Cold Spring Harbor Symposia on Quantitative Biology 

About: Cold Spring Harbor Symposia on Quantitative Biology is an academic journal. The journal publishes majorly in the area(s): Gene & DNA. It has an ISSN identifier of 0091-7451. It is also open access. Over the lifetime, 5212 publications have been published receiving 236924 citations.

Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction

Abstract: The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods. The de novo organic synthesis of oligonucleotides and the development of methods for their assembly into long double-stranded DNA molecules (Davies and Gassen 1983) have removed, at least theoretically, the minor limitations imposed by the availability of natural sequences with fortuitously unique flanking restriction sites. However, de novo synthesis, even with automated equipment, is not easy; it is often fraught with peril due to the inevitable indelicacy of chemical reagents (Urdea et al. 1985; Watt et al. 1985; Mullenbach et al. 1986), and it is not capable of producing, intentionally, a sequence that is not yet fully known. We have been exploring an alternative method for the synthesis of specific DNA sequences (Fig. 1). It involves the reciprocal interaction of two oligonucleotides and the DNA polymerase extension products whose synthesis they prime, when they are hybridized to different strands of a DNA template in a relative orientation such that their extension products overlap. The method consists of repetitive cycles of denaturation, hybridization, and polymerase extension and seems not a little boring until the realization occurs that this procedure is catalyzing a doubling with each cycle in the amount of the fragment defined by the positions of the 5' ends of the two primers on the template DNA, that this fragment is therefore increasing in concentration exponentially, and that the process can be continued for many cycles and is inherently very specific. The original template DNA molecule could have been a relatively small amount of the sequence to be synthesized (in a pure form and as a discrete molecule) or it could have been the same sequence embedded in a much larger molecule in a complex mixture as in the case of a fragment of a single-copy gene in whole human DNA. It could also have been a single-stranded DNA molecule or, with a minor modification in the technique, it could have been an RNA molecule. In any case, the product of the reaction will be a discrete double-stranded DNA molecule with termini corresponding to the 5' ends of the oligonucleotides employed. We have called this process polymerase chain reaction or (inevitably) PCR. Several embodiments have been devised that enable one not only to extract a specific sequence from a complex template and amplify it, but also to increase the inherent specificity of this process by using nested primer sets, or to append sequence information to one or both ends of the sequence as it is being amplified, or to construct a sequence entirely from synthetic fragments.

Physical Principles in the Construction of Regular Viruses

Abstract: Florida Press, Gainesville, Florida, pp. 267-291. Caspar, D. L. D. (1965) \"Design Principles in Virus Particle Construction.\" In Viral and Rickettsial Infections of Man, 4th edition (Horsfall, F. L., Jr. and Tamm, I., eds.), J. B. Lippincott Co., Philadelphia, pp. 51-93. Caspar, D. L. D. (1966) \"An Analogue for Negative Staining.\" \"Physical principles in the construction of regular viruses.\" Cold Spring Harb Symp Quant Biol 27 (1962): 1-24. b) Liddington, R. C., Y. Yan, J. Moulai, R. Sahli, T. L. Benjamin, and S. C. Harrison. c) Flint S.J., Enquist L.W., Racaniello V. R., Skalka A. M. “Principles of virology”, Chapter “Packaging the nucleic acid genome”, pp 109-112. d) Flint S.J., Enquist L.W., Racaniello V. R., Skalka A. M. “Principles of virology”, Chapter “Viruses with envelopes”, pp 114-121. 4. Nucleic Acid Packaging and Ejection. a) D'Souza, V., and M. F. Summers. \"Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.\" How do the architectures of our designs compare to those of virus capsids and other icosahedral protein complexes found in nature? Our designs obey strict icosahedral symmetry, with the asymmetric unit in each case containing a heterodimer that comprises one subunit from each of the two components. The most similar naturally occurring structures of which we are aware are cowpea mosaic virus (CPMV) and related 120-subunit capsids with pseudo T = 3 symmetry [T refers to the triangulation number 27)]. , Physical principles in the construction of regular viruses. Cold Spring Harb. Symp. Home About Archive Purchase Advertise Alerts Contact Privacy Policy Help