Abstract: A method is described for constructing double-stranded DNA using messenger RNA templates. DNA strands complementary to the templates are formed using a non-specific 3' hydroxy terminus primer. After separation, the DNA single strands are disabled from self priming by blocking the free 3' hydroxy. Primer segments are then provided complementary to a portion of the amino terminus-coding end of the desired single strands. These primer segments are then extended with a suitable polymerase to complete the complementary strands.

Abstract: A method for altering a single nucleotide at a predetermined position in a gene. The method involves the isolation of a single strand gene fragment extending up to the position before the nucleotide to be altered. A ribonucleotide or a protected deoxyribonucleotide corresponding to the desired altered nucleotide is attached at the end of this fragment. The fragment is then annealed to a complementary template which extends beyond the end of the fragment. The fragment is then extended complementary to the remainder of the template. The resulting partially mismatched double-stranded DNA is used to produce a pure DNA gene containing an altered deoxyribonucleotide at the single desired position.

Abstract: Synthetic oligonucleotides have been designed and prepared which are useful in the molecular cloning of a variety of DNA molecules. By means of such oligonucleotides, genetic informational material, e.g., DNA, can be joined to a cloning vehicle and transferred into host cells by transformation. Additionally, a method for determining whether genetic informational material has been transferred into transformed host cells has been developed.

Abstract: Recombinant plasmids are described that carry the structural gene for human proinsulin in expressible form. The human proinsulin structural gene is fused to a functional transport sequence. Host organisms transformed by the recombinant plasmids express a preproinsulin in vivo, and is transported across the host organism's cell wall. Following its recovery, in vitro methods are used to convert the human proinsulin to human insulin.