After studying this chapter, you should be able to:
Understand 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.
Describe 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 or RNA.
The development of recombinant DNA techniques, high-density DNA microarrays, high-throughput screening, low-cost genome-scale DNA and RNA sequencing, and other molecular genetic methodologies has revolutionized biology and is having an increasing impact on clinical medicine*. Although 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 cellular DNA and RNA molecules for information. Manipulation of a DNA sequence and the construction of chimeric molecules—so-called genetic engineering—provide a means of studying how a specific segment of DNA controls cellular function. New biochemical and molecular genetic tools allow investigators to query and manipulate genomic sequences as well as to examine the entire complement of cellular RNA, protein, and protein PTM status at the molecular level, even in single cells.
Understanding molecular genetics technology is important for several reasons: (1) it offers a rational approach to understanding the molecular basis of disease. 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, Alzheimer disease, cancer, obesity, and diabetes. (2) Human proteins can be produced in abundance for therapy (eg, insulin, growth hormone, tissue plasminogen activator). (3) Proteins for preparation of vaccines (eg, hepatitis B) and for diagnostic testing (eg, Ebola and AIDS tests) can be readily 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 pharmacologic therapeutics—so called personalized medicine. (5) Special techniques have led to remarkable advances in forensic medicine, which have allowed for the molecular diagnostic analysis of DNA from single cells. (6) Finally, in extremely well understood diseases, potentially curative gene therapy for diseases caused by a single-gene deficiency such as sickle cell disease, the thalassemias, adenosine deaminase deficiency, and others may be devised.
RECOMBINANT DNA TECHNOLOGY INVOLVES ISOLATION & MANIPULATION OF DNA TO MAKE CHIMERIC MOLECULES
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 ...