Information

3.1: Recombinant DNA, Polymerase Chain Reaction and Applications to Eukaryotic Gene Structure and Function - Biology


Methods to purify some abundant proteins were developed early in the 20th century, and some of the experiments on the fine structure of the gene (colinearity of gene and protein for trpA and tryptophan synthase) used microbial genetics and proteins sequencing. However, methods to isolate genes were not developed until the 1960’s, and the were applicable to only a few genes.

All this changed in the late 1970’s with the development of recombinant DNA technology, or molecular cloning. This technique enabled researchers to isolate any gene from any organism from which one could isolate intact DNA (or RNA). The full potential to provide access to all genes of organisms is now being realized as full genomes are sequenced. One of the by-products of the intense investigation of individual DNA molecules after the advent of recombinant DNA was a procedure to isolate any DNA for which one knows the sequence. This technique, called the polymerase chain reaction (PCR), is far easier than traditional molecular cloning methods, and it has become a staple of many laboratories in the life sciences. After covering the basic techniques in recombinant DNA technology and PCR, their application to studies of eukaryotic gene structure and function will be discussed.

Like many advances in molecular genetics, recombinant DNA technology has its roots in bacterial genetics.

Transducing Phage

The first genes isolated were bacterial genes that could be picked up by bacteriophage. By isolating these hybrid bacteriophage, the DNA for the bacterial gene could be recovered in a highly enriched form. This is the basic principal behind recombinant DNA technology.

Some bacteriophage will integrate into a bacterial chromosome and reside in a dormant state (Figure 3.1). The integrated phage DNA is called a prophage, and the bacterium is now a lysogen. Phage that do this are lysogenic. Induction of the lysogen will result in excision of the prophage and multiplication to produce many progeny, i.e. it enters a lytic phasein which the bacteria are broken open and destroyed. The nomenclature is descriptive. The bacteria carrying the prophage show no obvious signs of the phage (except immunity to superinfection with the same phage, covered later in Part Four), but when induced (e.g. by stress or UV radiation) they will generate a lytic state, hence they are called lysogens. Induced lysogens make phage from the prophage that was integrated. Phage that always multiply when they infect a cell are called lytic.

Excision of a prophage from a lysogen is notalways precise. Usually only the phage DNA is cut out of the bacterial chromosome, but occassionally some adjacent host DNA is included with the excised phage DNA and encapsidated in the progeny. These transducing phage are usually biologically inactive because the piece of the bacterial chromosome replaces part of the phage chromosome; these can be propagated in the presence of helper phage that provide the missing genes when co-infected into the same bacteria. When DNA from the transducing phage is inserted into the newly infected cell, the bacterial genes can recombine into the host chromosome, thereby bringing in new alleles or even new genes and genetically altering the infected cell. This process is called transduction.

Figure 3.1. Transfer of bacterial genes by transduction: A lac+ transducing phage can convert a lac‑ strain to lac+ by infection (and subsequent crossing over).

Note that the transducing phage are carrying one or a small number of bacterial genes. This is a way of isolating the genes. The bacterial gene in the transducing phage has been separated from the other 4000 bacterial genes (in E. coli). By isolating large numbers of the transducing phage, the phage DNA, including the bacterial genes, can be obtained in large quantitiesfor biochemical investigation. One can isolate mg or mg quantities of a single DNA molecule, which allows for precise structural determination and detailed investigation.

A generalized transducing phagecan integrate at many different locations on the bacterial chromosome. Imprecise excision from any of those locations generates a particular transducing phage, carrying a short sections of the bacterial genome adjacent to the integration site. Thus a generalized transducing phage such as P1 can pick up many different parts of the E. coli genome.

A specialized transducing phageintegrates into only one or very few sites in the host genome. Hence it can carryonly a few specific bacterial genes, e.g., l lac(Figure 3.2).

Figure 3.2. An example of a l transducing phage carrying part of the lacoperon.

This process of isolating a particular bacterial gene on a transducing phage is mimicked in recombinant DNA technology, in which a gene or genome fragment from any organism is isolated on a recombinant phage or plasmid.


17.1 Biotechnology

In this section, you will explore the following questions:

  • What are examples of basic techniques used to manipulate genetic material (DNA and RNA)?
  • What is the difference between molecular and reproductive cloning?
  • What are examples of uses of biotechnology in medicine and agriculture?

Connection for AP ® Courses

Did you eat cereal for breakfast or tomatoes in your dinner salad? Do you know someone who has received gene therapy to treat a disease such as cancer? Should your school, health insurance provider, or employer have access to your genetic profile? Understanding how DNA works has allowed scientists to recombine DNA molecules, clone organisms, and produce mice that glow in the dark. We likely have eaten genetically modified foods and are familiar with how DNA analysis is used to solve crimes. Manipulation of DNA by humans has resulted in bacteria that can protect plants from insect pests and restore ecosystems. Biotechnologies also have been used to produce insulin, hormones, antibiotics, and medicine that dissolve blood clots. Comparative genomics yields new insights into relationships among species, and DNA sequences reveal our personal genetic make-up. However, manipulation of DNA comes with social and ethical responsibilities, raising questions about its appropriate uses.

Nucleic acids can be isolated from cells for analysis by lysing cell membranes and enzymatically destroying all other macromolecules. Fragmented or whole chromosomes can be separated on the basis of size (base pair length) by gel electrophoresis. Short sequences of DNA or RNA can be amplified using the polymerase chain reaction (PCR). Recombinant DNA technology can combine DNA from different sources using bacterial plasmids or viruses as vectors to carry foreign genes into host cells, resulting in genetically modified organisms (GMOs). Transgenic bacteria, agricultural plants such as corn and rice, and farm animals produce protein products such as hormones and vaccines that benefit humans. (It is important to remind ourselves that recombinant technology is possible because the genetic code is universal, and the processes of transcription and translation are fundamentally the same in all organisms.) Cloning produces genetically identical copies of DNA, cells, or even entire organisms (reproductive cloning). Genetic testing identifies disease-causing genes, and gene therapy can be used to treat or cure an inheritable disease. However, questions emerge from these technologies including the safety of GMOs and privacy issues.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A Heritable information provides for continuity of life.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
Learning Objective 3.5 The student can justify the claim that humans can manipulate heritable information by identifying an example of a commonly used technology.
Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.C The processing of genetic information is imperfect and is a source of genetic variation.
Essential Knowledge 3.C.1 Changes in genotype can result in changes in phenotype.
Science Practice 7.2 The student can connect concepts in and across domain(s) to generalize or extrapolate in and/or across enduring understandings and/or big ideas.
Learning Objective 3.24 The student is able to predict how a change in genotype, when expressed as a phenotype, provides a variation that can be subject to natural selection.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.13][APLO 3.23][APLO 3.28][APLO 3.24][APLO 1.11][APLO 3.5][APLO 4.2][APLO 4.8]

Biotechnology is the use of biological agents for technological advancement. Biotechnology was used for breeding livestock and crops long before the scientific basis of these techniques was understood. Since the discovery of the structure of DNA in 1953, the field of biotechnology has grown rapidly through both academic research and private companies. The primary applications of this technology are in medicine (production of vaccines and antibiotics) and agriculture (genetic modification of crops, such as to increase yields). Biotechnology also has many industrial applications, such as fermentation, the treatment of oil spills, and the production of biofuels.

Basic Techniques to Manipulate Genetic Material (DNA and RNA)

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.

DNA and RNA Extraction

To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells. Various techniques are used to extract different types of DNA (Figure 17.2). Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution which is mostly a detergent) lysis means “to split.” These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. The DNA samples can be stored frozen at –80°C for several years.

RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size, using this charge. The nucleic acids can be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a semisolid, porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules this difference in the rate of migration separates the fragments on the basis of size. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size (Figure 17.3). A mixture of genomic DNA fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction

Although genomic DNA is visible to the naked eye when it is extracted in bulk, DNA analysis often requires focusing on one or more specific regions of the genome. Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis (Figure 17.4). PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the detection of genetic diseases.

DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR) . The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

LINK TO LEARNING

Deepen your understanding of the polymerase chain reaction by clicking through this interactive exercise.

  1. The process of PCR can isolate a particular piece of DNA for copying, which allows scientists to copy millions of strands of DNA in a short amount of time.
  2. The process of PCR can purify a particular piece of DNA, and very small amounts of DNA can be used for purification.
  3. The process of PCR separates and analyzes DNA and its fragments, which requires very little DNA.
  4. The process of PCR anneals DNA molecules to complementary DNA strands, which maintains the same amount of DNA.

Hybridization, Southern Blotting, and Northern Blotting

Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting (Figure 17.5). The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting , and when RNA is transferred to a nylon membrane, it is called northern blotting . Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.

Molecular Cloning

In general, the word “cloning” means the creation of a perfect replica however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.

Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a "folder" in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA , or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA .

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS) . The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease (Figure 17.6).

Recombinant DNA Molecules

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins . Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.


Recombinant DNA and Genetic Engineering Chapter 16 Impacts

Impacts, Issues: Golden Rice or Frankenfood? § Scientists created transgenic rice (Golden Rice) as a vitamin A supplement for undernourished nations is the benefit worth the risk in these gene-manipulated food sources?

16. 1 Cloning DNA § Process to add genes to food or other cell types is simple in principle § Researchers cut up DNA from different sources, then paste the resulting fragments together § Cloning vectors can carry foreign DNA into host cells

Cut and Paste for New DNA Combos § Restriction enzymes • Bacterial enzymes that cut DNA wherever a specific nucleotide sequence occurs § Single-stranded DNA tails produced by the same restriction enzyme base-pair together • DNA ligase bonds “sticky ends” together § Recombinant DNA • Composed of DNA from two or more organisms

Making Recombinant DNA This is especially useful to introduce genes into a sequence in research.

DNA Cloning § DNA cut into fragments by restriction enzymes is inserted into cloning vectors (plasmids) cut with the same enzyme § Cloning vectors with foreign DNA are placed in host cells, which divide and produce many clones, each with a copy of the foreign DNA

c. DNA Cloning § Complementary DNA (c. DNA) • DNA made from an m. RNA template § Reverse transcriptase transcribes m. RNA to DNA, forming a hybrid molecule • DNA polymerase builds a double-stranded DNA molecule that can be cloned • Especially useful to obtain DNA without introns.

c. DNA Cloning by Reverse Transcriptase

16. 1 Key Concepts DNA Cloning by Lab and Plasmids § Researchers routinely make recombinant DNA by cutting and pasting together DNA from different species § Plasmids and other vectors can carry foreign DNA into host cells

Genomes and DNA Libraries § Genome • The entire set of genetic material of an organism § DNA libraries are sets of cells containing various cloned DNA fragments • Genomic libraries (all DNA in a genome) • c. DNA libraries (all active genes in a cell)

Probes Used for ID of DNA § Probe • A fragment of DNA labeled with a tracer • Used to find a specific clone carrying DNA of interest in a library of many clones § Nucleic acid hybridization • Base pairing between DNA from different sources • A probe hybridizes with the targeted gene

Big-Time DNA Amplification: PCR § Polymerase chain reaction (PCR) • A cycled reaction that uses a heat-tolerant form of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment • This is how a single drop of blood at a crime scene can become expanded to enough to make necessary tests and still be available for future testing if needed

PCR in Overview § DNA to be copied is mixed with DNA polymerase, nucleotides and primers that basepair with certain DNA sequences § Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

16. 2 Key Concepts Needles in Haystacks § Researchers manipulate targeted genes by isolating and making many copies of particular DNA fragments

16. 3 DNA Sequencing § DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

DNA Sequencing § DNA is synthesized with normal nucleotides and dideoxynucleotides tagged with different colors • When a tagged base is added, DNA synthesis stops fragments of all lengths are made § Electrophoresis separates the fragments of DNA, each ending with a tagged base, by length • Order of colored bases is the sequence of DNA • Finished sequence is basis for comparison

16. 4 DNA Fingerprinting § One individual can be distinguished from all others on the basis of DNA “fingerprints” § Confidence here in results is extremely high, in the usually stated range of one in many millions

DNA Fingerprints § DNA fingerprint • A unique array of DNA sequences used to identify individuals § Short tandem repeats (STRs) • Many copies of the same 2 - to 10 -base-pair sequences in a series along a chromosome • Types and numbers of STRs vary greatly among individuals

Creating DNA Fingerprints § PCR is used to amplify DNA from regions of several chromosomes that have STRs § Electrophoresis is used to separate the fragments and create a unique DNA fingerprint § DNA fingerprints have many applications • Legal cases, forensics, population studies

DNA Fingerprints: Forensics Case Example You are on the jury. You are shown this prepared comparison of DNA fingerprints, with ID as shown. See if you can match suspect with sample from the crime scene.

16. 3 -16. 4 Key Concepts Deciphering DNA Fragments § Sequencing reveals the linear order of nucleotides in a fragment of DNA § A DNA fingerprint is an individual’s unique array of DNA sequences

16. 5 Studying Genomes § Comparing the sequence of our genome with that of other species is giving us insights into how the human body works § You already know of 98 percent same human sequences with that of chimpanzes § How about 49 percent the same between a banana and a human?

The Human Genome Project § Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome § 28, 976 genes have been identified, but not all of their products or functions are known § As of 2010, distinct gene numbers down to about 23, 000 by best estimates from work

Sequencing the Human Genome Computers have greatly speeded process up and also increased accuracy.

Genomics is a Growing Application § Genomics: The study of genomes • Structural genomics • Comparative genomics § Analysis of the human genome yields new information about genes and how they work • Applications in medicine and other fields • Example: APOA 5 mutations and triglycerides

DNA Chips Have a Future § DNA chips • Microarrays of many different DNA samples arranged on a glass plate • Used to compare patterns of gene expression among cells of different types or under different conditions • May be used to screen for genetic abnormalities, pathogens, or cancer

16. 6 Genetic Engineering § Genetic engineering • A laboratory process by which deliberate changes are introduced into an individual’s genome § Today’s most common genetically modified organisms are bacteria and yeast • Are used in research, medicine, and industry • Example: production of human insulin

GMOs – Now and Later § Genetically modified organisms (GMOs) • Individuals containing modified genes from the same species or a different species • Future will have major control problems as the developer of GMO usually patents process/result § Transgenic organisms • Individuals containing genes transferred from a different species (also GMOs) • Example: Bacteria with jellyfish genes

16. 7 Designer Plants by GM § Genetically engineered crop plants are widespread in the United States § But can their designed change(s) “jump” to other plant life or end up incorporated in animals eating the modified plants?

The Ti Plasmid – a GMO Mechanism § Ti plasmid • Plasmid of bacteria Agrobacterium tumefaciens • Contains tumor-inducing (Ti) genes • Used as a vector to transfer foreign or modified genes into plants, including some food crops

Ti Plasmid Transfer Steps

Genetically Engineered Plants § Crop plants are genetically modified to produce more food at lower cost • • Resistance to disease or herbicides Increased yield Plants that make pesticides (Bt protein gene) Drought resistance

GMO Controversies § 73 GMO crops are approved for use in US, with hundreds more pending • Corn, sorghum, cotton, soy, canola, alfalfa • Big problem of just a few companies doing nearly all the research and manufacturing – can lead to a monopoly problem in future § Facts and controversy – real life • In crops engineered for herbicide resistance, weeds are becoming resistant to herbicides • Engineered genes are spreading into wild plants and nonengineered crops

Some Genetically Modified Plants

16. 8 Biotech Barnyards § Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering § This can be really good for endangered animals § Genetically engineered animals are used in research, medicine, and industry

Of Mice and Men § 1982: The first transgenic animals – mice with genes for rat growth hormone

Examples of Transgenic Animals § Genetically modified animals are used as models of many human diseases • Mice used in knockout experiments § Genetically modified animals make proteins with medical and industrial applications • Goats and rabbits that make human proteins • Farms animals with desirable characteristics

Some Genetically Modified Animals That silly-looking featherless chicken is easily the most commercially viable possibility shown here. It would eliminate a costly part of chicken processing and could enable very warm climate poultry farms.

Knockout Cells and Organ Factories § Transgenic pigs with human proteins are a potential source of organs and tissues for transplants in humans • May prevent rejection by immune system § Xenotransplantation • Transplantation of a tissue or organ from one species to another • Pig heart valves used for many years.

16. 10 Modified Humans? § We as a society continue to work our way through the ethical implications of applying new DNA technologies § The manipulation of individual genomes continues even as we are weighing the risks and benefits of this research

Gene Therapy – Helping the Individual § Gene therapy • Transfer of recombinant DNA into body cells to correct a genetic defect or treat a disease • Viral vectors or lipid clusters insert an unmutated gene into an individual’s chromosomes • Examples: Cystic fibrosis, SCID-X 1

Getting Better by Gene Therapy § 1998: A viral vector was used to insert unmutated IL 2 RG genes into boys with severe combined immunodeficiency disease (SCID-X 1) – most recovered immune function

Getting Worse by Gene Therapy § No one can predict where a virus-injected gene will insert into a chromosome – several boys from the SCID-X 1 study developed cancer § In other studies, severe allergic reactions to the viral vector itself have resulted in death

Getting Perfect Over Time § Eugenic engineering • Engineering humans for particular desirable traits, not associated with treatment of disorders

16. 6 -16. 10 Key Concepts Using the New Technologies of GM § Genetic engineering, the directed modification of an organism’s genes, is now used in research, and it is being tested in medical applications § Many questions must be answered about the ethics and consequences of manipulating the human genome – some of these can be answered by our government but many will remain answerable only by the individual as he or she agrees to their usage personally.


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Genetic Engineering

Genetic engineering is the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering. The organism that receives the recombinant DNA is called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic. Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes. In the US, GMOs such as Roundup-ready soybeans and borer-resistant corn are part of many common processed foods.

Gene Targeting

Although classical methods of studying the function of genes began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: “What does this gene or DNA element do?” This technique, called reverse genetics, has resulted in reversing the classic genetic methodology. This method would be similar to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the function of the wing is flight. The classical genetic method would compare insects that cannot fly with insects that can fly, and observe that the non-flying insects have lost wings. Similarly, mutating or deleting genes provides researchers with clues about gene function. The methods used to disable gene function are collectively called gene targeting. Gene targeting is the use of recombinant DNA vectors to alter the expression of a particular gene, either by introducing mutations in a gene, or by eliminating the expression of a certain gene by deleting a part or all of the gene sequence from the genome of an organism.


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