria with mutations that compromise the structure of their cell wall, permitting the uptake of the large DNA molecules.

Yeast Artificial Chromosomes (YACs) E. coli cells are by no means the only hosts for genetic engineering. Yeasts are particularly convenient eukaryotic organisms for this work. As with E. coli, yeast genetics is a well-developed discipline. The genome of the most commonly used yeast, Saccharomyces cerevisiae, contains only 14x10⁶bp  (a simple genome by eukaryotic standards, less than four times the size of the E. coli chromosome), and its entire sequence is known. Yeast is also very easy to maintain and grow on a large scale in the laboratory. Plasmid vectors have been constructed for yeast, employing the same principles that govern the use of E. coli vectors described above. Convenient methods are now available for moving DNA into and out of yeast cells, facilitating the study of many aspects of eukaryotic cell biochemistry. Some recombinant plasmids incorporate multiple replication origins and other elements that al low them to be used in more than one species (for ex ample, yeast or E. coli). Plasmids that can be propagated in cells of two or more different species are called shuttle vectors.

FIGURE 9–7 (above right) Bacterial artificial chromosomes (BACs) as cloning vectors. The vector is a relatively simple plasmid, with a repli cation origin (ori) that directs replication. The par genes, derived from a type of plasmid called an F plasmid, assist in the even distribution of plasmids to daughter cells at cell division. This increases the likelihood of each daughter cell carrying one copy of the plasmid, even when few copies are present. The low number of copies is useful in cloning large segments of DNA because it limits the opportunities for unwanted recombination reactions that can unpredictably alter large cloned DNAs over time. The BAC includes selectable markers. A lacZ gene (required for the production of the enzyme β -galactosidase) is situated in the cloning region such that it is inactivated by cloned DNA inserts. Introduction of recombinant BACs into cells by electroporation is promoted by the use of cells with an altered (more porous) cell wall. Recombinant DNAs are screened for resistance to the antibiotic chloramphenicol (Cm^R). Plates also contain a substrate for β -galactosidase that yields a colored product. Colonies with active β -galactosidase and hence no DNA insert in the BAC vector turn blue; colonies without β-galactosidase activity—and thus with the desired DNA inserts—are white.

Research work with large genomes and the associated need for high-capacity cloning vectors led to the development of yeast artificial chromosomes (YACS; Fig. 9–8). YAC vectors contain all the elements needed to maintain a eukaryotic chromosome in the yeast nucleus: a yeast origin of replication, two selectable markers, and specialized sequences (derived from the telomeres and centromere, regions of the chromo some discussed in Chapter 24) needed for stability and proper segregation of the chromosomes at cell division. Before being used in cloning, the vector is propagated as a circular bacterial plasmid. Cleavage with a restriction endonuclease (BamH1 in Fig. 9–8) removes a length of DNA between two telomere sequences (TEL), leaving the telomeres at the ends of the linearized DNA. Cleavage at another internal site (EcoRI in Fig. 9–8) di vides the vector into two DNA segments, referred to as vector arms, each with a different selectable marker. The genomic DNA is prepared by partial digestion with restriction endonucleases (EcoRI in Fig. 9–8) to obtain a suitable fragment size. Genomic fragments are then separated by pulsed field gel electrophoresis, a variation of gel electrophoresis (see Fig. 3–19) that allows the separation of very large DNA segments. The DNA fragments of appropriate size (up to about 2x10⁶ bp) are mixed with the prepared vector arms and ligated. The ligation mixture is then used to trans form treated yeast cells with very large DNA molecules. Culture on a medium that requires the presence of both selectable marker genes ensures the growth of only those yeast cells that contain an artificial chromosome with a large insert sandwiched between the two vector arms (Fig. 9–8). The stability of YAC clones increases with size (up to a point). Those with inserts of more than 150,000 bp are nearly as stable as normal cellular chromosomes, whereas those with inserts of less than 100,000 bp are gradually lost during mitosis (so generally there are no yeast cell clones carrying only the two vector ends ligated together or with only short inserts). YACs that lack a telomere at either end are rapidly degraded.

FIGURE 9–8 Construction of a yeast artificial chromosome (YAC). A YAC vector includes an origin of replication (ori), a centromere (CEN), two telomeres (TEL), and selectable markers (X and Y). Digestion with BamH1 and EcoRI generates two separate DNA arms, each with a telomeric end and one selectable marker. A large segment of DNA (e.g., up to 2x10⁶  bp from the human genome) is ligated to the two arms to create a yeast artificial chromosome. The YAC transforms yeast cells (prepared by removal of the cell wall to form spheroplasts), and the cells are selected for X and Y; the surviving cells propagate the DNA insert.

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المجمع العلمي ينظّم سفرة دينية لطلبة مشاريعه القرآنية في كربلاء

عبر مجلّة (تراث الجنوب) المحكّمة.. قسم شؤون المعارف يوثق إرث مدن جنوب العراق

في ذكرى تأسيسه.. متحف الكفيل يستعرض مسيرته في حفظ التراث الإنساني والإسلامي

قسم الشؤون الفكرية وجامعة ميسان يبحثان تعزيز التعاون العلمي المشترك

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الآخبار الصحية

السجائر الإلكترونية والإقلاع عن التدخين.. نتائج متباينة تثير الجدل العلمي

مصر تحقق إنجازاً دولياً جديداً في منظمة الصحة العالمية

دراسة: السباحة فعّالة أكثر من الجري في الحفاظ على صحة القلب

عامل خطر في بداية الحمل يؤخر تطور الكلام والمهارات الحركية لدى الرضع

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الآخبار التكنلوجية

أحلامك ليست عشوائية.. إليك ما يفعله دماغك أثناء نومك

روسيا.. العلماء يضاعفون الاستقرار الحراري للألواح الشمسية ثلاث مرات

علماء يكشفون سر سرعة الدلافين في السباحة

سلمي أو دموي.. هكذا تنتقل السلطة في مجتمع الجرذان

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مواضيع ذات صلة

From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins

DNA Cloning: The Basics:- Expression of Cloned Genes Produces Large Quantities of Protein

DNA Cloning: The Basics:- Specific DNA Sequences Are Detectable by Hybridization

DNA Cloning: The Basics:- Restriction Endonucleases and DNA Ligase Yield Recombinant DNA

Other Functions of Nucleotides:- Adenine Nucleotides Are Components of Many Enzymes Cofactors

Other Functions of Nucleotides:- Nucleotides Carry Chemical Energy in Cells