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Published December 1, 2004 | public
Journal Article

Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis

Abstract

A wealth of genetic sequence information is available publicly and can be used to support investigations in organic geochemistry. Here, we present a new method to separate ribosomal RNA in order to use this rRNA as a "biomarker" for molecular isotopic studies. The primary goal is to obtain pure fractions that reflect selected phylogenetic groups. We demonstrate the ability to separate rRNA of a target organism from RNA representing a mixture of species. In this approach, an oligonucleotide probe containing a poly-d(GGGT) tail is hybridized to RNA in solution. Simultaneously, an aliquot of oligo-dT paramagnetic beads is hybridized to an oligonucleotide made of poly-d(CCCA) with a poly-dA tail. The two solutions are combined and a high-affinity GCAT complex is formed. The magnetic beads are captured and re-suspended in fresh solution. Careful determination of the melting temperatures of all three hybrids permits melting of the captured rRNA while leaving the majority of the oligonucleotides bound to the beads. Authenticity of the captured product is determined by reverse-transcriptase (RT)-PCR on a sub-sample; the remainder is washed, re-suspended in H_2O, and saved for subsequent isotopic analysis.

Additional Information

© 2004 Elsevier B.V. Received 4 November 2003; received in revised form 12 April 2004; accepted 30 June 2004; Available online 22 October 2004. The authors are grateful for the tremendous amount of valuable advice given by E. DeLong. K.T. Scott and C. Cavanaugh provided samples of bivalves and vestimentiferans. M. Fisher assisted with samples from Lake Mishwam. This work was supported by a WHOI-RCRC Grant to A.P., J.M.H., and K. Edwards; by NASA Astrobiology Award NCC2-1053 to J.M.H.; and by NSF Grant EAR-0311937 to A.P. This is WHOI contribution #11141.

Additional details

Created:
August 22, 2023
Modified:
October 23, 2023