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6 Determination of the Primary Structure of Nucleic Acids

6.1 Introduction

One of the most spectacular achievements of molecular biology in the past decade was the development of very rapid methods for determining the primary structure of DNA and, later, RNA, which are known as sequencing that is, determining the sequence of nucleotides in these biopolymers. Although the currently used sequencing techniques appeared as recently as 1977, their origin is to be found in the various branches of nucleic acid chemistry and enzymology, which were already well developed in the sixties.

In the early seventies, all those involved in sequencing could not help being fascinated by what became known as the block method of RNA sequencing. Developed in the mid sixties to determine the nucleotide sequence in tRNA, this method had made it possible to establish the structure of hundreds of tRNAs, many 5S RNAs, and even some high-molecular weight RNAs within mere five to eight years.

Naturally, the first DNA sequencing methods (early seventies) were closely similar to the block method. Radical changes had taken place by the mid seventies, when it became clear that the block method of RNA sequencing could not be applied to DNA. The decisive factor was the absence of enzymes hydrolysing DNA at one or only two of the four bases (analogs of guanyl and pyrimidyl RNases). The cul-de-sac into which scientists had worked themselves forced them to think about drastically new approaches. The problem had boiled down to developing a new concept of sequencing. Soviet scientists were involved in its elaboration at the very outset (see, e. g., the paper by Ye. D. Sverdlov and coworkers, published in FEBS Lett. in 1973). The new principle of determining the primary structure of DNA may be formulated as follows: label the ends, cleave the long polynucleotide statistically at one of the four nucleotides, fractionate the fragments, and measure the length of each labeled fragment. However, before this concept was embodied in a working method for sequencing high-molecular weight DNAs then RNAs, two methodological difficulties had to be obviated. First of all, it was necessary to develop a technique of fractionation along the length of oligo- and polynucleotides differing by a single nucleotide link (or a single negative charge). Sets of such fragments are formed when the new sequencing concept is put to practical use. Although homochromatography was used as recently as the late sixties to separate small oligonucleotides, the problem was solved only with the advent of such a powerful technique as polyacrylamide gel electrophoresis (PAGE).

The other problem was finding a way to isolate individual DNA fragments (individual high-molecular weight RNAs in the case of RNA) necessary for analysis. Used initially for the purpose were bacterial plasmids, bacteriophages, or animal viruses (e. g., SV40) containing homogeneous DNAs, which made it possible to easily produce several hundreds of micrograms or 100 picomoles of DNA for analysis. However, it was only advances in genetic engineering, especially in molecular cloning, that permitted experimenters to isolate in any amounts and easily identify any fragments of genomic DNAs under investigation. State-of-the-art methods allow one to sequence nucleic acid fragments consisting of no more than 500 nucleotides within a single experiment. Such a limitation is imposed by the current possibilities of gel electrophoresis. This is why molecules containing a thousand and more pairs of nucleotides are usually split into so-called restriction fragments with the aid of restriction enzymes.

The methodological difficulties were obviated along with elaboration of the new sequencing concept. The first seminal work outlining the polymerase copying method was published by F. Sanger in 1975 under the title "A Rapid Method for Determining Sequences in DNA by Primed Synthesis with DNA Polymerase" (the updated sequencing technique was described in 1977). It was followed by another work of equal importance, based on chemical destruction of DNA, which appeared in 1977: "A New Method for Sequencing DNA" by A. Maxam and W Gilbert.

Thus, the DNA sequencing methods belong essentially to two types of techniques differing in the exact way to obtain statistical sets of labeled oligo- and polynucleotides, which allow the nucleotide sequence in the DNA under investigation to be "read" immediately after fractionation. One of the techniques (Sanger's method) resides in enzymatic synthesis of DNA copies (cDNAs) terminating at one of the four nucleotides; the other technique is based on chemical cleavage of DNA at one or only two of the four nucleotides (Maxam-Gilbert method). In spite of the Sanger's method appearing before that developed by Maxam and Gilbert, the chemical method will be considered first in keeping with the general purpose of this textbook to cover as fully as possible recent developments in the chemistry of nucleic acids.

The revolutionary impact of the new methods on DNA sequencing was felt immediately by those involved in the sequencing of high-molecular weight RNAs.

The traditional block method for determining the primary structure of RNA had reached its limit by the late seventies during sequencing of single-stranded RNA of phage MS-2, containing 3500 nucleotides. It became evident at that time that rapid sequencing methods must be applied to RNA as well. This book also covers the rapid RNA sequencing methods. The solid-phase technique for sequencing DNA and RNA will be discussed in what follows along with the use of computers in this area.