Method for synthesizing cDNA
View Patent ↗A method for synthesizing cDNA characterized by preparing a reaction solution that does not allow an endodeoxyribonuclease to show its activity, without thermal deactivation of the endodeoxyribonuclease or removal of the endodeoxyribonuclease, and carrying out a reverse transcription reaction, wherein the reaction solution contains a treated sample and a reverse transcriptase, the treated sample being formed by treating a sample comprising RNA and DNA with the endodeoxyribonuclease to degrade DNA in the sample. The method and the kit for synthesizing cDNA of the present invention are widely useful in genetic engineering fields.
1. A method for synthesizing cDNA which comprises the following steps (a) to (c):
(a) obtaining a composition comprising a sample comprising RNA and DNA, and an endodeoxyribonuclease;
(b) treating the composition obtained in step (a) under conditions sufficient to degrade the DNA; and
(c) adding an additive comprising at least dithiothreitol and ammonium ion, and a reverse transcriptase to the composition treated in step (b) to prepare a reaction solution wherein activity of the endodeoxyribonuclease is inhibited without heat denaturation at 60° C. or higher, and carrying out a reverse transcription reaction with the reverse transcriptase to synthesize cDNA,
wherein the concentration of dithiothreitol in the reaction solution ranges from 11 mM to 30 mM, and
wherein the concentration of ammonium ion in the reaction solution ranges from 20 mM to 60 mM.
2. The method according to claim 1 , wherein the endodeoxyribonuclease is deoxyribonuclease I.
3. The method according to claim 1 , wherein the reverse transcriptase is a reverse transcriptase derived from Moloney murine leukemia virus.
4. The method according to claim 1 , further comprising the step of carrying out a gene amplification reaction with the synthesized cDNA as a template.
5. The method according to claim 2 , further comprising the step of carrying out a gene amplification reaction with the synthesized cDNA as a template.
6. The method according to claim 3 , further comprising the step of carrying out a gene amplification reaction with the synthesized cDNA as a template.
7. The method according to claim 1 , wherein the activity of the endodeoxyribonuclease is inhibited by at least 71%.
8. The method according to claim 1 , wherein the concentration of dithiothreitol in the reaction solution ranges from 11.25 to 20 mM.
9. The method according to claim 8 , wherein the additive further comprises a potassium ion.
10. The method according to claim 9 , wherein the additive further comprises magnesium ion in a concentration of 2 mM.
11. The method according to claim 1 , wherein the concentration of ammonium ion in the reaction solution ranges from 28 mM to 56 mM.