REVIEW
DNA Technology Rena S. Wong, MD, Edward Passaro, Jr., MD, Los Angeles, California With the availability of DNA recombinant technology and DNA and RNA sequencing techniques, diseases can now be studied and treated at a molecular level, while unlimited quantities of a pure protein product can be produced through gene cloning. Before the end of this century, gene therapy will be used to repair genetic defects. This article explains these advances in genetic technology and suggests their relevance in clinical problems and practice.
ith the rapid advances in DNA recombinant technology and DNA and RNA sequencing techW niques, scientists now have the tools to study and treat disease at a molecular level. Gene cloning enables production of unlimited quantities of a pure protein product. Human insulin [1], growth hormone [2], interferon [3,4], and factor VIII [5] produced by genetically engineered bacteria have been used clinically in the treatment of diabetes mellitus, growth hormone deficiency, cancer, and hemophilia. The ability to produce in quantity proteins such as somatostatin [6], or interferon, which are normally present in minute amounts, has helped elucidate their normal physiologic structures and functions. DNA technology has allowed us to discover the molecular basis of many human diseases such as sickle cell anemia [7], thalassemia [8,9], and some cancers [10,11]. DNA mapping and sequencing have revealed that viral oncogenes show remarkable similarity to normal cellular genes and, in fact, may be derived from them [12]. This new technology is being used to sequence the entire human genome [13]. Gene therapy, or the introduction of a therapeutic gene to repair a genetic defect, will become a reality in this century [14,15]. This rapid growth in genetic technology has also resuited in a widening of the intellectual gap between the clinician and the basic scientist. In order to apply the new technology fully, it must also be fully understood. This brief review highlights the advances in genetic technology and suggests their relevance to surgical problems and practice.
DEOXYRIBONUCLEIC ACID Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins make up the structural and functional elements of a cell. DNA contains the genetic information of a cell. It is composed of long, linear sequences of nueleotide bases called adenine (A), thymine (T), guanine (G), and cytosine (C). DNA has a helical, doublestranded structure [16]. Adenine is always paired opposite thymine, guanine opposite cytosine. This is base pairing. Thus, the nucleotide sequence of one strand can be deduced from the other, complementary strand. Two strands of DNA can be separated or denatured by high temperature, but will rejoin to re-form the double helix as the temperature drops. The process by which one strand of DNA (or RNA) finds its complementary strand is dependent on base pairing and is called hybridization. Hybridization and base pairing allow accurate copies of DNA to be made during replication and are also important in DNA molecular cloning [17]. RIBONUCLEIC ACID Ribonucleic acid (RNA) comes in three forms: messenger R N A (mRNA), ribosomal R N A (rRNA), and transfer RNA (tRNA) [18]. Messenger RNA, like DNA, is a polymer of nucleotides. It differs from DNA in that it exists in a single-stranded structure and that ribose replaces deoxyribose. In addition, uracil (U) replaces thymine (T). Like thymine, uracil is complementary to adenine. Messenger RNA is formed opposite a DNA strand or template by the enzyme RNA polymerase; the order of nucleotides in the m R N A is complementary to the order of nucleotides in the DNA [19]. After m R N A is formed in the nucleus, it enters the cytoplasm and associates with ribosomes in the endoplasmic reticulum, the site of protein synthesis [20]. There, the sequence of nucleotides is decoded, and the corresponding protein is made. Thus, m R N A carries the message from DNA in the nucleus to the ribosome in the cytoplasm. Ribosomal RNA is found only in ribosomes and gives them their structural and functional capacities. Transfer RNA is the adapter molecule in protein synthesis; it decodes the mRNA.
PROTEINS Proteins are the working molecules of an organism. They provide structure (collagen), alter cellular permeability (calcium channels), recognize messenger molecules (receptors), enable motion (flagella), and regulate gene expression (repressor protein). Enzymes are proteins that are catalysts for a cell's chemical reactions. Antibodies are specialized proteins that function as high Fromthe Departmentof Surgery,Divisionof GeneralSurgery,UCLA Schoolof Medicine,and the SurgicalService,VeteransAdministration specificity recognition molecules in the immune system. MedicalCenter,WestLos Angeles,Los Angeles,California. Proteins are linear chains of amino acids. There are 20 Requests for reprints shouldbe addressedto EdwardPassaro,Jr., amino acids, and their order in a protein determines its MD, SurgicalService(W112),VeteransAdministrationMedicalCenstructure and function. All proteins are coded for by ter, Los Angeles,California90073. ManuscriptsubmittedJanuary24, 1989,and acceptedFebruary9, genes in the DNA. The protein corresponding to a gene is called the gene product. 1989. 610
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Protein synthesis: Although all the genetic information of a cell is stored in its DNA, it is the proteins that actually perform all cellular functions. Thus, a method by which the information stored in the DNA is translated into different proteins is required. Protein synthesis begins in the nucleus. The enzyme RNA polymerase copies the DNA gene into a linear, complementary mRNA strand. The copying of DNA into RNA is called transcription [19]. Messenger RNA strands then associate with the ribosomes in the cytoplasm. Messenger RNA is composed of only four nucleotides (A,U,G,C); proteins are composed of 20 amino acids. A group of three consecutive nucleotides codes for each amino acid. Each of these triplets is called a codon [21]. There are 64 (4 to the third power) possible codons, and thus some redundancy, with one amino acid corresponding to several different codons. Other codons serve as start and stop codons [22-25]. The correlation of codons to amino acids is called the genetic code and is universal in all organisms [26]. A bacterium such as Escherchia coB, therefore, can decode the message from a human gene and make the human gene product (e.g., human insulin). Because of the genetic code, if either the DNA or RNA sequence is known, the protein sequence is also known. Conversely, if a protein has been sequenced, the possible DNA sequences that code for that protein can be deduced. The first human protein made by E. coli through genetic engineering was somatostatin, which is 14 amino acids in length. The gene was actually synthesized, one nucleotide at a time, by deduction of its nucleotide sequence from its known amino acid sequence, and then cloned into E. coli in 1977 [6]. Messenger RNA is decoded by tRNA. Transfer RNAs are small molecules with two functional ends [27]. One end contains three consecutive nucleotides that are complementary to the mRNA codons. These are the anticodons. An amino acid is attached at the other end of a tRNA molecule. Protein synthesis occurs in the following steps: (1) mRNA is transcribed in the nucleus; (2) mRNA associates with the ribosomes where tRNA molecules recognize the codons and base pairing between complementary codon-anticodons occurs; (3) the individual amino acids carried by each tRNA are joined by chemical bonding [28]. The order of amino acids is determined by the order of nucleotides in the mRNA, which is ultimately determined by the sequence in the DNA. The process by which the nucleotide sequence of a mRNA is decoded into the amino acid sequence of the corresponding protein is called translation. Translation of a single mRNA by multiple ribosomes results in the formation of many identical proteins. Similarly, during transcription, multiple mRNA copies can be made of a single DNA gene. A gene in the DNA can therefore be expressed in multiple copies, although the gene itself is read or copied only several times. GENE CLONING A clone is a large number of identical molecules or cells with a common ancestor. Cloning of a gene requires isolation, amplification, and identification of that gene. The techniques used for the biologic separation and pro-
TECHNOLOGY
duction of a piece of DNA (gene) are collectively called recombinant DNA technology. Each chromosome is a long molecule of DNA with genes arranged linearly on it. Each gene is (generally) present in only one copy. Therefore, a single gene represents only a small fraction of the total DNA. Since all genes are composed of the same four nucleotides, they are chemically indistinguishable. Biologic isolation is therefore necessary. In order to separate a particular gene, DNA must be cut into small, gene-length fragments, which can then be amplified. This is done by restriction enzymes [29,30]. A restriction enzyme recognizes and cleaves DNA at certain nucleotide sequences called restriction sites. Restriction enzymes are ordinarily found in bacteria and "restrict" entry of foreign DNA into a bacterial cell by cutting it into pieces. The bacterium protects its own DNA from destruction by modifying each of its own restriction sites to prevent cleaving by the restriction enzyme [31]. For example [32], the enzyme Eco RI recognizes the DNA sequence -GAATTC- and cleavesit: -G AATTC-CTTAAG-CTTAA GThe cut, unpaired, single-stranded ends are able to recognize each other, or any other piece of DNA cut by the same enzyme, by base pairing. They can be rejoined using the enzyme DNA ligase [33]. They are the sticky ends. Incubation of chromosomal (genomal) DNA with a restriction enzyme cleaves the DNA into multiple, small fragments suitable for cloning. Since a restriction enzyme always cuts at the same places, the pattern of DNA fragments is reproducible and is characteristic of a particular chromosome and restriction enzyme. This is restriction enzyme mapping. Restriction enzyme mapping of certain areas of human DNA called mini-satellite regions results in a DNA fragment pattern that is different in every person. This is called restriction fragment-length polymorphism (RFLP) [34]. RFLP, or DNA fingerprinting, can accurately identify the individual origin of any tissue or body fluid [35]. It is useful in forensic medicine, where DNA fingerprinting has been used as evidence in rape cases [36]. It is used for genetic screening and in the prenatal diagnosis of various genetic disorders such as muscular dystrophy [37], sickle cell anemia [38], and thalassemia
[391. Restriction enzymes are used in DNA cloning for cutting long pieces of DNA into segments that can be separated. These pieces of DNA with sticky ends must then be amplified. This is accomplished using cloning vectors. A vector is a piece of DNA capable of independent growth. The two most commonly used cloning vectors are bacterial plasmids [40,41] and phage lambda [42]. A phage is a virus that infects bacteria. A plasmid is a circular piece of extrachromosomal DNA found in bacteria that multiplies independently of chromosomal DNA. Up to 3,000 copies of a plasmid can be present in a single bacterium. Often a plasmid contains genes encoding antibiotic resistance [43]. Bacteria with plasmids can then be selected by growing them in media containing
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antibiotics. If a plasmid is cut with a restriction enzyme and then incubated with DNA fragments cut with the same enzyme, the complementary sticky ends will join and a new plasmid containing both plasmid and foreign D N A will be formed. Combined foreign (e.g., human) and plasmid (bacterial) DNA is recombinant DNA [44]. If this recombinant DNA is introduced into bacteria, multiple copies of both plasmid and foreign DNA will be made, resulting in a D N A clone. Plasmids are useful then for amplifying the number of copies of a single piece of DNA. It is difficult for technical reasons, however, to screen large numbers of bacteria for the plasmid containing the desired gene. This is done more efficiently using the vector phage lambda. Phage lambda is a commonly used vector. It has the advantage over plasmids in that larger pieces of DNA can be cloned and that screening of a large number of clones is possible. Foreign pieces of DNA are recombined with lambda phage using DNA ligase. The phage are then spread over a bacterial lawn, an agar plate with a thin layer of bacteria covering it. The phage then infect the bacteria, multiply, and then lyse the bacteria, forming clear zones called plaques [45]. Each plaque corresponds to a single phage carrying a piece of recombinant DNA. A piece of nitrocellulose filter paper is placed over the agar plate, allowing the phage particles to adhere. The filter paper is treated with alkaline solution, which lyses the phage and denatures both the phage and the foreign DNA. The paper is then incubated with a labeled DNA probe, which will hybridize with the desired gene [46]. Once this gene is identified, one can go back to the original agar plate and find the phage or plasmid carrying the D N A clone. Gene cloning then involves three basic steps: (1) D N A is cut into small pieces by a restriction enzyme; (2) the restriction fragments are inserted into a cloning vector and amplified; (3) the desired D N A clone is identified using a DNA probe [44,47]. Once a gene is cloned, it can be produced, and its structure, function, and regulation studied. Its pure gene product (protein) can be synthesized in large quantities and can be analyzed (e.g., hemoglobin in the hemoglobinopathies) or used clinically (e.g., interferon). The cloning of human growth hormone (HGH) is a classic example of the application of these molecular techniques to a clinical problem. Growth hormone deficiency causes pituitary dwarfism. Prior to the cloning of H G H , the hormone was obtained from the pituitaries of 60,000 human cadavers per year to treat the 1,600 hypopituitary dwarfs in the United States. Extraction was an expensive, time-consuming process [48]. In addition, human tissues were a source of infectious diseases such as Creutzfeldt-Jakob disease [49]. Cloning of H G H was accomplished in 1979. Now an unlimited supply of pure H G H is available for treatment of growth hormone deficiency, as well as for studies in wound healing and aging. DNA P R O B E S A probe is necessary to identify a DNA gene after it has been isolated by restriction enzymes and amplified cloning vectors. A probe is a sequence of nucleotides (DNA or RNA) that is complementary to the gene of 612
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interest and will hybridize to it. A probe will select a complementary piece of DNA out of millions of noncomplementary sequences. If a probe is labeled (usually with radioactive isotope), the gene that hybridizes to that probe can then be found by simply locating the label. Most probes are made of complementary D N A (eDNA) [50]. Complementary DNA is made from m R N A by the enzyme reverse transcriptase [51,52], which is normally found in retroviruses. Construction of a cDNA probe proceeds as follows: (1) a cell line that produces the protein (gene product) of interest is found; (2) m R N A from that cell line is isolated and purified; (3) this m R N A is incubated with reverse transcriptase, which synthesizes a eDNA molecule on the m R N A template; (4) this piece of eDNA is inserted into a vector and amplified; (5) the cDNA is labeled with radioisotope [44,50]. A probe composed of nucleotides complementary to the desired gene is thus synthesized and has many uses. DNA probes can be used to screen large amounts of DNA. If the entire human genome is cut with restriction enzymes and all the pieces cloned into lambda phage, the entire collection is called the DNA library or DNA bank [53]. Lambda plaques can be screened with the probe and the phage carrying the gene of interest identified. Identification of the desired gene is the final step in gene cloning. If each piece of human genome DNA library were sequenced, this would result in the sequencing of the entire human genome. This ambitious project is underway, and the Senate has just passed a bill to oversee the federal genome project [13]. S O U T H E R N BLOT TECHNIQUE Probes can be used to screen DNA before it is amplified by vectors. A technique called Southern blot, named after its inventor, Dr. Edwin Southern, can be used to detect the presence of a specific sequence of DNA [54]. The DNA to be screened is treated with restriction enzymes. The DNA fragments are then separated by gel electrophoresis. Gel electrophoresis employs a polyacrylamide matrix. The D N A pieces are placed at one end and an electric field is placed across the gel. DNA will migrate according to size. The different pieces of DNA can therefore be separated into discrete bands. The D N A is blotted onto nitrocellulose paper, which is then exposed to the radiolabeled probe. The probe hybidizes and is timed to any cDNA sequences. The excess probe is washed off. By exposing the nitrocellulose sheet to x-ray film, the location of the hybridized radioactive probe and DNA fragment can be seen. The process of labeling with radioisotope and visualization by exposure to x-ray film is called autoradiography [55]. The presence or absence of any gene for which one has a probe can be detected in a very small amount of DNA by this Southern blotting technique. Similarly, m R N A can be isolated from a cell and be screened by these probes. The technique of detection of specific m R N A sequences is called Northern blotting (a laboratory joke, since it is patterned on Southern blotting). There are various techniques available to the scientist
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for manipulation of the organism of a molecular level. Genes can be isolated, amplified, and sequenced; their protein products can be produced and purified. Genes and their protein products are used in the diagnosis, treatment, and prevention of disease. Proteins such as insulin [1], interferon [3,4], and tissue plasminogen activator [56] can be synthesized and used therapeutically in the treatment of diabetes, cancer, and coronary artery disease. Gene cloning has enabled scientists to pinpoint the exact defect that causes such diseases as sickle cell anemia [7,37] and some forms of cancer [10,11]. DNA fingerprinting or RFLP is used in the presymptomatic diagnosis of hemophilia [5], thalassemia [38], and muscular dystrophy [38]. As more probes become available and more genes are located on the chromosome, DNA probing will become the most sensitive and specific test for a variety of diseases. Genetic engineering techniques have been used to synthesize hepatitis B antigens [57], and may also be useful in the development of a vaccine for the acquired immunodeficiency syndrome. Gene therapy, or insertion of a normal gene to replace a defective one, is in its nascent stages but may soon become the treatment of choice for various hematologic diseases [14,15]. Genetic engineering has influenced the treatment and diagnosis of such varying diseases as diabetes mellitus, coronary artery disease, pituitary dwarfism, and cancer. It is therefore essential that the clinician have an understanding of the underlying molecular principles. POLYMERASE CHAIN REACTION As a measure of the rapidity with which this field has progressed, since this paper was submitted, a new and important technology has been developed. Polymerase chain reaction (PCR) has been heralded as one of the most important technical advances in the past decade. This technique makes possible the amplification (or production) of segments of DNA from 50 to 2,000 base pairs in length, in vitro, within a few hours. Heretofore, as explained earlier, it was necessary to obtain a relatively pure portion of DNA for replication in a biologic system (gene cloning), a process both technically demanding and protracted. PCR has permitted amplification of relatively minute quantities of DNA obtained from old and new tissue, fixed pathologic tissue and fresh tissue, and pure and impure preparations. The power and simplicity of the technique lie in the fact that everything occurs within a test tube that is repeatedly heated and cooled. The tube contains a minute quantity of the DNA material to be replicated. This material may be in an impure form. Added are specific primers to ends of the D N A strand, a heat-stable polymerase enzyme, and a large excess of bases (deoxyribonucleoside triphosphates) from which to construct the DNA. In the initial heating, DNA is separated into single strands. The temperature is then reduced, and DNA primers become attached to their complementary sequences at opposite ends of the DNA strands. The specific amplification of a portion of DNA contained within the test tube mixture occurs because of the specific binding of the primers. The heat-stable polymerase enzyme now synthesizes a single strand of DNA beginning from each
primer attached to the strand of DNA to be duplicated. A large excess of bases ensures that the reaction will go to completion. The test tube is then reheated, again converting the newly formed double-stranded DNA to singlestranded DNA, and the cycle is repeated many more times. Thus, there is an exponential increase in duplicated specific new DNA strands. REFERENCES 1. Goeddel DV, Kleid DG, Bolivar F, et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 1979; 76: 106-10. 2. Goeddel DV, Heyneker HL, Hozumi T, et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 1979; 281: 544-8. 3. Nagata S, Taira H, Hall A, et al. Synthesis in E. coli of a polypeptidewith human leukocyte interferon activity. Nature 1980; 284: 316-20. 4. Goeddel DV, Yelverton E, Ullrich A, et al. Human leukocyte interferon produced by E. coli is biologically active. Nature 1980; 287:411-6. 5. Wood WF, Capon D, Simonsen C, et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-7. 6. Itakura K, Hirose T, Crea R, et al. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 1977; 198: 1056-63. 7. Ingram VM. Gene mutation in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin. Nature 1957; 180: 326-8. 8. Orkin SH, Old J, Lazarus H, et al. The molecular basis of alpha thalassemias: frequent occurrence of dysfunctional alpha loci among non-Asians with Hb H disease. Cell 1979; 17: 33-42. 9. Weatherall DJ, Clegg JB. Thalessemia revisited. Cell 1982; 29: 7-9. 10. Ready EP, Reynolds RK, Santos E, Barbacid M. A point mutation is responsible for the acquisition of transforming properties by the T24 B human bladder carcinoma oncogene. Nature 1982; 30: 149-52. 11. Erkison J, ar-Rushdi A, Drwinga HL, Nowell PC, Croce CM. Transcriptional activation of the translocated c-myc oncogene in Burkltt lymphoma. Proc Natl Acad Sci USA 1983; 80: 820-4. 12. Bishop JM. Cellular oncogenes and retroviruses. Annu Rev Biochem 1983; 52: 301-54. 13. Federal Genome Project: Biotechnology Competitiveness Act of 1987. Congressional Record. 100th Cong. 2d Sess., 1988. 14. Miller AD, Palmer TD, Hock RA. Transfer of genes into human somatic cells using retrovirus vectors. Cold Spr Harbor Symp Quant Biol 1986; 51: 1013-20. 15. Roberts L. Human gene therapy test. Science 1988; 241: 419. 16. Watson JD, Crick FHC. Molecular structure of nucleic acids, Nature 1953; 171: 737-8. 17. Watson JD, Crick FHC. Genetical implications of the structure of deoxyribonucleic acid. Nature 1953; 171: 964-7. 18. Watson JD. Involvement of RNA in the synthesis of proteins. Science 1963; 140: 17-26. 19. Chamberlin M. Transcription of genetic material. Cold Spr Harbor Symp Quant Biol 1970; 35: 851-73. 20. Prescott DM. Cellular sites of RNA synthesis. Prog Nucleic Acid Res Mol Biol 1964; 3: 35-57. 21. Crick FHC. The genetic code. Sci Am, Oct 1962: 66-74. 22. Adams JM, Capecchi MR. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc Natl Acad Sci USA 1966; 55: 147-55. 23. Ghosh HP, Soil D, Ichorana HG. Initiationof protein synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide sequences as messengers. J Mol Biol 1967; 25: 275-98. 24. Weigert MG, Garen A. Base compositionof nonsensecodons in E. coil Nature 1965; 206: 992-4.
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25. Brenner S, Stretton AOW, Kaplan S. Genetic code: the "nonsense" triplets for chain termination and their suppression.Nature 1965; 206: 994-8. 26. Crick FHC. The genetic code. Cold Spr Harbor Symp Quant Biol 1966; 31: 1-9. 27. Schimmel P, Soil D, Abelson J, eds. Transfer RNA. Cold Spr Harbor Laboratory, 1979. 28. Mechanisms of protein biosynthesis. Cold Spr Harbor Symp Quant Biol 1969; 34: 828-41. 29. Arber W, Linn S. DNA modification and restriction. Annu Rev Biochem 1969; 38: 467-500. 30. Nathans D, Smith HO. Restriction endonucleasesin the analysis and restructuring of DNA molecules.Annu Rev Biochem 1975; 44: 273-93. 31. Rubin R_A,Modrich P. Eco RI methylase. J Biol Chem 1977; 252: 7265-72. 32. Hedgepeth J, Goodman HM, Boyer HW. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci USA 1972; 69: 3448-52. 33. Sgaramella V, van de Sande JH, Khorana HG. Studies on polynucleotides. C.*: a novel joining reaction catalyzed by the T4polynucleotideligase. Proc Natl Acad Sci USA 1970; 66: 1460-75. 34. Botstein D, White R, Skolnic M, Davis R. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980; 32: 314. 35. Jeffreys A J, Wilson V, Thein SL. Individual-specific"fingerprints" of human DNA. Nature 1985; 316: 76-8. 36. Marx JL. DNA fingerprinting takes the witness stand. Science 1988; 240: 1616-8. 37. Kunkel LM. Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy. Nature 1986; 322: 737. 38. Orkin SH, Little PFR, Kazazian HH, Boehm CD. Improved detection of the sickle mutation by DNA analysis. N Engl J Med 1982; 307: 32-6. 39. Boehm CD, Antonarakis SE, Phillips JA, Stetten G, Kazazian HH. Prenatal diagnosis using DNA polymorphisms.N Engl J Med 1983; 308: 1054-8. 40. Broda P. Plasmids. San Francisco: W.H. Freeman and Co, 1979. 41. Helsinkl DR. Plasmids as vehicles for gene cloning: impact on basic and applied research. Trends Biochem Sci 1978; 3: 10-4. 42. Cameron JR, Panesenko SM, Lehman IR, Davis RW. In vivo construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the
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gene for DNA ligase. Proc Natl Acad Sci USA 1975; 72: 3416-20. 43. Clowes RC. The molecule of infectiousdrug resistance. Sci Am 1973; 228: 18-27. 44. Abelson J, Butz E, eds. Recombinant DNA. Science 1980;209: 1317-1438. 45. Benton WD, Davis RW. Screening lambda-gt recombinant clones by hybridization to single plaques in situ. Science 1977; 196: 180-3. 46. Grunstein M, Hogness DS. Colony hybridization: a method for the isolation of cloned DNAs that contain a specificgene. Proc Natl Acad Sci USA 1975; 72: 3961-5. 47. Scott WA, Werner R. Molecular cloning of recombinant DNA. Duluth, MN: Academic Press, 1977. 48. Seeburg PH. Human growth hormone: from clone to clinic. Cold Spr Harbor Symp Quant Biol 1986; 51: 669-77. 49. Brown P, Gajdusek DC, Gibbs CJ Jr, Asher DM. Potential epidemic of Creutzfeldt-Jacob disease from human growth hormone therapy. N Engl J Med 1985; 313: 728-31. 50. Okayama H, Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol 1982; 2: 161-70. 51. Baltimore D. Viral RNA-dependent DNA polymerase.Nature 1970; 226: 1209-11. 52. Temin HM, Mizutani S. RNA-dependent DNA polymerase on virions of Rous Sarcoma virus. Nature 1970; 226: 1211-3. 53. Maniatis T, Hardison C, Lacy E, et al. The isolation of structural genes from libraries of eukaryotic DNA. Cell 1978; 15: 687701. 54. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98. 503-7. 55. Rogers AW. Techniques of autoradiography, 3rd ed. Amsterdam, North Holland: Elsevier, 1979. 56. Vehar GA, Spellman BA, Keyt CK, et al. Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology. Cold Spr Harbor Symp Quant Biol 1986; 51: 551-62. 57. Christman JK, Gerber M, Price PM, Flordellis C, Edelman J. Amplification of expression of hepatitis-B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc Natl Acad Sci USA 1982; 79: 1815-9. 58. Mullis KB, Faloona FA. Specific synthesisof DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987; 155: 335-50. 59. Eisenstein BI. The polymerase chain reaction. N Engl J Med 1990; 322: 178-83.
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DNA Technology Rena S. Wong, MD, Edward Passaro, Jr., MD, Los Angeles, California With the availability of DNA recombinant technology and DNA and RNA sequencing techniques, diseases can now be studied and treated at a molecular level, while unlimited quantities of a pure protein product can be produced through gene cloning. Before the end of this century, gene therapy will be used to repair genetic defects. This article explains these advances in genetic technology and suggests their relevance in clinical problems and practice.
ith the rapid advances in DNA recombinant technology and DNA and RNA sequencing techW niques, scientists now have the tools to study and treat disease at a molecular level. Gene cloning enables production of unlimited quantities of a pure protein product. Human insulin [1], growth hormone [2], interferon [3,4], and factor VIII [5] produced by genetically engineered bacteria have been used clinically in the treatment of diabetes mellitus, growth hormone deficiency, cancer, and hemophilia. The ability to produce in quantity proteins such as somatostatin [6], or interferon, which are normally present in minute amounts, has helped elucidate their normal physiologic structures and functions. DNA technology has allowed us to discover the molecular basis of many human diseases such as sickle cell anemia [7], thalassemia [8,9], and some cancers [10,11]. DNA mapping and sequencing have revealed that viral oncogenes show remarkable similarity to normal cellular genes and, in fact, may be derived from them [12]. This new technology is being used to sequence the entire human genome [13]. Gene therapy, or the introduction of a therapeutic gene to repair a genetic defect, will become a reality in this century [14,15]. This rapid growth in genetic technology has also resuited in a widening of the intellectual gap between the clinician and the basic scientist. In order to apply the new technology fully, it must also be fully understood. This brief review highlights the advances in genetic technology and suggests their relevance to surgical problems and practice.
DEOXYRIBONUCLEIC ACID Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins make up the structural and functional elements of a cell. DNA contains the genetic information of a cell. It is composed of long, linear sequences of nueleotide bases called adenine (A), thymine (T), guanine (G), and cytosine (C). DNA has a helical, doublestranded structure [16]. Adenine is always paired opposite thymine, guanine opposite cytosine. This is base pairing. Thus, the nucleotide sequence of one strand can be deduced from the other, complementary strand. Two strands of DNA can be separated or denatured by high temperature, but will rejoin to re-form the double helix as the temperature drops. The process by which one strand of DNA (or RNA) finds its complementary strand is dependent on base pairing and is called hybridization. Hybridization and base pairing allow accurate copies of DNA to be made during replication and are also important in DNA molecular cloning [17]. RIBONUCLEIC ACID Ribonucleic acid (RNA) comes in three forms: messenger R N A (mRNA), ribosomal R N A (rRNA), and transfer RNA (tRNA) [18]. Messenger RNA, like DNA, is a polymer of nucleotides. It differs from DNA in that it exists in a single-stranded structure and that ribose replaces deoxyribose. In addition, uracil (U) replaces thymine (T). Like thymine, uracil is complementary to adenine. Messenger RNA is formed opposite a DNA strand or template by the enzyme RNA polymerase; the order of nucleotides in the m R N A is complementary to the order of nucleotides in the DNA [19]. After m R N A is formed in the nucleus, it enters the cytoplasm and associates with ribosomes in the endoplasmic reticulum, the site of protein synthesis [20]. There, the sequence of nucleotides is decoded, and the corresponding protein is made. Thus, m R N A carries the message from DNA in the nucleus to the ribosome in the cytoplasm. Ribosomal RNA is found only in ribosomes and gives them their structural and functional capacities. Transfer RNA is the adapter molecule in protein synthesis; it decodes the mRNA.
PROTEINS Proteins are the working molecules of an organism. They provide structure (collagen), alter cellular permeability (calcium channels), recognize messenger molecules (receptors), enable motion (flagella), and regulate gene expression (repressor protein). Enzymes are proteins that are catalysts for a cell's chemical reactions. Antibodies are specialized proteins that function as high Fromthe Departmentof Surgery,Divisionof GeneralSurgery,UCLA Schoolof Medicine,and the SurgicalService,VeteransAdministration specificity recognition molecules in the immune system. MedicalCenter,WestLos Angeles,Los Angeles,California. Proteins are linear chains of amino acids. There are 20 Requests for reprints shouldbe addressedto EdwardPassaro,Jr., amino acids, and their order in a protein determines its MD, SurgicalService(W112),VeteransAdministrationMedicalCenstructure and function. All proteins are coded for by ter, Los Angeles,California90073. ManuscriptsubmittedJanuary24, 1989,and acceptedFebruary9, genes in the DNA. The protein corresponding to a gene is called the gene product. 1989. 610
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Protein synthesis: Although all the genetic information of a cell is stored in its DNA, it is the proteins that actually perform all cellular functions. Thus, a method by which the information stored in the DNA is translated into different proteins is required. Protein synthesis begins in the nucleus. The enzyme RNA polymerase copies the DNA gene into a linear, complementary mRNA strand. The copying of DNA into RNA is called transcription [19]. Messenger RNA strands then associate with the ribosomes in the cytoplasm. Messenger RNA is composed of only four nucleotides (A,U,G,C); proteins are composed of 20 amino acids. A group of three consecutive nucleotides codes for each amino acid. Each of these triplets is called a codon [21]. There are 64 (4 to the third power) possible codons, and thus some redundancy, with one amino acid corresponding to several different codons. Other codons serve as start and stop codons [22-25]. The correlation of codons to amino acids is called the genetic code and is universal in all organisms [26]. A bacterium such as Escherchia coB, therefore, can decode the message from a human gene and make the human gene product (e.g., human insulin). Because of the genetic code, if either the DNA or RNA sequence is known, the protein sequence is also known. Conversely, if a protein has been sequenced, the possible DNA sequences that code for that protein can be deduced. The first human protein made by E. coli through genetic engineering was somatostatin, which is 14 amino acids in length. The gene was actually synthesized, one nucleotide at a time, by deduction of its nucleotide sequence from its known amino acid sequence, and then cloned into E. coli in 1977 [6]. Messenger RNA is decoded by tRNA. Transfer RNAs are small molecules with two functional ends [27]. One end contains three consecutive nucleotides that are complementary to the mRNA codons. These are the anticodons. An amino acid is attached at the other end of a tRNA molecule. Protein synthesis occurs in the following steps: (1) mRNA is transcribed in the nucleus; (2) mRNA associates with the ribosomes where tRNA molecules recognize the codons and base pairing between complementary codon-anticodons occurs; (3) the individual amino acids carried by each tRNA are joined by chemical bonding [28]. The order of amino acids is determined by the order of nucleotides in the mRNA, which is ultimately determined by the sequence in the DNA. The process by which the nucleotide sequence of a mRNA is decoded into the amino acid sequence of the corresponding protein is called translation. Translation of a single mRNA by multiple ribosomes results in the formation of many identical proteins. Similarly, during transcription, multiple mRNA copies can be made of a single DNA gene. A gene in the DNA can therefore be expressed in multiple copies, although the gene itself is read or copied only several times. GENE CLONING A clone is a large number of identical molecules or cells with a common ancestor. Cloning of a gene requires isolation, amplification, and identification of that gene. The techniques used for the biologic separation and pro-
TECHNOLOGY
duction of a piece of DNA (gene) are collectively called recombinant DNA technology. Each chromosome is a long molecule of DNA with genes arranged linearly on it. Each gene is (generally) present in only one copy. Therefore, a single gene represents only a small fraction of the total DNA. Since all genes are composed of the same four nucleotides, they are chemically indistinguishable. Biologic isolation is therefore necessary. In order to separate a particular gene, DNA must be cut into small, gene-length fragments, which can then be amplified. This is done by restriction enzymes [29,30]. A restriction enzyme recognizes and cleaves DNA at certain nucleotide sequences called restriction sites. Restriction enzymes are ordinarily found in bacteria and "restrict" entry of foreign DNA into a bacterial cell by cutting it into pieces. The bacterium protects its own DNA from destruction by modifying each of its own restriction sites to prevent cleaving by the restriction enzyme [31]. For example [32], the enzyme Eco RI recognizes the DNA sequence -GAATTC- and cleavesit: -G AATTC-CTTAAG-CTTAA GThe cut, unpaired, single-stranded ends are able to recognize each other, or any other piece of DNA cut by the same enzyme, by base pairing. They can be rejoined using the enzyme DNA ligase [33]. They are the sticky ends. Incubation of chromosomal (genomal) DNA with a restriction enzyme cleaves the DNA into multiple, small fragments suitable for cloning. Since a restriction enzyme always cuts at the same places, the pattern of DNA fragments is reproducible and is characteristic of a particular chromosome and restriction enzyme. This is restriction enzyme mapping. Restriction enzyme mapping of certain areas of human DNA called mini-satellite regions results in a DNA fragment pattern that is different in every person. This is called restriction fragment-length polymorphism (RFLP) [34]. RFLP, or DNA fingerprinting, can accurately identify the individual origin of any tissue or body fluid [35]. It is useful in forensic medicine, where DNA fingerprinting has been used as evidence in rape cases [36]. It is used for genetic screening and in the prenatal diagnosis of various genetic disorders such as muscular dystrophy [37], sickle cell anemia [38], and thalassemia
[391. Restriction enzymes are used in DNA cloning for cutting long pieces of DNA into segments that can be separated. These pieces of DNA with sticky ends must then be amplified. This is accomplished using cloning vectors. A vector is a piece of DNA capable of independent growth. The two most commonly used cloning vectors are bacterial plasmids [40,41] and phage lambda [42]. A phage is a virus that infects bacteria. A plasmid is a circular piece of extrachromosomal DNA found in bacteria that multiplies independently of chromosomal DNA. Up to 3,000 copies of a plasmid can be present in a single bacterium. Often a plasmid contains genes encoding antibiotic resistance [43]. Bacteria with plasmids can then be selected by growing them in media containing
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antibiotics. If a plasmid is cut with a restriction enzyme and then incubated with DNA fragments cut with the same enzyme, the complementary sticky ends will join and a new plasmid containing both plasmid and foreign D N A will be formed. Combined foreign (e.g., human) and plasmid (bacterial) DNA is recombinant DNA [44]. If this recombinant DNA is introduced into bacteria, multiple copies of both plasmid and foreign DNA will be made, resulting in a D N A clone. Plasmids are useful then for amplifying the number of copies of a single piece of DNA. It is difficult for technical reasons, however, to screen large numbers of bacteria for the plasmid containing the desired gene. This is done more efficiently using the vector phage lambda. Phage lambda is a commonly used vector. It has the advantage over plasmids in that larger pieces of DNA can be cloned and that screening of a large number of clones is possible. Foreign pieces of DNA are recombined with lambda phage using DNA ligase. The phage are then spread over a bacterial lawn, an agar plate with a thin layer of bacteria covering it. The phage then infect the bacteria, multiply, and then lyse the bacteria, forming clear zones called plaques [45]. Each plaque corresponds to a single phage carrying a piece of recombinant DNA. A piece of nitrocellulose filter paper is placed over the agar plate, allowing the phage particles to adhere. The filter paper is treated with alkaline solution, which lyses the phage and denatures both the phage and the foreign DNA. The paper is then incubated with a labeled DNA probe, which will hybridize with the desired gene [46]. Once this gene is identified, one can go back to the original agar plate and find the phage or plasmid carrying the D N A clone. Gene cloning then involves three basic steps: (1) D N A is cut into small pieces by a restriction enzyme; (2) the restriction fragments are inserted into a cloning vector and amplified; (3) the desired D N A clone is identified using a DNA probe [44,47]. Once a gene is cloned, it can be produced, and its structure, function, and regulation studied. Its pure gene product (protein) can be synthesized in large quantities and can be analyzed (e.g., hemoglobin in the hemoglobinopathies) or used clinically (e.g., interferon). The cloning of human growth hormone (HGH) is a classic example of the application of these molecular techniques to a clinical problem. Growth hormone deficiency causes pituitary dwarfism. Prior to the cloning of H G H , the hormone was obtained from the pituitaries of 60,000 human cadavers per year to treat the 1,600 hypopituitary dwarfs in the United States. Extraction was an expensive, time-consuming process [48]. In addition, human tissues were a source of infectious diseases such as Creutzfeldt-Jakob disease [49]. Cloning of H G H was accomplished in 1979. Now an unlimited supply of pure H G H is available for treatment of growth hormone deficiency, as well as for studies in wound healing and aging. DNA P R O B E S A probe is necessary to identify a DNA gene after it has been isolated by restriction enzymes and amplified cloning vectors. A probe is a sequence of nucleotides (DNA or RNA) that is complementary to the gene of 612
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interest and will hybridize to it. A probe will select a complementary piece of DNA out of millions of noncomplementary sequences. If a probe is labeled (usually with radioactive isotope), the gene that hybridizes to that probe can then be found by simply locating the label. Most probes are made of complementary D N A (eDNA) [50]. Complementary DNA is made from m R N A by the enzyme reverse transcriptase [51,52], which is normally found in retroviruses. Construction of a cDNA probe proceeds as follows: (1) a cell line that produces the protein (gene product) of interest is found; (2) m R N A from that cell line is isolated and purified; (3) this m R N A is incubated with reverse transcriptase, which synthesizes a eDNA molecule on the m R N A template; (4) this piece of eDNA is inserted into a vector and amplified; (5) the cDNA is labeled with radioisotope [44,50]. A probe composed of nucleotides complementary to the desired gene is thus synthesized and has many uses. DNA probes can be used to screen large amounts of DNA. If the entire human genome is cut with restriction enzymes and all the pieces cloned into lambda phage, the entire collection is called the DNA library or DNA bank [53]. Lambda plaques can be screened with the probe and the phage carrying the gene of interest identified. Identification of the desired gene is the final step in gene cloning. If each piece of human genome DNA library were sequenced, this would result in the sequencing of the entire human genome. This ambitious project is underway, and the Senate has just passed a bill to oversee the federal genome project [13]. S O U T H E R N BLOT TECHNIQUE Probes can be used to screen DNA before it is amplified by vectors. A technique called Southern blot, named after its inventor, Dr. Edwin Southern, can be used to detect the presence of a specific sequence of DNA [54]. The DNA to be screened is treated with restriction enzymes. The DNA fragments are then separated by gel electrophoresis. Gel electrophoresis employs a polyacrylamide matrix. The D N A pieces are placed at one end and an electric field is placed across the gel. DNA will migrate according to size. The different pieces of DNA can therefore be separated into discrete bands. The D N A is blotted onto nitrocellulose paper, which is then exposed to the radiolabeled probe. The probe hybidizes and is timed to any cDNA sequences. The excess probe is washed off. By exposing the nitrocellulose sheet to x-ray film, the location of the hybridized radioactive probe and DNA fragment can be seen. The process of labeling with radioisotope and visualization by exposure to x-ray film is called autoradiography [55]. The presence or absence of any gene for which one has a probe can be detected in a very small amount of DNA by this Southern blotting technique. Similarly, m R N A can be isolated from a cell and be screened by these probes. The technique of detection of specific m R N A sequences is called Northern blotting (a laboratory joke, since it is patterned on Southern blotting). There are various techniques available to the scientist
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for manipulation of the organism of a molecular level. Genes can be isolated, amplified, and sequenced; their protein products can be produced and purified. Genes and their protein products are used in the diagnosis, treatment, and prevention of disease. Proteins such as insulin [1], interferon [3,4], and tissue plasminogen activator [56] can be synthesized and used therapeutically in the treatment of diabetes, cancer, and coronary artery disease. Gene cloning has enabled scientists to pinpoint the exact defect that causes such diseases as sickle cell anemia [7,37] and some forms of cancer [10,11]. DNA fingerprinting or RFLP is used in the presymptomatic diagnosis of hemophilia [5], thalassemia [38], and muscular dystrophy [38]. As more probes become available and more genes are located on the chromosome, DNA probing will become the most sensitive and specific test for a variety of diseases. Genetic engineering techniques have been used to synthesize hepatitis B antigens [57], and may also be useful in the development of a vaccine for the acquired immunodeficiency syndrome. Gene therapy, or insertion of a normal gene to replace a defective one, is in its nascent stages but may soon become the treatment of choice for various hematologic diseases [14,15]. Genetic engineering has influenced the treatment and diagnosis of such varying diseases as diabetes mellitus, coronary artery disease, pituitary dwarfism, and cancer. It is therefore essential that the clinician have an understanding of the underlying molecular principles. POLYMERASE CHAIN REACTION As a measure of the rapidity with which this field has progressed, since this paper was submitted, a new and important technology has been developed. Polymerase chain reaction (PCR) has been heralded as one of the most important technical advances in the past decade. This technique makes possible the amplification (or production) of segments of DNA from 50 to 2,000 base pairs in length, in vitro, within a few hours. Heretofore, as explained earlier, it was necessary to obtain a relatively pure portion of DNA for replication in a biologic system (gene cloning), a process both technically demanding and protracted. PCR has permitted amplification of relatively minute quantities of DNA obtained from old and new tissue, fixed pathologic tissue and fresh tissue, and pure and impure preparations. The power and simplicity of the technique lie in the fact that everything occurs within a test tube that is repeatedly heated and cooled. The tube contains a minute quantity of the DNA material to be replicated. This material may be in an impure form. Added are specific primers to ends of the D N A strand, a heat-stable polymerase enzyme, and a large excess of bases (deoxyribonucleoside triphosphates) from which to construct the DNA. In the initial heating, DNA is separated into single strands. The temperature is then reduced, and DNA primers become attached to their complementary sequences at opposite ends of the DNA strands. The specific amplification of a portion of DNA contained within the test tube mixture occurs because of the specific binding of the primers. The heat-stable polymerase enzyme now synthesizes a single strand of DNA beginning from each
primer attached to the strand of DNA to be duplicated. A large excess of bases ensures that the reaction will go to completion. The test tube is then reheated, again converting the newly formed double-stranded DNA to singlestranded DNA, and the cycle is repeated many more times. Thus, there is an exponential increase in duplicated specific new DNA strands. REFERENCES 1. Goeddel DV, Kleid DG, Bolivar F, et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 1979; 76: 106-10. 2. Goeddel DV, Heyneker HL, Hozumi T, et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 1979; 281: 544-8. 3. Nagata S, Taira H, Hall A, et al. Synthesis in E. coli of a polypeptidewith human leukocyte interferon activity. Nature 1980; 284: 316-20. 4. Goeddel DV, Yelverton E, Ullrich A, et al. Human leukocyte interferon produced by E. coli is biologically active. Nature 1980; 287:411-6. 5. Wood WF, Capon D, Simonsen C, et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-7. 6. Itakura K, Hirose T, Crea R, et al. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 1977; 198: 1056-63. 7. Ingram VM. Gene mutation in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin. Nature 1957; 180: 326-8. 8. Orkin SH, Old J, Lazarus H, et al. The molecular basis of alpha thalassemias: frequent occurrence of dysfunctional alpha loci among non-Asians with Hb H disease. Cell 1979; 17: 33-42. 9. Weatherall DJ, Clegg JB. Thalessemia revisited. Cell 1982; 29: 7-9. 10. Ready EP, Reynolds RK, Santos E, Barbacid M. A point mutation is responsible for the acquisition of transforming properties by the T24 B human bladder carcinoma oncogene. Nature 1982; 30: 149-52. 11. Erkison J, ar-Rushdi A, Drwinga HL, Nowell PC, Croce CM. Transcriptional activation of the translocated c-myc oncogene in Burkltt lymphoma. Proc Natl Acad Sci USA 1983; 80: 820-4. 12. Bishop JM. Cellular oncogenes and retroviruses. Annu Rev Biochem 1983; 52: 301-54. 13. Federal Genome Project: Biotechnology Competitiveness Act of 1987. Congressional Record. 100th Cong. 2d Sess., 1988. 14. Miller AD, Palmer TD, Hock RA. Transfer of genes into human somatic cells using retrovirus vectors. Cold Spr Harbor Symp Quant Biol 1986; 51: 1013-20. 15. Roberts L. Human gene therapy test. Science 1988; 241: 419. 16. Watson JD, Crick FHC. Molecular structure of nucleic acids, Nature 1953; 171: 737-8. 17. Watson JD, Crick FHC. Genetical implications of the structure of deoxyribonucleic acid. Nature 1953; 171: 964-7. 18. Watson JD. Involvement of RNA in the synthesis of proteins. Science 1963; 140: 17-26. 19. Chamberlin M. Transcription of genetic material. Cold Spr Harbor Symp Quant Biol 1970; 35: 851-73. 20. Prescott DM. Cellular sites of RNA synthesis. Prog Nucleic Acid Res Mol Biol 1964; 3: 35-57. 21. Crick FHC. The genetic code. Sci Am, Oct 1962: 66-74. 22. Adams JM, Capecchi MR. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc Natl Acad Sci USA 1966; 55: 147-55. 23. Ghosh HP, Soil D, Ichorana HG. Initiationof protein synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide sequences as messengers. J Mol Biol 1967; 25: 275-98. 24. Weigert MG, Garen A. Base compositionof nonsensecodons in E. coil Nature 1965; 206: 992-4.
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