Patent ID: RE39031

Claim:
A method for the amplification of RNA, in a sample, comprising: a) obtaining a starting solution by adding to a container comprising the sample, a buffer, a first primer, a second primer, a plurality of nucleotide triphosphates, a sufficient amount of an enzyme system having reverse transcriptase activity and a heat stable enzyme system having DNA polymerase activity, and closing the countainer, wherein said sufficient amount is an amount which, after the heat treatment of step b) below, will retain sufficient reverse transcriptase activity to permit performance of step c) d) hereafter; b) heating the solution obtained in a) to a temperature sufficient to permit denaturation, said temperature not to exceed 75 ° C., and maintaining said temperature for a sufficient time to provide denaturation of said RNA without inactivating the enzyme system having reverse transcriptase activity; c) permitting the first primer to hybridize with said RNA in said solution; d) bringing the solution obtained in b) c) to a predetermined temperature from 55 ° to 75 ° C. and maintaining said temperature for sufficient time whereby a first cDNA strand is synthesized and a RNA-cDNA heteroduplex is formed; d) e) heating the solution obtained in c) d) to a predetermined temperature whereby said RNA-cDNA heteroduplex is denatured to form an RNA single strand and a first cDNA single strand; e) f) bringing the solution obtained in d) e) to a predetermined temperature and maintaining said temperature for a sufficient time whereby the second primer hybridizes with the first cDNA single strand; f) g) bringing the solution obtained in e) f) to a predetermined temperature and maintaining said temperature for a sufficient time whereby a second cDNA strand is synthesized to form a double-stranded cDNA; and g) h) denaturing the double-stranded cDNA to form cDNA single strands and subjecting the cDNA single strands to a sufficient number of amplification cycles to obtain a desired amount of amplified product ; wherein after step a), all steps are performed without subsequent addition of any ingredients.