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Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis

Pearson, Ann and Sessions, Alex L. and Edwards, Katrina J. and Hayes, John M. (2004) Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis. Marine Chemistry, 92 (1-4). pp. 295-306. ISSN 0304-4203. doi:10.1016/j.marchem.2004.06.032.

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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.

Item Type:Article
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URLURL TypeDescription
Pearson, Ann0000-0003-2785-8405
Sessions, Alex L.0000-0001-6120-2763
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.
Funding AgencyGrant Number
Woods Hole Oceanographic InstitutionUNSPECIFIED
Subject Keywords:Analytical techniques; Carbon isotopes; Nucleic acids; RNA
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Other Numbering System NameOther Numbering System ID
Woods Hole Oceanographic Institution (WHOI)11141
Issue or Number:1-4
Record Number:CaltechAUTHORS:20130604-074054886
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Official Citation:Ann Pearson, Alex L. Sessions, Katrina J. Edwards, John M. Hayes, Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis, Marine Chemistry, Volume 92, Issues 1–4, 1 December 2004, Pages 295-306, ISSN 0304-4203, 10.1016/j.marchem.2004.06.032.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38769
Deposited By: Tony Diaz
Deposited On:05 Jun 2013 17:37
Last Modified:09 Nov 2021 23:39

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