Connecting Concepts Across Chapters 9

This chapter has focused on recombinant DNA technology, a set of methods to isolate, study, and manipulate DNA sequences. Before the development of this technology, geneticists were forced to study genes by examining the phenotypes produced by the genes under study. The power of recombinant DNA technology is that it allows geneticists to read and alter genetic information directly, leading to an entirely new approach to the study of heredity in which genes are studied by altering DNA sequences and observing the associated change in phenotype.

A major theme of this chapter has been that working at the molecular level requires special approaches because

DNA and other molecules are too small to see and manipulate directly. A number of recombinant DNA techniques are available and can be mixed and matched in different combinations or strategies; the particular set of methods used depends both on the sequences being manipulated and on the ultimate goal of the researcher.

Mastering the information in this chapter requires an understanding of material presented in many of the preceding chapters, particularly those on molecular genetics. A detailed understanding of DNA structure (Chapter 10), replication (Chapter 12), and the genetic code (Chapter 15) are essential for grasping the details of recombinant DNA technology. Knowledge of bacterial and viral genetics (Chapter 8) is helpful, because much of gene cloning takes place in bacteria, and plasmids and viruses are commonly used as cloning vectors. Knowledge of gene regulation (Chapter 16) is useful for understanding expression vectors and recombinant DNA applications where proteins are produced.

The information presented in this chapter will complement and enhance much of the material presented in the remaining chapters of the book. Chapter 19 deals with the use of recombinant DNA technology to compare the organization, content, and expression of genomes of different organisms.

CONCEPTS SUMMARY]_

• Recombinant DNA technology is a set of molecular techniques for locating, cutting, joining, analyzing, and altering DNA sequences and for inserting the sequences into a cell.

• Restriction endonucleases are enzymes that make double-stranded cuts in DNA at specific base sequences.

• DNA fragments can be separated with the use of gel electrophoresis and visualized by staining the gel with a dye that is specific for nucleic acids or by labeling the fragments with a radioactive or chemical tag.

• Individual genes can be studied by transferring DNA fragments from a gel to nitrocellulose or nylon and applying complementary probes.

• Gene cloning refers to placing a gene or a DNA fragment into a bacterial cell, where it will be multiplied as the cell divides.

• Plasmids, small circular pieces of DNA, are often used as vectors to ensure that a cloned gene is stable and replicated within the recipient cells.

• Bacteriophage X offers several advantages over plasmids: it can hold larger fragments of foreign DNA and transfers DNA to cells with higher efficiency.

• Cosmids, which combine properties of plasmids and phage vectors, hold even larger amounts of foreign DNA. Yeast and bacterial artificial chromosomes can accommodate large inserts more than 100,000 bp in length.

• Expression vectors contain promoters, ribosome-binding sites, and other sequences necessary for foreign DNA to be transcribed and translated.

• Genes can be isolated by creating a DNA library, a set of bacterial colonies or viral plaques that each contain a different cloned fragment of DNA. A genomic library contains the entire genome of an organism, cloned as a set of overlapping fragments; a cDNA library contains DNA fragments complementary to all the different mRNAs in a cell.

• DNA libraries can be screened with probes complementary to particular genes or DNA fragments in the library can be cloned into an expression vector and screened by looking for the associated protein product.

• Genes can also be located by chromosome walking, in which a neighboring gene is used to make a probe; a genomic library is screened with this probe to find a clone that overlaps the gene. A probe is made from the end of this clone, and the probe is used to screen the library for a second clone that overlaps the first. The process is continued until the gene of interest is reached.

• The cloning strategy depends on the purpose of the cloning experiment, what is known about the gene, the size of the gene to be cloned, the size of the genome from which it is isolated, and the organism into which it will be cloned.

• The polymerase chain reaction is a method for amplifying DNA enzymatically without cloning. A solution containing DNA is heated, so that the two DNA strands separate, and then quickly cooled, allowing primers to attach to the template DNA. The solution is then heated again, and DNA polymerase synthesizes new strands from the primers. Each time the cycle is repeated, the amount of DNA doubles.

• In situ hybridization can be used to determine the chromosomal location of a gene and the distribution of the mRNA produced by a gene. DNA footprinting reveals the nucleotides that are covered by DNA-binding proteins. Site-directed mutagenesis can be used to produce mutations at specific sites in DNA, allowing genes to be tailored for a particular purpose. Transgenic animals, produced by injecting DNA into fertilized eggs, contain foreign DNA that is integrated into a chromosome. Knockout mice are transgenic mice that have a normal gene disabled.

• Recombinant DNA technology has many applications, including not only the production of pharmaceuticals and other biological substances in bacteria but also the creation of bacteria that are genetically engineered for economically or medically important tasks. It is also being used in agriculture to transfer particular traits, such as disease and pest resistance, to crop plants. Transgenic domestic animals can be produced with desirable traits. Oligonucleotide drugs—short nucleotide sequences for treating diseases—are another application of recombinant DNA technology.

• In gene therapy, diseases are being treated by altering the genes of human cells.

• Restriction fragment length polymorphisms and variable number tandem repeats facilitate gene mapping by making available numerous genetic markers and are being used to identify people by their DNA sequences (DNA fingerprinting).

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