CH. 20- DNA Tools and BiotechnologyCH. 20- DNA Tools and Biotechnology- The DNA Toolbox o Recently the genome sequences of two extinct species—Neanderthals and woolymammoths— have been completed. o Advances in sequencing techniques make genome sequencing increasingly fasterand less expensive. o Biotechnology is the manipulation of organisms or their components to make useful products. o The applications of DNA technology affect everything from agriculture, to criminal law, to medical research. o The complementarity of the two DNA strands is the basis for nucleic acid hybridization, the base pairing of one strand of nucleic acid to the complementary sequence on another strand. o Genetic engineering is the direct manipulation of genes for practical purposes. - DNA Sequencing o Researchers can exploit the principle of complementary base pairing to determine a gene’s complete nucleotide sequence, called DNA sequencing. o The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger o “Next-generation sequencing” techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments  Thousands or hundreds of thousands of fragments (400–1,000 nucleotides long) are sequenced in parallel  This is a type of “high-throughput” technology  In “third-generation sequencing,” the techniques used are even faster andless expensive than the previouso To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning. o Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome. o Researchers can insert DNA into plasmids to produce recombinant DNA, a molecule with DNA from two different sources. o The production of multiple copies of a single gene is a type of DNA cloning called gene cloning. o A plasmid used to clone a foreign gene is called a cloning vector. - Using Restriction Enzymes to Make a Recombinant DNA Plasmido Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites. o A restriction enzyme usually makes many cuts, yielding restriction fragments. o The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends”. - Amplifying DNA o The polymerase chain reaction, PCR, can produce many copies of a specific targetsegment of DNA.  PCR uses a pair of primers specific for the sequence to be amplified. PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells.  PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors. - Determining Gene Function o One way to determine function is to disable the gene and observe the consequences. o Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function.o When the mutated gene is returned to the cell, the normal gene’s function mightbe determined by examining the mutant’s phenotype.  Gene expression can also be silenced using RNA interference (RNAi).  Synthetic double-stranded RNA molecules matching the sequence of a gene is used to break down or block the gene’s mRNA.- Cloning Plants: Single-Cell Cultureso In plants, cells can dedifferentiate and then give rise to all the specialized cell types of the organism. o A totipotent cell, such as this, is one that can generate a complete new organism  Plant cloning is used extensively in agriculture. - Human Gene Therapyo Gene therapy is the alteration of an afflicted individual’s genes. o Gene therapy holds great potential for treating disorders traceable to a single defective gene. o Vectors are used for delivery of genes into specific types of cells, for example bone marrow. o Gene therapy provokes both technical and ethical