Chapter Capsule

A. Amazing DNA

1. DNA readily anneals (becomes temporarily denatured) when heat breaks the hydrogen bonds holding the double helix together, and it separates into two strands. Slow cooling causes single DNA strands to rejoin (renature) at complementary sites.

2. DNA can also be clipped crosswise at specific sites by means of bacterial enzymes called restriction endonucleases. These enzymes leave short tails called sticky ends that base-pair with complementary tails on linear DNA or plasmids. Endonucleases allow splicing of genes into specific sites and create fragments that circularize into a plasmid.

3. Fragments produced by restriction endonucleases—restriction fragment length polymorphisms (RFLPs)—vary in length and are inherited in predictable patterns that make them useful markers of unique genetic characteristics.

4. Ligases rejoin sticky ends made by endonucleases for final splicing of genes into plasmids and chromosomes.

5. Reverse transcriptase can convert RNA into DNA. Complementary, or cDNA, of messenger, transfer, and ribosomal RNA provide a valuable means of synthesizing eucaryotic genes from mRNA transcripts.

6. Gel electrophoresis can produce a readable pattern of DNA fragments. When samples in gel are subjected to an electrical current, the DNA pieces migrate in the gel substrate toward the positive pole, forming a pattern based on fragment size. The pattern is analyzed by comparing it against known standards to characterize genetic similarities.

7. Oligonucleotides are short fragments of DNA or RNA usually made by DNA synthesizers. The exact sequence of the base pairs in DNA is automatically analyzed by sequencers. Sequencing is critical for detailed gene analysis.

B. Nucleic Acid Hybridization and Probes

1. The ability of single-stranded nucleic acids to hybridize or join together at complementary sites makes it possible to use gene or hybridization probes to detect specific nucleotide sequences.

a. Hybridization probes can identify unknown samples or isolate precise fragments from a complex mixture. In the Southern blot method, probes are used to label a gene or nucleotide sequence on an electrophoretic gel; the blot hybridization is a rapid, direct probe of a sample. Probes are used for diagnosing the cause of an infection from a patient’s specimen and identifying a culture of an unknown bacterium or virus with a microbe-specific probe.

b. In situ hybridization uses probes on intact cells to visualize the presence and location of specific nucleic acids such as genes on chromosomes or RNA in cells and tissues.

c. DNA sequencing machines can produce highly accurate maps of the exact sequence of DNA. The Sanger sequencer synthesizes and analyzes DNA fragments using fluorescent dyes to label bases.

2. The polymerase chain reaction (PCR) is a valuable tool that can amplify the amount of DNA in a sample from a few copies to billions of copies in a few hours.

a. The PCR technique operates by repetitive cycling of three basic steps: (1) denaturation—heating target DNA to separate it into two strands, called amplicons; (2) addition of primers (synthetic oligonucleotides) to serve as guides for positioning the start of DNA amplification; (3) extension—thermostable DNA polymerase synthesizes the complementary strand by adding appropriate nucleotides.

b. After the first cycle, the DNA strands will be denatured to be primed and extended in the second cycle, thereby doubling the number of copies to four. This doubling can be continued for 20 to 30 cycles.

A. Technical aspects of recombinant DNA (rDNA) technology methods deliberately remove genetic material from one organism and combine it with that of a different organism.

1. An important objective of rDNA is the formation of genetic clones, or duplicates. Cloning involves the selection and removal of a foreign gene from an animal, plant, or microorganism (the genetic donor) followed by its propagation in a different host organism. Insertion of the foreign gene is accomplished by means of a vector (usually a plasmid or virus) that will carry the DNA into a cloning host, usually a bacterium or yeast.

2. A target gene can be isolated or prepared by (1) analysis of the genetic donor’s chromosomes through Southern blotting and screening techniques; (2) synthesis by reverse transcription from an mRNA transcript; and (3) synthesis by machines. Cloned genes are maintained in vectors, producing genomic libraries of donor genes.

B. Characteristics of Cloning Vectors and Cloning Hosts

1. The best recombinant vectors are plasmids and bacteriophages that carry a significant piece of donor DNA and can be readily transferred into appropriate host cells.

2. Examples include E. coli plasmids; a modified phage vector, the Charon phage; and a hybrid vector formed by merging a plasmid and a phage, called a cosmid. Other systems for cloning large pieces of foreign DNA are yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs).

3. The best cloning hosts have a rapid growth rate, are nonpathogenic, have a well-delineated, simple genome, can function with plasmids and virus vectors, and will replicate and express foreign genes. Common cloning hosts are special strains of E. coli and Saccharomyces cerevisiae.

C. Construction of a Chimera—The Recombination Process

1. In the first cloning step, the foreign gene is excised with an endonuclease and spliced into a plasmid that has been cut with the same endonuclease so that the terminal nucleotides of the two will properly mate; the bonds are sealed by a ligase.

2. The recombinant plasmid, or chimera, can be introduced by transformation into the proper bacterial cloning host. Recombinant clones are identified by their antibiotic resistance.

3. Progeny cells containing the chimera gene can express the foreign gene by synthesizing the polypeptide it codes for and secreting it.

4. After final processing, a relatively pure product is left. This process may be done on an industrial scale to mass-produce a variety of hormones, enzymes, and agricultural products.

A. Recombinant Bacteria and Viruses

1. Recombinant DNA is used to genetically alter Pseudomonas syringae to make a commercial product called Frostban that can prevent ice crystals from forming on plants in the field. A natural insecticide gene was added to Pseudomonas fluorescens to colonize plant roots. Biotechnologists have developed bacteria to clean up oil spills and degrade pollutants.

2. Viral vectors have been developed for testing drug resistance in Mycobacterium tuberculosis, gene therapy, an experimental AIDS vaccine, and to introduce resistance genes into crops.

B. Transgenic Plants

Agrobacterium bacteria can invade plant cells and integrate their DNA into the genome causing a tumor called crown gall disease. This ability makes them valuable for inserting foreign genes into plant genomes through a special Ti plasmid using rDNA techniques. Infection of the plant with the recombinant bacteria automatically transfers the foreign gene into its cells. Transfection of dicot plants has led to built-in pesticide and pathogen resistance in many species. Other plants are transfected with gene guns that forcefully impel genes into plant embryos.

C. Transgenic Animals

1. Several hundred strains of transgenic animals have been introduced by research and industry to study genetic diseases, to test new genetic therapies, and to become “factories” to manufacture proteins and other products.

2. Common transgenic animals are mice transfected by inserting genes into the embryo (gene line engineering). Mice have been engineered with human genes for growth hormone and to create animal models for human diseases (refer to table 10.4 for details).

A. Gene therapy has the potential to cure genetic diseases by replacing a faulty gene. The ex vivo method uses virus vectors containing the normal gene to transfect human tissues in a test tube. The transfected cells are then reintroduced into the patient’s body. In in vivo therapy, the body is directly infected with virus vectors.

B. Antisense and Triplex DNA Technology

1. Antisense and triplex agents are oligonucleotide drugs delivered into cells to block undesirable expression of genes. Antisense refers to a nucleic acid strand that is complementary to the sense, or translatable, strand. Antisense drugs (usually DNA) are chemically modified agents that bind to a target mRNA and interfere with its reading on ribosomes.

2. Triplex DNA is a triple helix formed when a third strand of DNA forms hydrogen bonds with the purine bases on one of the helices. This extra strand can make the DNA template inaccessible to normal transcription.

A. Gene mapping is a way to demarcate the nature of a genome. Location, or physical maps, indicate sites of the genes on chromosomes, and sequence maps provide the order of base pairs in a gene.

1. The Human Genome Project is a long-term attempt to map the 40,000 genes in the human genome.

2. The completed map will present detailed genetic information on inherited diseases and will allow genetic screening, enabling families to know their risks for certain diseases.

B. DNA Fingerprinting

The exact way DNA nucleotides are combined is unique for each individual, and DNA technology can be used to array the genome in patterns for comparison. It involves releasing DNA from cells, isolating it, and exposing it to restriction endonucleases that cleave the DNA into a set of relatively unique fragments. After the fragments have been separated by gel electrophoresis, probes are applied to highlight specific restriction landmarks (RFLPs).

1. The pattern of bands is called a DNA fingerprint. This technique can identify hereditary relationships and inheritance patterns of genetic diseases.

2. It is also used to keep genetic records in the military and to analyze ancient DNA for comparative and evolutionary studies. Anthropologists and historians have applied the technology in tracing the possible origins of humans.