Chapter 20 Flashcards
• Recently the genome sequences of two extinct species—Neanderthals and wooly mammoths—have been completed
• Advances in sequencing techniques make genome sequencing increasingly faster and
less expensive
Biotechnology is the manipulation of organisms or their components to make useful products
• The applications of DNA technology affect everything from agriculture, to criminal law,
to medical research
• Concept 20.1: DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
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
- Genetic engineering is the direct manipulation of genes for practical purposes
- DNA Sequencing
Researchers can exploit the principle of complementary base pairing to determine a gene’s complete nucleotide sequence, called DNA sequencing
The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger
• “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 and less expensive than
the previous
• Making Multiple Copies of a Gene or Other DNA Segment
•
To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
• Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
• Researchers can insert DNA into plasmids to produce recombinant DNA, a molecule with
DNA from two different sources
• Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
•
This results in the production of multiple copies of a single gene
• The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
• A plasmid used to clone a foreign gene is called a cloning vector
• Bacterial plasmids are widely used as cloning vectors because they are readily obtained, easily manipulated, easily introduced into bacterial cells, and once in the bacteria they multiply rapidly
•
Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes
• Using Restriction Enzymes to Make a Recombinant DNA Plasmid
• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction fragments
• The most useful restriction enzymes cut DNA
in a staggered way, producing fragments with “sticky ends”
•
- Sticky ends can bond with complementary sticky ends of other fragments
- DNA ligase is an enzyme that seals the bonds between restriction fragments
• To check the recombinant plasmid, researchers might cut the products again using the same restriction enzyme
• To separate and visualize the fragments produced, gel electrophoresis would be carried out
• This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size, charge, or other physical properties
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Figure 20.7
• Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules
• The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase
• 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
• Figure 20.8
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PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
• The resulting clones are sequenced and
error-free inserts selected
• Figure 20.9
• Expressing Cloned Eukaryotic Genes
• After a gene has been cloned, its protein product can be produced in larger amounts for research
• Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
• Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes
• Even yeasts may not possess the proteins required to modify expressed mammalian
proteins properly
• In such cases, cultured mammalian or insect
cells may be used to express and study proteins
•
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One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes
• Alternatively, scientists can inject DNA into cells using microscopically thin needles
• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination
• Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
- Reverse transcriptase is added to mRNA to make complementary DNA (cDNA), which serves as a template for PCR amplification of the gene of interest
- The products are run on a gel and the mRNA of interest is identified
• Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
• With rapid and inexpensive sequencing methods available, researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
•
By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
