Molecular Biotechnology: Principles and Applications - Glick, B. R., Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Recombinant DNA Technology
Restriction Endonucleases

Recombinant DNA technology (also known as molecular cloning or Introduction/32.html">Genetic Engineering) is a set of experimental Procedures that enables The transfer of genetic material (deoxyribonucleic acid, DNA) from one Organism to another. While there is no single, universal set of techniques for this, recombinant DNA experiments most commonly follow the Procedure outlined below (Fig. 4.1).

✵ Native DNA (cloned DNA, insert DNA, target DNA, foreign DNA) is extracted from the donor organism—the source of the desired genes—subjected to Enzymatic Hydrolysis (cleaved, cut), and joined (ligated, spliced) with another DNA molecule (a cloning vector) to form a new recombinant molecule (the cloning vector–insert DNA construct).

✵ This construct is introduced into a host Cell (recipient), where it replicates and is passed on to progeny. This process is known as transformation.

✵ Cells carrying the recombinant DNA (transformed cells) are identified and selected.

✵ A specific protein product synthesized by the host cells is obtained, confirming the successful cloning of the target Gene.

The Development of recombinant DNA technology was made possible by numerous breakthroughs in molecular biology, nucleic acid enzymology, and the Molecular Genetics of bacterial Viruses and bacterial extrachromosomal elements (Plasmids). The construction of recombinant molecules relies on a whole arsenal of Enzymes—indispensable and essential tools for virtually every step of this highly complex process. Chief among these are restriction enzymes (Restriction Endonucleases, restrictionases), which recognize and cleave specific nucleotide sequences within double-stranded DNA molecules.

In molecular cloning, it is crucial that the Cleavage of both donor and vector DNA occurs at strictly defined sites, yielding a discrete and reproducible set of fragments. If chromosomal DNA is forced through a small-gauge syringe needle or treated with ultrasound, fragments ranging from 0.3 to 5 kbp in length are obtained. Unfortunately, these simple mechanical operations cause random breaks in double-stranded molecules, meaning that every preparation of DNA yields a completely new set of fragments. Molecular cloning only became feasible with the isolation of highly specific bacterial enzymes that recognize specific base sequences in double-stranded DNA and cleave both strands. These enzymes are known as type II restriction endonucleases. One of the first type II restriction endonucleases was isolated from the bacterium Escherichia coli and named EcoRI. This enzyme recognizes a DNA site containing a specific palindromic sequence (an inverted sequence, identical in both strands when read in the 5'→3' direction) of six Base Pairs and introduces a break between the guanine and adenine residues in each strand (Fig. 4.2), cleaving the bond between the 3'-carbon oxygen atom of one nucleotide sugar residue and the phosphate group attached to the 5'-carbon atom of the adjacent nucleotide sugar residue. The breaks in the DNA strands are offset from one another, resulting in single-stranded complementary ends with four-nucleotide "tails" (sticky ends). Each single-stranded "tail" terminates in a 5'-phosphate group, while the 3'-hydroxyl group of the opposite strand is somewhat recessed.

Class="center">

Fig. 4.1. Recombinant DNA Cloning. Donor DNA is cleaved with a restriction endonuclease and inserted into a cloning vector. The resulting construct is introduced into a population of host cells, those containing the recombinant DNA are identified, and they are cultured. If necessary, expression of the cloned gene can be induced in the host cells to yield the encoded protein.

Fig. 4.2. Cleavage of a short DNA fragment by the type II restriction endonuclease EcoRI, yielding sticky ends. Arrows indicate the bonds cleaved within the sugar-phosphate backbone. S stands for deoxyribose, P for phosphate group, and OH for hydroxyl group. The sequence recognized by EcoRI is highlighted by a dashed line.

Fig. 4.3. Cleavage of a short DNA fragment by the type II restriction enzyme HindII, yielding blunt ends. Arrows indicate the bonds cleaved within the sugar-phosphate backbone. Letter designations are the same as in Fig. 4.2. The sequence recognized by the HindII restriction enzyme is highlighted by a dashed line.

In addition to EcoRI, hundreds of other type II restriction endonucleases have been isolated from bacterial cells. These endonucleases are named following the same principle as EcoRI: the genus of the microorganism is designated by a capital letter, and the species by the first two lowercase letters, while the strain is usually omitted. Roman numerals indicate the chronological order of discovery of that particular restriction enzyme from the given microorganism. For example, HpaI and HpaII are the First and Second type II restriction endonucleases isolated from Haemophilus parainfluenzae, respectively.

The palindromic sequences recognized by type II restriction endonucleases where DNA cleavage occurs are called recognition sites. Besides restriction enzymes that hydrolyze (cleave) the polynucleotide chain to produce sticky ends, there are restriction enzymes that introduce breaks directly opposite each other in both strands, generating DNA fragments with blunt ends (Fig. 4.3). Recognition sites may consist of four, five, six, eight, or more base pairs (Table 4.1). The frequency of a site within a DNA molecule depends on the length of its recognition sequence; in most cases, restriction enzymes that recognize tetra- and hexanucleotides are used.

Type II restriction endonucleases play a pivotal role in Gene cloning.

Table 4.1. Nucleotide sequences recognized by selected restriction enzymes

Digestion of a DNA sample with a specific restriction enzyme always yields the same set of fragments, provided that cleavage goes to completion at all recognition sites. By using multiple restriction enzymes—digesting the DNA first with each enzyme individually and then with combinations thereof—one can construct a physical map of the DNA, i.e., determine the linear order of restriction sites along the molecule. Determining the sizes of the resulting fragments by gel Electrophoresis allows the positions of the restriction sites to be mapped (Box 4.1). Fig. 4.4A shows the sizes of fragments obtained by digesting DNA with various restriction enzymes and their mixtures. These data indicate that this DNA region contains two sites each for BamHI and EcoRI. To construct a restriction map, the fragment sizes obtained from single digests must be compared with those from double digests. The results of such a comparison are presented in Fig. 4.4B. When DNA is hydrolyzed with each of the two restriction enzymes (EcoRI and BamHI) separately and each yields three fragments, it means the original DNA fragment contained two recognition sites for each of the restriction enzymes used. The 300 bp fragment generated by EcoRI digestion is not cleaved by the EcoRI/BamHI double digest, unlike the 850 bp and 500 bp EcoRI fragments. This indicates that the two EcoRI sites are located 300 bp apart with no BamHI site between them, whereas the 850 bp and 500 bp EcoRI fragments each contain one BamHI site. The 950 bp fragment produced by BamHI digestion is cleaved by EcoRI into three fragments during double digestion (250 + 300 + 400 = 950 bp). This means that the two BamHI sites are located 250 bp and 400 bp away on opposite sides of the EcoRI sites. BamHI cleaves the 850 bp EcoRI fragment into 250 bp and 600 bp fragments; since one of the EcoRI sites lies 250 bp away from the BamHI site, the 600 bp fragment must contain one of the ends of the original DNA molecule. Furthermore, we see that BamHI cleaves the 500 bp EcoRI fragment into two fragments of 100 bp and 400 bp, and one of the EcoRI sites is separated from the BamHI site by 400 bp, meaning that the 100 bp fragment must contain the other end of the original molecule. The map in Fig. 4.4B illustrates the clear correlation between the positions of restriction sites and the sizes of the fragments produced by each digest.

Fig. 4.4. Restriction site mapping. A. Results of gel electrophoresis of DNA fragments generated by digestion with the indicated enzymes. Purified DNA was hydrolyzed with EcoRI and BamHI individually and then with a mixture of both, subjected to gel electrophoresis, and visualized by ethidium bromide staining. Numbers to the left of the horizontal bands indicate fragment lengths in base pairs. B. Restriction map constructed from electrophoretic data. Numbers represent the distances between the recognition sites of the respective enzymes.

Cleavage by restriction endonucleases has yet another application. When two different DNA samples are treated with the same restriction enzyme to generate fragments with sticky ends and these samples are then mixed, new gene combinations—recombinant DNAs—can form via complementary base pairing between the sticky ends of fragments from the different samples (Fig. 4.5). However, restriction enzymes alone are not sufficient to accomplish molecular cloning. First, the Hydrogen Bonds between the four bases forming the sticky ends are not strong enough to hold two joined DNA fragments together. A tool is needed to seal the nick in the sugar-phosphate backbone of the molecule, i.e., to restore the bond between the 3'-hydroxyl terminal group of one strand and the 5'-phosphate group of the other. T4 bacteriophage DNA ligase serves this purpose. This enzyme catalyzes The formation of phosphodiester bonds between the ends of polynucleotide chains that are already held together by sticky-end base pairing. In addition, T4 DNA ligase joins blunt ends that are brought into close proximity after the fragments bind to the enzyme (Fig. 4.6). Second, joining different DNA molecules is useless in itself unless the newly formed combinations (recombinant DNAs) can replicate within the host cell. Thus, while one part of the recombinant DNA molecule carries the desired gene to be cloned, another part must contain the information necessary for the recombinant DNA to replicate in the host cell. Cloning vectors are used to solve this problem. Third, DNA Restriction produces a mixture of diverse fragments, and following their ligation with vector DNA, a multitude of different combinations are generated. It is therefore essential to be able to identify those recipient cells that contain DNA with the desired nucleotide sequence. Various screening systems are employed for this purpose.

Fig. 4.5. Annealing of complementary sticky ends of fragments generated by digestion of two different DNA samples with the BamHI restriction endonuclease. The four fragments shown in the figure can join together to form six different DNA molecules (not all possible combinations are shown). The fragments are held together by hydrogen bonds formed between the four bases of the sticky ends, but these bonds are not strong enough to keep the molecules stable in solution for a prolonged period.