Kary B. Mullis

The Polymerase chain reaction

Edited by the inventor of polymerase chain reaction (PCR) and the 1993 Nobel Prize winner in Chemistry, Kary Mullis, as well as two experts in the field, this handbook provides up-to-date methodological protocols from the worlds leading laboratories, in addition to new techniques and enhanced applications not yet available in book form. Nearly 40 chapters inform the novice and experienced PCR user on how to optimize their results. In the chapters on applications, researchers provide not only protocols, but also descriptions of how PCR has revolutionized their particular field. Future enhancements of PCR, as well as new potential uses, are discussed. Readers can learn how PCR has changed the face of diagnostic testing, cancer research, genetics, forensics, plant biology, DNA sequencing and gene therapy. Special sections include the latest on QPCR, non-isotopic detection, genetic analysis, and PCR and the world of business, which includes a behind-the-scenes look at the legal battles between biotech giants Cetus and DuPont. It also provides insights into the origins of PCR and the history of nucleic acid research.

Opiate antagonist receptor binding in vivo: evidence for a new receptor binding model.

The in vivo accumulation and retention of the opiate antagonist tracers [3H]diprenorphine and [3H]naloxone at cerebral opiate receptor sites in rats exceed that expected from their known in vitro receptor affinities. The [3H]diprenorphine serum and brain levels can be stimulated with a pharmacokinetic model that contains the receptors in a micro-compartment. The receptor micro-compartment consists of a population of binding sites next to a diffusion boundary which restricts ligand diffusion away from the receptor. Such an arrangement introduces a delay in the binding equilibrium of potent antagonists with the receptor sites and an increase in the apparent in vivo receptor affinity at subsaturating doses of the ligand; at saturating ligand concentrations these functions of the receptor micro-compartment are abolished. A physiological interpretation of the receptor micro-compartment could be the location of clustered opiate receptor sites on the exterior cell surface next to the synaptic cleft as the diffusion boundary. This kinetic approach involving a combination of pharmacokinetics and drug-receptor interactions permits the quantitative analysis of receptor site availability in the intact animal. Our results support the hypothesis that only one receptor population affects the in vivo disposition of the antagonist tracers, while they do not exclude the presence of low affinity binding sites that have been observed with the use of [3H]naloxone in vitro. Moreover, the binding site population observed in vivo may be responsible for mediating opiate agonist analgesia.

The Effect of Chronic Alveolar Hypoxia on Lung and Serum Angiotensin I Converting Enzyme Activity

Summary Mice exposed to chronic alveolar hypoxia showed elevations of serum and lung angiotensin I converting enzyme activity during the second week of exposure. The increment of activity of the lung and serum converting enzyme was of the same order of magnitude and occurred at approximately the same time. An increase in renal renin granules was closely correlated with both lung and serum converting enzyme activity.

Angiotensin-I-converting enzyme activity in idiopathic respiratory distress syndrome of the newborn infant and in experimental alveolar hypoxia in mice*

Serum angiotensin-I-converting enzyme activity was found to be elevated in infants with idiopathic respiratory distress syndrome when compared with healthy premature infants, normal infants, and acutely ill full-term infants. Serum and lung CE activity has been found to be elevated in mice exposed to hypobaric alveolar hypoxia which also stimulated renal renin production. These findings suggest that alveolar hypoxia stimulates the renin-angiotensin-aldosterone system and this system may be involved in the response to the stress of IRDS.

PCR and Scientific Invention: The Trial of DuPont vs. Cetus

Had I known my way around in what they call the real world, I could have easily been bringing home a million dollars. I had no idea that my market value had soared temporarily to the level of a fairly good pitcher until the trial was over, and then it was too late.

9 – Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction

Publisher Summary This chapter discusses specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. A source of DNA including the desired sequence is denatured in the presence of a large molar excess of two oligonucleotides and the four deoxyribonucleoside triphosphates. Oligonucleotides were synthesized using an automated DNA synthesis machine using phosphoramidite chemistry. Mispriming can be usefully employed to make intentional in vitro mutations or to add sequence information to one or both ends of a given sequence. Amplifications of other human loci have resulted in varying degrees of specificity and efficiency. The polymerase chain reaction has thus found immediate use in developmental DNA diagnostic procedures and in molecular cloning from genomic DNA.

STUDY OF OPIATE RECEPTOR MULTIPLICITY IN RATS BY IN VIVO BINDING ASSAYS

Reproducible methods have been developed for the determination of the binding of H labeled opiate tracers to rat cerebral binding sites in the intact animal. The results support the existence of multiple opiate receptor sites and indicate that agonist binding studies performed in vivo may yield data different from in vitro receptor binding results.