Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Sequencing end-labeled DNA with base-specific chemical cleavages.

https://www.cell.com/cell/pdf/0092-8674(93)90530-4.pdf

Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

3.7. Palladium Nanoparticles as Catalysts 888 3.8. Other Transition-Metal Complexes 888 3.9. Transition-Metal-Free Reactions 889 4. Applications 889 4.1. Alkynylation of Arenes 889 4.2. Alkynylation of Heterocycles 891 4.3. Synthesis of Enynes and Enediynes 894 4.4. Synthesis of Ynones 896 4.5. Synthesis of Carbocyclic Systems 897 4.6. Synthesis of Heterocyclic Systems 898 4.7. Synthesis of Natural Products 903 4.8. Synthesis of Electronic and Electrooptical Molecules 906

https://www.chem.ccu.edu.tw/~joyce/referance/ericyang/Chem.%20Rev/Chem.%20Rev.%202007,%20107,%20874-922.pdf

The Sonogashira Reaction: A Booming Methodology in Synthetic Organic Chemistry†

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics

Publisher Summary This chapter reviews the current state of the knowledge of transcription and translation in Caenorhabditis elegans. Transcription in eukaryotes is accomplished by three RNA polymerases, each transcribing a particular type of RNA; RNA polymerase I transcribes ribosomal RNA, RNA polymerase II transcribes mRNAs and snRNAs, and RNA polymerase III transcribes tRNAs and other low-molecular-weight RNAs. The chapter focuses on RNA polymerase II transcription. The C. elegans gene for the large subunit of RNA polymerase II has been cloned, and has shown to encode a protein that resembles its vertebrate counterparts. For most polymerase II transcribed genes in C. elegans, a sequence fitting the consensus TATA element about 30 bp upstream of the transcriptional start site is found. Recently, a cDNA encoding the TATA box binding protein, or TATA binding protein (TBP) subunit of transcription factor II D (TFIID), has been identified in the nematode. This protein shows extended regions of sequence similarity to vertebrate TBP, binds to a TATA box sequence and can substitute for vertebrate TFIID basal activity in in vitro extracts. These results illustrate a high degree of evolutionary conservation in the basic transcription machinery.

Transcription and translation.

Synthesis and duplex stability of oligonucleotides containing adenine-guanine analogues

Analytical study of phosphorothioate analogues of oligodeoxynucleotides using high-performance liquid chromatography

Selective reaction of methoxyamine with cytosine bases in tyrosine transfer ribonucleic acid.

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

/pdf/lethal-mutagenesis-of-poliovirus-mediated-by-a-mutagenic-192b3c9qdn.pdf

Daniel M. Brown

Papers

Synthesis and duplex stability of oligonucleotides containing adenine-guanine analogues

Analytical study of phosphorothioate analogues of oligodeoxynucleotides using high-performance liquid chromatography

Selective reaction of methoxyamine with cytosine bases in tyrosine transfer ribonucleic acid.

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

The structure of triphosphoinositide from beef brain.