Biotechnology Quiz

PDF page 473: Biotechnology is the use of biological agents for technological advancement. Biotechnology was used for breeding livestock and crops long before people understood the scientific basis of these techniques. Since the discovery of the structure of DNA in 1953, the biotechnology field has grown rapidly through both academic research and private companies. The primary applications of this technology are in medicine (vaccine and antibiotic production) and agriculture (crop genetic modification in order to increase yields). Biotechnology also has many industrial applications, such as fermentation, treating oil spills, and producing biofuels (Figure 17.2).

PDF page 474: Scientists perform RNA analysis 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 using 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, an electric field can mobilize them. Gel electrophoresis is a technique that scientists use to separate molecules on the basis of size, using this charge. One can separate the nucleic acids as whole chromosomes or fragments. The nucleic acids load into a slot near the semisolid, porous gel matrix's negative electrode, and pulled toward the positive electrode at the gel's opposite end. Smaller molecules move through the gel's pores faster than larger molecules. This difference in the migration rate separates the fragments on the basis of size. There are molecular weight standard samples that researchers can run alongside the molecules to provide a size comparison. We can observe nucleic acids in a gel matrix using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the gel's top (the negative electrode end) on the basis of their size (Figure 17.4). 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 gel's top.

PDF page 474: Scientists perform RNA analysis 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 using 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, an electric field can mobilize them. Gel electrophoresis is a technique that scientists use to separate molecules on the basis of size, using this charge. One can separate the nucleic acids as whole chromosomes or fragments. The nucleic acids load into a slot near the semisolid, porous gel matrix's negative electrode, and pulled toward the positive electrode at the gel's opposite end. Smaller molecules move through the gel's pores faster than larger molecules. This difference in the migration rate separates the fragments on the basis of size. There are molecular weight standard samples that researchers can run alongside the molecules to provide a size comparison. We can observe nucleic acids in a gel matrix using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the gel's top (the negative electrode end) on the basis of their size (Figure 17.4). 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 gel's top.

PDF page 474: Scientists perform RNA analysis 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 using 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, an electric field can mobilize them. Gel electrophoresis is a technique that scientists use to separate molecules on the basis of size, using this charge. One can separate the nucleic acids as whole chromosomes or fragments. The nucleic acids load into a slot near the semisolid, porous gel matrix's negative electrode, and pulled toward the positive electrode at the gel's opposite end. Smaller molecules move through the gel's pores faster than larger molecules. This difference in the migration rate separates the fragments on the basis of size. There are molecular weight standard samples that researchers can run alongside the molecules to provide a size comparison. We can observe nucleic acids in a gel matrix using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the gel's top (the negative electrode end) on the basis of their size (Figure 17.4). 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 gel's top.

PDF page 475: under UV light; and b) a researcher from International Rice Research Institute, reviewing DNA profiles using UV light. (credit: a: James Jacob, Tompkins Cortland Community College b: International Rice Research Institute) Nucleic Acid Fragment Amplification 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 genome regions. Polymerase chain reaction (PCR) is a technique that scientists use to amplify specific DNA regions for further analysis (Figure 17.5). Researchers use PCR for many purposes in laboratories, such as cloning gene fragments to analyze genetic diseases, identifying contaminant foreign DNA in a sample, and amplifying DNA for sequencing. More practical applications include determining paternity and detecting genetic diseases.

PDF page 476: pieces of DNA complementary to each end of the target sequence combine 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 that scientists use in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park. Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins.

PDF page 476: 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.

PDF page 477: size. Scientists then transfer the fragments in the gel onto a nylon membrane in a procedure we call blotting (Figure 17.6). Scientists can then probe the nucleic acid fragments that are bound to the membrane's surface with specific radioactively or fluorescently labeled probe sequences. When scientists transfer DNA to a nylon membrane, they refer to the technique as Southern blotting. When they transfer the RNA to a nylon membrane, they call it Northern blotting. Scientists use Southern blots to detect the presence of certain DNA sequences in a given genome, and Northern blots to detect gene expression.

PDF page 477: size. Scientists then transfer the fragments in the gel onto a nylon membrane in a procedure we call blotting (Figure 17.6). Scientists can then probe the nucleic acid fragments that are bound to the membrane's surface with specific radioactively or fluorescently labeled probe sequences. When scientists transfer DNA to a nylon membrane, they refer to the technique as Southern blotting. When they transfer the RNA to a nylon membrane, they call it Northern blotting. Scientists use Southern blots to detect the presence of certain DNA sequences in a given genome, and Northern blots to detect gene expression.

PDF page 477: fragments on a gel, transfer them to a nylon membrane, and incubate them with a DNA probe complementary to the sequence of interest. Northern blotting is similar to Southern blotting, but scientists run RNA on the gel instead of DNA. In Western blotting, scientists run proteins on a gel and detect them using antibodies.

PDF page 477: 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 genome fragments allows researchers to manipulate and study specific genes (and their protein products), or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, scientists can use the plasmid molecules 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, scientists call the DNA fragment from the human genome (or the genome of another studied organism) foreign DNA, or a transgene, to differentiate it from the bacterium's DNA, or 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). Scientists have repurposed and engineered plasmids 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 scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. 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 DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA's two ends that has been cut with the same restriction endonuclease (Figure 17.7). 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 molecule parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. We call proteins that are expressed from recombinant DNA molecules recombinant

PDF page 477: 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 genome fragments allows researchers to manipulate and study specific genes (and their protein products), or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, scientists can use the plasmid molecules 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, scientists call the DNA fragment from the human genome (or the genome of another studied organism) foreign DNA, or a transgene, to differentiate it from the bacterium's DNA, or 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). Scientists have repurposed and engineered plasmids 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 scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. 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 DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA's two ends that has been cut with the same restriction endonuclease (Figure 17.7). 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 molecule parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. We call proteins that are expressed from recombinant DNA molecules recombinant