Abstract: A gene having a DNA sequence complementary to that of the glucoamylase polypeptide mRNA from a fungal species, preferably Aspergillus awamori, is prepared. The mRNA is an approximately 2.2 kilobase poly A RNA obtained from fungal cells grown under conditions of glucoamylase induction. Reverse transcription of the mRNA provides a glucoamylase probe used to identify genomic digest fragments containing glucoamylase gene regions, which are sequenced to locate the introns and exons. The genomic fragments are spliced together to form a gene having a DNA sequence with altered or deleted introns which codes for fungal glucoamylase protein and is capable, when correctly combined with a cleaved DNA expression vector, of expressing a non-native protein having glucoamylase enzyme activity upon transformation of a host organism by the vector. The host is preferably bacteria or yeast. The transformed yeast host may be used to produce ethanol.

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.