17: Biotechnology and Genomics Flashcards
Biotechnology, Mapping Genomes, Whole-Genome Sequencing, Applying Genomics, Genomics and Proteomics
What is antibiotic resistance?
The ability of an organism to be unaffected by the actions of an antibiotic.
What is biotechnology?
The use of biological agents for technological advancement.
What is cellular cloning?
The production of identical cell populations by binary fission.
What is a clone?
An exact replica.
What is foreign DNA?
DNA that belongs to a different species or DNA that is artificially synthesized. AKA transgene.
What is gel electrophoresis?
A technique used to separate molecules on the basis of size using electric charge.
What is gene targeting?
A method for altering the sequence of a specific gene by introducing the modified version on a vector.
What is gene therapy?
A technique used to cure inheritable diseases by replacing mutant genes with good genes.
What is genetic diagnosis?
Diagnosis of the potential for disease development by analyzing disease-causing genes.
What is genetic engineering?
Alteration of the genetic makeup of an organism.
What is genetic testing?
The process of testing for the presence of disease-causing genes.
What is a genetically modified organism (GMO)?
An organism whose genome has been artificially changed.
What is host DNA?
DNA that is present in the genome of the organism of interest.
What is a lysis buffer?
A solution used to break the cell membrane and release cell contents.
What is molecular cloning?
Cloning of DNA fragments.
What is a multiple cloning site (MCS)?
A site that can be recognized by multiple restriction endonucleases.
What is northern blotting?
The transfer of RNA from a gel to a nylon membrane.
What is polymerase chain reaction (PCR)?
A technique used to amplify DNA.
What is a probe?
A small DNA fragment used to determine if the complementary sequence is present in a DNA sample.
What is a protease?
An enzyme that breaks down proteins.
What is recombinant DNA?
Combination of DNA fragments generated by molecular cloning that does not exist in nature.
What is a recombinant protein?
A protein product of a gene derived by molecular cloning (recombinant DNA).
What is reproductive cloning?
Cloning of entire organisms.
What is a restriction endonuclease?
An enzyme that can recognize and cleave specific DNA sequences.
What are reverse genetics?
A method of determining the function of a gene by starting with the gene itself instead of starting with the gene product.
What is reverse transcriptase PCR (RT-PCR)?
A PCR technique that involves converting RNA to DNA (cDNA) by reverse transcriptase.
What is a ribonuclease?
An enzyme that breaks down RNA.
What is southern blotting?
The transfer of DNA from a gel to a nylon membrane.
What is a Ti plasmid?
A plasmid system derived from Agrobacterium tumifaciens that has been used by scientists to introduce foreign DNA into plant cells.
What is a transgenic organism?
An organism that receives DNA from a different species.
What are some examples of the uses of biotechnology?
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.
How is DNA extracted?
Various techniques are used to extract different types of DNA. 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. 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.
Why is RNA analysis performed?
RNA analysis is performed to study gene expression patterns in cells.
How is RNA extracted?
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.
How does gel electrophoresis work?
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. 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.
What is PCR used for?
Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis. 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 determination of paternity and detection of genetic diseases.
How does PCR work?
Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA. Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures used in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park.
The PCR cycle consists of three steps–denaturation, annealing, and DNA synthesis–that occur at high, low, and intermediate temperatures, respectively. The cycle is repeated again and again, resulting in a doubling of DNA molecules each time. After several cycles, the vast majority of strands produced are the same length as the distance between the two primers.
In step 1, denaturation, the sample is heated to a high temperature so the DNA strands separate.
In step 2, annealing, the sample is cooled so the primer can anneal to the DNA.
In step 3, DNA synthesis, the sample is warmed. Taq polymerase synthesizes new strands of DNA.
How does reverse transcriptase PCR work?
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.
How does northern and southern blotting work?
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. 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.
What is northern and southern blotting used for?
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.
What is western blotting?
The transfer of protein from a gel to a nylon membrane and detected using antibodies.
What is the difference between reproductive and molecular cloning?
The re-creation of a whole organism is referred to as “reproductive cloning.” The reproduction of regions or fragments of the genome is referred to as molecular cloning.
What is molecular cloning used for?
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation.
What is a plasmid?
A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal 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).
How are plasmids used in molecular cloning?
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 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).
How do restriction endonucleases work?
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.
What is another term for recombinant DNA?
Recombinant DNA molecules 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.
When will genes not be expressed using recombinant DNA?
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.
What is parthenogenesis?
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction.
What are some examples of parthenogenesis in nature?
An example of parthenogenesis occurs in species in which the female lays an egg and if the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.
How does reproductive cloning work?
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. Therefore, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor.
What is a technique for reproductive cloning?
Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
How has reproductive cloning been used in animals?
The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications. There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals such as horses, bulls, and goats have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities.
How has reproductive cloning been used in humans?
There have been attempts at producing cloned human embryos as sources of embryonic stem cells, sometimes referred to as cloning for therapeutic purposes. Therapeutic cloning produces stem cells to attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations.
How is genetic engineering performed?
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.
What are some examples of GMOs?
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.
How is gene targeting performed?
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.
How is genetic testing used?
The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, women diagnosed with breast cancer are usually advised to have a biopsy so that the medical team can determine the genetic basis of cancer development. Treatment plans are based on the findings of genetic tests that determine the type of cancer. If the cancer is caused by inherited gene mutations, other female relatives are also advised to undergo genetic testing and periodic screening for breast cancer. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.
How is gene therapy used?
Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA. More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID)
How are vaccines produced?
Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory. Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.
How are antibiotics produced?
Antibiotics are a biotechnological product. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.
What are some examples of recombinant DNA used for hormone production?
Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. In addition, human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector.
How have transgenic animals been used?
Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing. For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals. Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.
How have transgenic plants been used?
Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life. Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established. Plants that have received recombinant DNA from other species are called transgenic plants.
