Polymerase chain reaction | EPFL Graph Search

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993. PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics. The

Access to long-chain polyketides by double-elongation reactions

Christopher Flowers

Due to the spongistatins' fascinating and complex molecular architectures, and their important biological activities, these marine macrolides have been the subject of intensive efforts directed towards their total synthesis. As an attractive alternative to isolation from marine microorganisms, which usually provides only minuscule quantities of this family of macrolides, total synthesis aims to deliver sizable amounts of the natural product, along with enabling access to novel structural analogues, for further biological evaluation. These factors, combined with the impressive molecular architectures themselves, present compelling and formidable challenges to contemporary organic synthesis with regard both to strategy and methodology for their total synthesis in the laboratory. To this end, efforts targeting such complex macrolides provide an arena in which the synthetic chemist can develop new and more efficient methodologies and display such advances to the ultimate task of complex target synthesis. Through this process, new levels of stereocontrol and novel strategies to streamline the entire synthetic route help evolve the field as a whole. The synthetic efforts directed at the AB and CD spiroacetals of the northern fragment are herein disclosed. In conjunction with the synthesis of the southern fragment within our group, this renders itself as an original and efficient formal total synthesis of spongistatin 1. The synthesis of these key fragments has been optimized to reduce protections, set up chirality in an efficient and absolute manner and avoid fluctuations in oxidation states. Achievements in these aspects, that are often used to define the art of modern day total synthesis, have resulted in a rapid synthesis of these key fragments. The synthesis of the AB and CD spiroacetals was completed in 7 and 11 steps, respectively, rendering the process competitive with the most rapid previous synthesis to date. The sequences are based around the use of 1,3-bis(trimethylsilyl)-2-methylidenepropane in double elongation Sakurai reactions with both aldehydes and ketones to form advanced functionalized polyketides.

EPFL2010

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms

Gerardo Turcatti

Different chemical methods used to attach oligonucleotides by their 5'-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5'-specific linkage that satisfyingly withstands PCR conditions and leaves the 3'-ends available for DNA polymerase activity. The best results were obtained with 5'-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5'-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.

Oxford University Press2000

SV40 activates transcription from the transferrin receptor promoter by inducing a factor which binds to the CRE/AP-1 recognition sequence

Peter Martin Beard, Nicole Paduwat

During the course of lytic infection by simian virus 40 (SV40), expression of both the viral late genes and certain host cellular genes is induced. The promoter of the cellular transferrin receptor (TR) gene contains a DNA sequence which is similar to the AP-1- and AP-4-binding region in SV40 which has been implicated in the control of the viral late promoter. Expression of TR is needed for cells to enter S-phase and is therefore expected to be important for the SV40 lytic cycle. Here we show that the level of TR mRNA in vivo was increased by SV40 infection. A factor which activates transcription from the TR promoter in vitro was specifically induced in SV40-infected cells. Gel mobility shift assays with an oligonucleotide comprising this part of the TR promoter showed three nucleoprotein complexes to be formed with proteins from CV-1 cells. Following SV40 infection, one of the complexes was increased ten-fold. Formation of this complex was specifically reduced by competition with the phorbol ester-responsive element of the collagenase gene, implying that the factor is a member of the AP-1/Jun/Fos family. Cross-linking of the complex by ultraviolet light showed major DNA-binding components to be proteins of about 55 kD and 47 kD. Removal of this factor by adding the oligonucleotide to in vitro transcription reactions with the TR promoter, abolished the activation of TR transcription. The factor which binds to the TR promoter co-sedimented with SV40 chromosomes extracted late in infection. This suggests that similar transcriptional regulatory proteins are involved in controlling transcription from both the SV40 and the TR promoters, and that the virus can use a common mechanism to induce viral and host cellular transcription.

1991

Access to long-chain polyketides by double-elongation reactions

Christopher Flowers

EPFL2010