9.1 Introduction to Biotechnology

• Biotechnology is the use of microorganisms, cells, or cell components to make a product.

• Microorganisms and plants are being used as “factories” to produce chemicals that the organisms don’t naturally make.

  • This is accomplished by inserting, deleting, or modifying genes with recombinant DNA (rDNA) technology, which is sometimes called genetic engineering.

9.2 Tools of Biotechnology

• Humans use artificial selection to select desirable breeds of animals or strains of plants to cultivate.

• Site-directed mutagenesis is more targeted and can be used to make a specific change in a gene.

• Recombinant DNA technology has its technical roots in the discovery of restriction enzymes, a special class of DNA-cutting enzymes that exist in many bacteria.

• The bacterial DNA is protected from digestion because the cell methylates (adds methyl groups to) some of the cytosines in its DNA.

• Some of these enzymes cut both strands of DNA in the same place, producing blunt ends, and others make staggered cuts in the two strands—cuts that are not directly opposite each other.

• These staggered ends, or sticky ends, are most useful in rDNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme.

• Some plasmids are capable of existing in several different species.

  • They are called shuttle vectors and can be used to move cloned DNA sequences among organisms, such as among bacterial, yeast, and mammalian cells, or among bacterial, fungal, and plant cells.

• The polymerase chain reaction (PCR) is a technique by which small samples of DNA can be quickly amplified, that is, increased to quantities that are large enough for analysis.

9.3 Techniques of Genetic Modification

• To modify a cell, a plasmid must be inserted into a cell by transformation, a procedure during which cells can take up DNA from the surrounding environment.

• A process called electroporation uses an electrical current to form microscopic pores in the membranes of cells; the DNA then enters the cells through the pores.

• Protoplasts are produced by enzymatically removing the cell wall, thereby allowing more direct access to the plasma membrane.

• The process of protoplast fusion also takes advantage of the properties of protoplasts.

  • Protoplasts in solution fuse at a low but significant rate; the addition of polyethylene glycol increases the frequency of fusion.

  • In the new hybrid cell, the DNA derived from the two “parent” cells may undergo natural recombination.

  • This method is especially valuable in the genetic manipulation of plant and algal cells.

• DNA can be introduced directly into an animal cell by microinjection.

  • This technique requires the use of a glass micropipette with a diameter that is much smaller than the cell.

  • The micropipette punctures the plasma membrane, and DNA can be injected through it.

• The collection of clones containing different DNA fragments is called a genomic library; each “book” is a bacterial or phage strain that contains a fragment of the genome.

• An artificial gene that contains only exons can be produced by using an enzyme called reverse transcriptase to synthesize complementary DNA (cDNA) from an mRNA template.

9.4 Applications of DNA Technology

• Subunit vaccines, consisting only of a protein portion of a pathogen, are being made by genetically modifying yeasts.

• DNA vaccines are usually circular plasmids that include a gene encoding a viral protein that’s under the transcriptional control of a promoter region active in human cells.

• Gene therapy may eventually provide cures for some genetic diseases.

• Gene editing is a promising new technology to correct genetic mutations at precise locations.

• Gene silencing is a natural process that occurs in a wide variety of eukaryotes and is apparently a defense against viruses and transposons.

• Following transcription, RNAs called small interfering RNAs (siRNAs) are formed after processing by an enzyme called Dicer.

  • The siRNA molecules bind to mRNA, which is then destroyed by proteins called the RNA-induced silencing complex (RISC), thus silencing the expression of a gene.

• New technology called RNA interference (RNAi) holds promise for gene therapy for treating genetic diseases.

• In shotgun sequencing, small pieces of a genome of a free- living cell are sequenced, and the sequences are then assembled using a computer.

• DNA sequencing has produced an enormous amount of information that has spawned the new field of bioinformatics, the science of understanding the function of genes through computer-assisted analysis.

• Proteomics is the science of determining all of the proteins expressed in a cell.

• Reverse genetics is an approach to discovering the function of a gene from a genetic sequence.

  • Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism.

• The fragments are called RFLPs , for restriction fragment length polymorphisms.

• The different fragments are then separated by gel electrophoresis.

• For several years, microbiologists have used RFLPs in a method of identification known as DNA fingerprinting to identify bacterial or viral pathogens.

• The new field of forensic microbiology developed because hospitals, food manufacturers, and individuals can be sued in courts of law and because microorganisms can be used as weapons.

• The suppression was accomplished by antisense DNA technology.

9.5 Safety Issues and the Ethics of Using DNA Technology

• Genetically modified organisms intended for use in the environment (in agriculture, for example) may be engineered to contain “suicide genes”—genes that eventually turn on to produce a toxin that kills the microbes, thus ensuring that they will not survive in the environment for very long after they have accomplished their task.

• The safety issues in agricultural biotechnology are similar to those concerning chemical pesticides: toxicity to humans and to nonpest species.