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

Particularly important to recombinant DNA technology is a set of enzymes (Table 9–1) made available through decades of research on nucleic acid metabolism. Two classes of enzymes lie at the heart of the general approach to generating and propagating a recombinant DNA molecule (Fig. 9–1). First, restriction endonucleases (also called restriction enzymes) recognize and cleave DNA at specific DNA sequences (recognition sequences or restriction sites) to generate a set of smaller fragments. Second, the DNA fragment to be cloned can be joined to a suitable cloning vector by using DNA ligases to link the DNA molecules together. The recombinant vector is then introduced into a host cell, which amplifies the fragment in the course of many genera tions of cell division. Restriction endonucleases are found in a wide range of bacterial species. Werner Arber discovered in the early 1960s that their biological function is to recognize and cleave foreign DNA (the DNA of an infecting virus, for example); such DNA is said to be restricted. In the host cell’s DNA, the sequence that would be recognized

FIGURE 9–1 Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

by its own restriction endonuclease is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase. The restriction endonuclease and the corresponding methylase are sometimes referred to as a restriction-modification system. There are three types of restriction endonucleases, designated I, II, and III. Types I and III are generally large, multisubunit complexes containing both the endonuclease and methylase activities. Type I restriction endonucleases cleave DNA at random sites that can be more than 1,000 base pairs (bp) from the recognition sequence. Type III restriction endonucleases cleave the DNA about 25 bp from the recognition sequence. Both types move along the DNA in a reaction that requires the energy of ATP. Type II restriction endonucleases, first isolated by Hamilton Smith in 1970, are simpler, re quire no ATP, and cleave the DNA within the recognition sequence itself. The extraordinary utility of this group of restriction endonucleases was demonstrated by Daniel Nathans, who first used them to develop novel methods for mapping and analyzing genes and genomes. Thousands of restriction endonucleases have been discovered in different bacterial species, and more than 100 different DNA sequences are recognized by one or more of these enzymes. The recognition sequences are usually 4 to 6 bp long and palindromic (see Fig. 8–20). Table 9–2 lists sequences recognized by a few type II restriction endonucleases. In some cases, the interaction between a restriction endonuclease and its target sequence has been elucidated in exquisite molecular de tail; for example, Figure 9–2 shows the complex of the type II restriction endonuclease EcoRV and its target sequence.

Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends (Fig. 9–3a), because they can base-pair with each other or with complementary sticky ends of other DNA fragments. Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9–3b).

The average size of the DNA fragments produced by cleaving genomic DNA with a restriction endonuclease depends on the frequency with which a particular re striction site occurs in the DNA molecule; this in turn depends largely on the size of the recognition sequence. In a DNA molecule with a random sequence in which all four nucleotides were equally abundant, a 6 bp sequence recognized by a restriction endonuclease such as BamHI would occur on average once every 4⁶ (4,096) bp, assuming the DNA had a 50%G≡C content. Enzymes that recognize a 4 bp sequence would produce smaller DNA fragments from a random-sequence DNA molecule; a recognition sequence of this size would be expected to occur about once every 4⁴ (256) bp. In natural DNA molecules, particular recognition sequences tend to occur less frequently than this because nucleotide sequences in DNA are not random and the four nucleotides are not equally abundant. In laboratory experiments, the average size of the fragments produced by restriction endonuclease cleavage of a large DNA can be increased by simply terminating the reaction before completion; the result is called a partial digest. Fragment size can also

be increased by using a special class of endonucleases called homing endonucleases (see Fig. 26–34). These recognize and cleave much longer DNA sequences (14 to 20 bp). Once a DNA molecule has been cleaved into fragments, a particular fragment of known size can be enriched by agarose or acrylamide gel electrophoresis or by HPLC (pp. 92, 90). For a typical mammalian genome, however, cleavage by a restriction endonuclease usually yields too many different DNA fragments to permit isolation of a particular fragment by electrophoresis or HPLC. A common intermediate step in the cloning of a specific gene or DNA segment is the construction of a DNA library (as described in Section 9.2). After the target DNA fragment is isolated, DNA lig ase can be used to join it to a similarly digested cloning vector—that is, a vector digested by the same restriction endonuclease; a fragment generated by EcoRI, for example, generally will not link to a fragment generated by BamHI. As described in more detail in Chapter 25 (see Fig. 25–16), DNA ligase catalyzes the formation of new phosphodiester bonds in a reaction that uses ATP

FIGURE 9–2 Interaction of EcoRV restriction endonuclease with its target sequence. (a) The dimeric EcoRV endonuclease (its two subunits in light blue and gray) is bound to the products of DNA cleavage at the sequence recognized by the enzyme. The DNA backbone is shown in two shades of blue to distinguish the segments separated by cleavage (PDB ID 1RVC). (b) In this view, showing just the DNA, the DNA segment has been turned 180. The enzyme creates blunt ends; the cleavage points appear staggered on the two DNA strands because the DNA is kinked. Bound magnesium ions (orange) play a role in catalysis of the cleavage reaction.

FIGURE 9–3 Cleavage of DNA molecules by restriction endonucleases. Restriction endonucleases recognize and cleave only specific sequences, leaving either (a) sticky ends (with protruding single strands) or (b) blunt ends. Fragments can be ligated to other DNAs, such as the cleaved cloning vector (a plasmid) shown here. This reaction is facilitated by the annealing of complementary sticky ends. Ligation is less efficient for DNA fragments with blunt ends than for those with complementary sticky ends, and DNA fragments with different (noncomplementary) sticky ends generally are not ligated. (c) A synthetic DNA fragment with recognition sequences for several restriction endonucleases can be inserted into a plasmid that has been cleaved by a restriction endonuclease. The insert is called a linker; an insert with multiple restriction sites is called a polylinker.

or a similar cofactor. The base-pairing of complementary sticky ends greatly facilitates the ligation reaction (Fig. 9–3a). Blunt ends can also be ligated, albeit less efficiently. Researchers can create new DNA sequences by inserting synthetic DNA fragments (called linkers) between the ends that are being ligated. Inserted DNA fragments with multiple recognition sequences for re striction endonucleases (often useful later as points for inserting additional DNA by cleavage and ligation) are called polylinkers (Fig. 9–3c).

The effectiveness of sticky ends in selectively join ing two DNA fragments was apparent in the earliest recombinant DNA experiments. Before restriction endo nucleases were widely available, some workers found they could generate sticky ends by the combined action of the bacteriophage exonuclease and terminal transferase (Table 9–1). The fragments to be joined were given complementary homopolymeric tails. Peter Lobban and Dale Kaiser used this method in 1971 in the first experiments to join naturally occurring DNA fragments.

Similar methods were used soon after in the laboratory of Paul Berg to join DNA segments from simian virus 40 (SV40) to DNA derived from bacteriophage , thereby creating the first recombinant DNA molecule with DNA segments from different species.

اخر الاخبار

اخبار العتبة العباسية المقدسة

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

المزيد

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

المزيد

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

المزيد

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:- Cloning Vectors Allow Amplification of Inserted DNA Segments

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

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