Just as an understanding of the structure and function of the atom led to the advent of laptops, cell phones, and Instagram, an understanding of the workings of DNA has led to an era where DNA fingerprints are commonplace, human proteins are created in animals, vaccines may be rapidly produced from genetic material, and genetic diseases may be cured.
The primary goals of this science are to isolate, identify, and manipulate DNA. Much of this work falls into the category of genetic engineering (sometimes referred to as bioengineering): the direct, deliberate modification of an organism’s genome. Genetic engineering itself is part of the larger science of synthetic biology and biotechnology, whose major aims are the design of biological systems that can synthesize new forms of molecules, cells, organs, and even organisms.
The subject of genetic engineering and its biotechnological ap plications are growing at such a rate that some new discovery or product is disclosed almost daily. To keep this subject somewhat manageable, we will organize the topics as follows:
● Genetic engineering tools and techniques
● Uses and products of recombinant DNA technology
● Genetically modified organisms
● Genetic treatment ∙ Genome analysis
Tools and Techniques of DNA Technology
All of the intrinsic properties of DNA that we touched on hold true whether the DNA is in a bacterium or a test tube. For example, the enzyme helicase is able to unwind the two strands of the double helix just as easily inside or outside a bacterial cell. But in the laboratory we can take advantage of our knowledge of DNA chemistry to make helicase unnecessary. It turns out that when DNA is heated to just be low boiling temperature (90°C to 95°C), the two strands separate. With the nucleotides exposed, DNA can be easily identified, replicated, or transcribed. If heat-denatured DNA is then slowly cooled, complementary nucleotides will hydrogen-bond with one another, and the strands will renature or regain their familiar double-stranded form (figure 1). As we shall see, this process is a necessary feature of the polymerase chain reaction and nucleic acid probes.
Fig1. Denaturation of the DNA double helix. When heated to temperatures just below boiling, the hydrogen bonds holding complementary bases together will break, and the two DNA strands will separate. Similarly, when DNA that has been rendered single stranded is cooled, complementary bases will bind to one another. This property allows for the binding of small DNA primers in the polymerase chain reaction, as well as the binding of labeled DNA probes involved in various hybridization processes.
Enzymes for Dicing, Splicing, and Reversing Nucleic Acids
DNA can also be clipped crosswise at selected positions by means of restriction endonucleases (or restriction enzymes) that are a natural product of bacterial cells. These enzymes recognize foreign DNA and are capable of breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA, leading to a break in the DNA strand. In the bacterial cell, these enzymes protect against the incompatible DNA from bacteriophages. In the laboratory, these enzymes allow DNA to be cut at specific locations.
So far, about 3,000 restriction endonucleases have been discovered in bacteria. Each restriction endonuclease has a sequence of 4 to 10 base pairs referred to as its recognition site or sequence, and every time the enzyme encounters that sequence, it will cut both strands of DNA. Interestingly, recognition sites tend to be palindromic1, meaning that the sequence of bases on one strand (in the 5' to 3' direction) is the same as the sequence of bases on the complementary strand (again, in the 5' to 3' direction).
Endonucleases are named by combining the first letter of the bacterial genus and the first two letters of the species. Roman numerals within the name of the enzyme are used to describe certain mechanisms of cleavage (which are truthfully unimportant to us). All restriction enzymes fall into one of four types and are therefore named I-IV. Thus, EcoRI is an endonuclease found in Escherichia coli’s R strain, and Hin dIII is an endonuclease discovered in Haemophilus influenzae Type d.
Endonucleases are used in the laboratory to cut DNA into smaller pieces for further study as well as to remove and insert it during recombinant DNA techniques. Usually the enzymes make staggered symmetrical cuts that leave short, single-stranded tails called “sticky ends” (table 1). Such adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids. This effect makes it possible to splice genes into specific sites (figure 2). A few endonucleases such as PvuII make straight, blunt cuts on DNA and do not form sticky ends.
Table1. Restriction Endonucleases
Fig2. Examples of cutting patterns by restriction endonucleases and how they are used in gene splicing. (a) The restriction endonuclease HindIII cuts both strands of the DNA whenever its recognition sequence, 5′ AAGCTT 3′, is encountered. For most restriction enzymes, the cut produces two overhanging strands of DNA called sticky ends (a few enzymes cut straight across the DNA and produce blunt ends). (b) Sticky ends can be used to join DNA from different organisms by cutting both with the same restriction enzyme, which provides complementary sticky ends on both fragments. C (a): Barry Chess/McGraw Hill
The separated segments of DNA produced by restriction endo nucleases are termed restriction fragments. Because DNA sequences vary, even among members of the same species, differences in the cutting pattern of specific restriction endonucleases give rise to restriction fragments of differing lengths, known as restriction fragment length polymorphisms (RFLPs). RFLPs allow the direct com parison of the DNA cutting patterns of two different organisms with the same endonuclease, which is one step in preparing genes from different organisms to be spliced together.
Another enzyme, called a ligase, is necessary to seal the sticky ends together by rejoining the phosphate-sugar bonds cut by endo nucleases. Its main application is in final splicing of genes into plasmids and chromosomes.
An enzyme called reverse transcriptase is best known for its role in the replication of HIV and other retroviruses. It also pro vides geneticists with a valuable tool for converting RNA into DNA. Copies called complementary DNA, or cDNA, can be made from RNA. The technique provides a valuable means of synthesizing eukaryotic genes from processed mRNA transcripts (figure 3). The bacteria often used to transcribe and translate eukaryotic genes in genetic engineering lack the cellular machinery to splice out introns found in unprocessed eukaryotic mRNA. So the advantage with synthesized cDNA genes is that they already have the introns removed and consist only of exons and can be readily transcribed and translated by bacteria. Reverse transcriptase is also used to convert the genetic material of RNA viruses, like coronavirus, to DNA for identification or further study.
Fig3. Synthesizing cDNA from eukaryotic mRNA. Bacterial cloning hosts cannot process intact eukaryotic genes. To provide a gene that will be transcribable and translatable by a bacterium, (a) first modified mRNA, lacking introns, is isolated from the source eukaryotic cell. (b) This strand is converted in a test tube to a single-stranded DNA by reverse transcriptase and double-stranded DNA by a polymerase. This copy of DNA (cDNA) can be transcribed and translated by bacteria.
Analysis of DNA
One way to produce a readable pattern of DNA fragments is through electrophoresis (figure 4). In this technique, samples are placed in compartments (wells) in an agar gel or similar soft medium and subjected to an electrical current. The phosphate groups in DNA give the entire molecule an overall negative charge, which causes the DNA to move toward the positive pole in the gel. The rate of movement is based primarily on the size of the fragments. The larger fragments are pulled into the gel more slowly and remain nearer the wells, whereas the smaller fragments migrate faster and are positioned farther from the wells. The gel can then be stained to reveal the position of the bands of DNA within the gel. (figure 5). Electrophoresis patterns can be quite distinctive and are very useful in displaying DNA fragments and comparing the degree of genetic similarities among samples in a Southern blot or DNA profile.
Fig4. Electrophoresis equipment. Electrophoresis uses an electrical current to separate DNA molecules of different sizes. The samples are loaded into small wells in an agarose gel submerged in a buffer solution. The gel contains tiny pores that allow the passage of molecules according to their size. DNA, which is negatively charged, is attracted to the positively charged end of the gel, with smaller fragments moving more rapidly. The result is that larger DNA fragments remain close to the wells at the positively charged end of the gel (red wire) while smaller fragments appear nearer the negatively charged end (black wire). (inset) DNA (along with a dye to enhance visibility) is loaded into a single well of a gel. (Electrophoresis setup): Barry Chess; (inset): red_moon_rise/Getty Images
Fig5. Separating DNA restriction fragments with electrophoresis. (a) A restriction endonuclease is used to cut a DNA sample (using sample 4 in this example). The restriction endonuclease cuts the DNA every time it encounters a recognition site. In this case, the DNA is cut twice, producing three fragments. The DNA fragments are then loaded into a well at one end of the gel and electrophoresed; small DNA samples move more quickly through the gel and end up closer to the positive electrode. (b) A stained gel reveals the position of the DNA fragments. The size of each fragment (each band) can be determined by comparing the distance traveled to the distance traveled by a set of DNA fragments of known size called a marker (lane 7), measured in number of base pairs. Different DNA sequences will produce different sets of DNA fragments. Identical fragments in lanes 1 and 4 are evidence that these samples likely came from the same individual. (b): Barry Chess
Nucleic Acid Hybridization and Probes
Two different nucleic acid strands can hybridize by uniting at their complementary sites. All different combinations are possible: Single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can hybridize with other RNA. This knowledge has led to the development of short, single strands of 6 to 25 nucleotides called oligonucleotides to be used as gene probes. These probes consist of a segment of DNA or RNA of known sequence that can base-pair with a stretch of DNA that has a complementary sequence, if one exists in a test sample. Hybridization probes have practical value because they can detect specific nucleotide sequences in unknown samples. So that areas of hybridization can be visualized, the probes carry reporter molecules, such as fluorescent dyes, which can be visualized with ultraviolet radiation (see figure 6), or radioactive labels, which are revealed by placing photographic film in contact with the test reaction.
Fig6. Fluorescent in situ hybridization (FISH) technique. This is a microscopic technique using fluorescently labeled probes to react with intact cells and chromosomes. The probe is mixed with test cells or chromosomes to hybridize any matching DNA sequences and observed with a UV microscope for fluorescence indicative of a particular location and type of gene. The inset displays chromosomes that have DNA from a papillomavirus integrated into chromosomes (yellow dots). Tammur, J., Sibul, H., Ustav, E., Ustav, M., & Metspalu. A. (2002). A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor In the receptor-deficient CHO-ldIA7 cell line. BMC Mol Biol., 3:5.
When probes hybridize with an unknown sample of DNA or RNA, they confirm the presence of that RNA or DNA sequence in a sample. In a method called the Southern blot, DNA fragments are first separated by electrophoresis and then denatured and transferred to a special filter. A DNA probe is then incubated with the sample, and wherever this probe encounters the segment for which it is complementary, it will attach and form a hybrid, showing up as one or more bands. This method is a sensitive and specific way to find specific gene sequences. Much of the information obtained through Southern blotting can be achieved in more detail from another hybridization procedure called a microarray.
Southern blots and similar hybridization techniques can be used to identify specific nucleotide sequences found only in one particular organism, facilitating identification. Commercially available diagnostic kits are now on the market for identifying intestinal pathogens such as E. coli, Salmonella, Campylobacter, Shigella, Clostridium difficile, rotaviruses, and adenoviruses. Other bacterial probes exist for Mycobacterium, Neisseria, Staphylococcus, Mycoplasma, Bordetella, and Chlamydia; probes are available for herpes simplex and zoster, papilloma, hepatitis A and B, and HIV viruses. DNA probes have also been developed for human genetic markers and some types of cancer.
With another method, called fluorescent in situ hybridization (FISH), probes are applied to intact cells in situ, meaning that the DNA is left intact in the cell and probed in place rather than being extracted. The preparation is then observed microscopically for signs of genetic marker sequences on genes that have hybridized with the probe. FISH is a very effective way to locate genes on chromosomes (figure6). In situ techniques can also be used to identify unknown bacteria living in natural habitats without having to culture them, and they can be used to detect RNA in cells and tissues.
DNA Sizing, Sequencing, and Synthesizing
The relative sizes of nucleic acids are usually denoted by the number of base pairs (bp) or nucleotides they contain. For example, the palindromic sequences recognized by endonucleases are usually 4 to 10 bp in length; an average gene in E. coli is approximately 1,300 bp, or 1.3 kilobases (kb); and its entire genome is approximately 4,600,000 bp, 4,600 kb, or 4.6 megabases (mb). The DNA of the human mitochondrion contains 16 kb, and the Epstein-Barr virus (a cause of infectious mononucleosis) has 172 kb. Humans have approximately 6.4 billion base pairs (bbp) arrayed along 46 chromosomes.
DNA Sequencing: Determining the Exact Genetic Code One of the most powerful kinds of genetic knowledge attainable for organisms is to decipher the order and types of base pairs in their genes and genome. This process, called DNA sequencing, can be used on all types of DNA, including genomic, cDNA, chromosomal, plasmid, and mitochondrial DNA. Many sequencing techniques are based on Frederick Sanger’s original idea, using the synthesis and analysis of a complementary strand of DNA (figure 7). Even though mostly auto mated techniques are used today, we will illustrate the basic principles behind this process using the Sanger type of sequencing as a model.
Fig7. Some basic techniques in DNA sequencing. (1–7) The sequencing of a 9-base strand using the Sanger method. The sequence in #6 is read from the bottom to the top and must be converted to its complement to get the true nucleotide order of TCGGCTAGG.
Because most DNA being sequenced is usually very long, it is made more manageable by cutting it into a number of shorter fragments, which are then separated. The test strands, typically several hundred nucleotides long, are denatured to expose single strands that will serve as templates to synthesize complementary strands. The concepts behind the sequencing procedure involve the same enzymes and raw materials as would be present for regular DNA synthesis as performed in a cell. A DNA primer is bound to the template, then a DNA polymerase adds the complementary nucleotides using the template strand as a guide.
Besides the DNA molecule to be sequenced, DNA polymerase III, and short DNA primers , the reaction tube also contains all of the normal nucleotides (A, T, C, G) and a small amount of dideoxynucleotides (ddNT) that have two special features: (1) They are color-labeled with four different fluorescent dyes that can report their identity, and (2) they are missing the hydroxyl group (OH−) bound to the 3′ carbon of deoxyribose that is required for the chemical bonding of a nucleotide to the growing DNA strand. The addition of a ddNT at any location terminates the further elongation of the strand. As the synthesis proceeds, most strands will elongate normally, except for a small percentage of fragments that will have a ddNT nucleotide incorporated and be terminated there. Late in the process, all possible positions in the sequence will have a terminal ddNT nucleotide. The reaction tube will thus have a series of strands reflecting the correct sequence that can be traced by its color from the first nucleotide to the last.
During analysis, the reaction products are subjected to electrophoresis in fine capillaries that separate strands differing by only a single nucleotide in length. This arrays the fragments in order of size, and only the fragments carrying the labeled nucleotides will be readable (figure 7). Automatic sequencing machines scan the electrophoresis pattern and record the DNA sequence by detecting the color of each band with a sensing device. In this way, the DNA sequence of the synthesized strand is analyzed and that of the template strand can easily be inferred. Most sequencers have computers that read the sequence and display it as a graphic figure. This method of sequencing is remarkably accurate, with only about one mistake in every 1,000 bases. All of these steps have been auto mated to obtain the speed necessary to sequence the entire genomes of humans and other organisms.
One of the remarkable discoveries in this huge enterprise has been how similar the genomes of relatively unrelated organisms can be. Humans share around 99% of their genome with chimpanzees, 80% with mice, about 50% with corn, and even 30% with the round worm Caenorhabditis elegans.
Although sequencing provides the ultimate genetic map, it does not immediately inform us about gene function. For that we need additional information about gene expression. Because the data consist of both biological and mathematical content, whole new disciplines have grown up around managing them. The fields of genomics and bioinformatics focus on analyzing, comparing, and classifying DNA; determining protein sequences; and ultimately understanding the functions of specific genes and proteins. Like other all-encompassing fields, genomics and bioinformatics can be subdivided into more manageable subspecialties:
● Transcriptomics: analysis of all RNA molecules in a cell. This includes mRNA, tRNA, rRNA, and the regulatory RNAs. It is an intermediate link between the activity of genes and the protein products of translation.
● Proteomics: the study of an organism’s complement of proteins (its “proteome”) and functions mediated by the proteins.
● Metagenomics: the study of all the genomes in a particular ecological niche, as opposed to individual genomes from single species.
● Metabolomics: the study of the complete complement of small chemicals present in a cell at any given time; provides a snapshot of the physiological state of the cell and the end products of its metabolism.
One of the fastest growing of these subspecialties is metage nomics, a field designed to survey the scope of organisms that live in various habitats. In the past it was difficult to study complex communities where millions of organisms coexist, and it has not been possible to isolate and culture most of them. Metagenomics provides microbiologists a window through which to view the diversity of such habitats and makes it possible to pull out the microbial genomes, study them, and begin to understand their interactions. This technique has been used for several years in the large-scale genetic sampling of the oceans. Other research efforts on dozens of habitats, including the human body, are yielding massive amounts of new genetic data.
Polymerase Chain Reaction: A Molecular Copy Machine for DNA Some of the techniques used to analyze DNA and RNA are limited by the small amounts of nucleic acid available. This problem was largely solved by the invention of a simple, versatile way to amplify DNA called the polymerase chain reaction (PCR). This technique rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques. It is so sensitive that it holds the potential to detect cancer from a single cell or to diagnose an infection from a single gene copy. It is comparable to being able to pluck a single DNA “needle” out of a “haystack” of other needles and make unlimited copies of the DNA. PCR makes it possible to replicate a target DNA from a few copies to billions of copies in a few hours. It can amplify DNA fragments that con sist of a few base pairs to whole genomes containing several million base pairs.
Initiating the reaction requires a few specialized ingredients (process figure 8). The primers are synthetic oligonucleotides (short DNA strands) of a known sequence of 15 to 30 bases that serve as landmarks to indicate where DNA amplification will begin. These take the place of RNA primers that would normally be provided by the cell. Depending upon the purposes and what is known about the DNA being replicated, the primers can be random, attaching to any sequence they may fit, or they may be highly specific and chosen to amplify a known gene. To keep the DNA strands separated, processing must be carried out at a relatively high temperature. This necessitates the use of heat-resistant DNA polymerases isolated from thermophilic bacteria. Examples of these unique enzymes are Taq polymerase obtained from Thermus aquaticus and Vent polymerase from Thermococcus litoralis. Enzymes isolated from these thermophilic organisms remain active at the elevated temperatures used in PCR. Another useful component of PCR is a machine called a thermal cycler that automatically initiates the cyclic temperature changes.
Process Fig8. Diagram of the polymerase chain reaction. (a) Details of the process over two cycles. (b) Yield after the cycle continues past the second cycle.
The PCR technique operates by repetitive cycling of three basic steps: denaturation, priming, and extension:
1. Denaturation. The first step involves heating target DNA to 94°C to separate it into two strands. Each strand will become a template DNA, or amplicon.
2. Priming. As the reaction is cooled to between 50°C and 65°C (depending on the exact sequence of the primers), the oligonucleotide primers base-pair with specific sites on the amplicons.
3. Extension. In the third phase, which proceeds at 72°C, the DNA polymerase is activated and begins to synthesize DNA from a pool of available nucleotides. Beginning at the free end of the primers on both strands, the polymerases move along the strands, which serve as templates for the synthesis of two complete double strands of DNA.
It is through cyclic repetition of these steps that DNA becomes amplified. When the DNAs formed in the first cycle are denatured, they become amplicons to be primed and extended in the second cycle. Each subsequent cycle converts the new DNAs to amplicons and doubles the number of copies. The number of cycles required to produce a million molecules is 20, but the process is usually carried out to 30 or 40 cycles. One significant advantage of this technique has been its natural adaptability to automation. A thermal cycler can perform 20 cycles on nearly 100 samples in 2 or 3 hours.
DNA amplified by PCR can be analyzed by any of the techniques discussed earlier. PCR can also be adapted to analyze RNA by initially converting an RNA sample to DNA with reverse transcriptase. This cDNA can then be amplified by PCR in the usual manner. It is by such means that ribosomal RNA and messenger RNA are readied for sequencing and RNA viruses are sequenced and identified. The PCR technique has found prominence as a powerful workhorse of molecular biology, medicine, and biotechnology. It often plays an essential role in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies.
For all of its advantages, PCR techniques can encounter problems. One concern is the introduction and amplification of nontarget DNA from the surrounding environment, such as a skin cell from the technician carrying out the PCR rather than material from the sample DNA that was supposed to be amplified. Such contamination can be minimized by using equipment and rooms dedicated for DNA analysis and maintained with the utmost degree of cleanliness. Problems with contaminants can also be reduced by using gene-specific primers and treating samples with enzymes that can degrade the contaminating DNA before it is amplified.
