Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorothioate-analogues.
The conditions for the stepwise synthesis of single- (ss) and double-stranded (ds) cDNA using thio-analogues instead of dNTPs are described in this paper. RNA of paramyxovirus Sendai (strain 6/94) serves as template in these experiments. The increased resistance of this alpha S-modified cDNA against several nucleases, like S1-Nuclease, DNase I, Exonuclease III, snake venom Phosphodiesterase (PDE) and the combination of DNase I and PDE is demonstrated.
G
G
Schreiber
E M
EM
Koch
W J
WJ
Neubert
D004247:DNA / Q000138:chemical synthesis*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000138:chemical synthesis; D003851:Deoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D010222:Parainfluenza Virus 1, Human / Q000235:genetics; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013698:Templates, Genetic; D013873:Thionucleotides / Q000378:metabolism*
D004277:DNA, Single-Stranded; D012367:RNA, Viral; D013873:Thionucleotides; D004247:DNA; D011062:Polynucleotide Adenylyltransferase; D004253:DNA Nucleotidylexotransferase; D012194:RNA-Directed DNA Polymerase; D003851:Deoxyribonucleases