Nucleotides poly-, synthesis

In the absence of deoxyribonucleoside triphosphates, DNA polymerases with associated 3 - 5 exonucleases would eventually degrade duplex DNA to mononucleotides. The replacement DNA synthesis method thus requires suf-ficiendy high levels of deoxyribonucleotides to maximize nucleotide poly-... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

This at once raises the question Could RNA ever have arisen without the help of enzymes, without replicases Experiments by Leslie Orgel and his co-workers12 suggest that this was possible. It was found that zinc ions, found today as cofactors in all replicases, are excellent catalysts for the 3 -5 union of nucleotides, thus allowing the template-instructed synthesis of polymers. This was first demonstrated with poly-C as template. If activated G and A nucleotides are offered, in equal concentration, then G is preferentially incorporated into the product by a factor, depending on reaction conditions, between 30 and 200. 

These early contributions were later supplemented by a comprehensive article on the mononucleotides by Ueda and Fox.4 The present article is an extension of these articles to chemical synthesis of oligo-and poly-nucleotides, and it is hoped that, in conjunction with the earlier articles, it will provide the organic chemist with a reasonable appreciation of some of the challenges, both past and present, of the synthetic chemistry of the nucleic acids. 

In view of the central role of the nicotinamide nucleotides in energy-yielding metabolism, and the fact that, at least in theory, the nicotinamide released by ADP-ribosyltransferase (Section 8.4.2) and poly(ADP-ribose) polymerase (Section 8.4.3) is available to be reutilized for nucleotide synthesis (although this may not occur when these enzymes are significantly activated), niacin requirements are conventionally calculated on the basis of energy expenditure. 

Reverse transcription PCR (RT-PCR) is a modification of the standard PCR technique that can be used to amplify mRNA. The first step is to convert isolated mRNA to a complementary DNA (cDNA) molecule using an RNA-dependent DNA polymerase (also known as reverse transcriptase) during a process called reverse transcription (RT). The complementary DNA can be used as any other DNA molecule for PCR amplification. The primers used for cDNA synthesis can be either nonsequence-specific primers (a mixture of random hexa-mers or oligo-dT primers) or sequence-specific primers (Fig. 2.4). Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool. Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules. Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process. 

Oligo- and poly-nucleotides 50 years of chemical synthesis , Reese, C. B., Org. Biomol. Chem., 2005, 3, 3851. 

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