1205 ed sequentially to the 3'end of the primer from the corresponding deoxynucleoside triphosphates, making a com-plementary copy of the template DNA. By using triphosphates containing 32p in the a position, the newly synthesized DNA can be labeled. In the early experi-ments synthetic oligonucleotides were used as primers, but after the discovery of restriction enzymes it was more con-venient to use fragments resulting from their action as they were much more easily obtained.

The copying procedure was used initially to prepare a short, specific region of labeled DNA which could then be subjected to partial digestion proce-dures. One of the difficulties of sequenc-ing DNA was to find specific methods for breaking it down into small frag-ments. No suitable enzymes were known that would recognize only one nucleotide. However, Berg, Fancher, and Chamberlin (7) had shown earlier that under certain conditions it was possible to incorporate ribonucleotides, in place of the normal deoxyribonucleotides, into DNA chains with DNA polymerase. Thus, for instance, if copying were car-ried out with ribo CTP (7a) and the other three deoxynucleoside triphosphates, a chain could be built up in which the C residues were in the ribo form. Bonds involving ribonucleotides could be broken by alkali under conditions where those involving the deoxynucleotides were not, so that a specific splitting at C residues could be obtained. Using this method we were able to extend our se-quencing studies to some extent (8). However extensive fractionations and analyses were still required.