Hybrid selection with cDNA-silica.
A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 micrograms/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29-162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.
Jarrett
H W
HW
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
D000255:Adenosine Triphosphate / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D003565:Cytidine Diphosphate / Q000032:analysis / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D009132:Muscles / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D013462:Sulfur Radioisotopes; D013696:Temperature; D013698:Templates, Genetic; D014544:Uridine Triphosphate / Q000032:analysis / Q000737:chemistry
D017931:DNA Primers; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D003565:Cytidine Diphosphate; D012313:RNA; D012822:Silicon Dioxide; D000255:Adenosine Triphosphate; D010047:Ovalbumin; D012194:RNA-Directed DNA Polymerase; D014544:Uridine Triphosphate