After studying this chapter, you should be able to:
- Explain the basic procedures and methods involved in recombinant DNA technology and genetic engineering.
- Appreciate the rationale behind the methods used to synthesize, analyze, and sequence DNA and RNA.
- Explain how to identify and quantify individual proteins, both soluble and insoluble (ie, membrane bound or compartmentalized intracellularly) proteins, as well as proteins bound to specific sequences of genomic DNA and RNA.
The development of recombinant DNA, high-density DNA microarrays, high-throughput screening, low-cost genome-scale analyses, DNA sequencing and other molecular genetic methodologies has revolutionized biology and is having an increasing impact on clinical medicine. Though much has been learned about human genetic disease from pedigree analysis and study of affected proteins, in many cases where the specific genetic defect is unknown, these approaches cannot be used. The new technologies circumvent these limitations by going directly to the DNA molecule for information. Manipulation of a DNA sequence and the construction of chimeric molecules—so-called genetic engineering—provides a means of studying how a specific segment of DNA works. Novel biochemical and molecular genetic tools and direct DNA sequencing allow investigators to query and manipulate genomic sequences as well as to examine the entire complement of cellular RNA, protein profiles and protein PTM status at the molecular level.
Understanding this technology is important for several reasons: (1) it offers a rational approach to understanding the molecular basis of a number of diseases. For example, familial hypercholesterolemia, sickle-cell disease, the thalassemias, cystic fibrosis, muscular dystrophy as well as more complex multifactorial diseases like vascular and heart disease, cancer, and diabetes. (2) Human proteins can be produced in abundance for therapy (eg, insulin, growth hormone, and tissue plasminogen activator). (3) Proteins for vaccines (eg, hepatitis B) and for diagnostic testing (eg, Ebola and AIDS tests) can be obtained. (4) This technology is used both to diagnose existing diseases as well as to predict the risk of developing a given disease and individual response to pharmacological therapeutics. (5) Special techniques have led to remarkable advances in forensic medicine. (6) Gene therapy for potentially curing diseases caused by a single-gene deficiency such as sickle-cell disease, the thalassemias, adenosine deaminase deficiency, and others may be devised.
*See glossary of terms at the end of this chapter.
Isolation and manipulation of DNA, including end-to-end joining of sequences from very different sources to make chimeric molecules (eg, molecules containing both human and bacterial DNA sequences in a sequence-independent fashion), is the essence of recombinant DNA research. This involves several unique techniques and reagents.
Restriction Enzymes Cleave DNA Chains at Specific Locations
Certain endonucleases—enzymes that cut DNA at specific DNA sequences within the molecule (as opposed to exonucleases, which digest from the ends of DNA molecules)—are a key tool in recombinant DNA research. These enzymes were called restriction enzymes because their presence in a given bacterium restricted the growth ...