RNA fingerprinting of total bacterial RNA obtained by in situ extraction of soil microcosms inoculated with strain Pseudomonas putida F1

Author

Wen-Hsiang Yao

Date of Award

12-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Gary S. Sayler

Abstract

The differential display RT-PCR (DDRT-PCR) technique has been successfully applied to obtain catabolic gene information from environmental bacterial strains. Pseudomonas putida F1 RNA was used as a model system to allow optimization of this technique. A 382-bp todC1 gene fragment was detected on a sequencing gel by using tod specific 13 oligonucleotides. The time course for tod transcripts induction and quantitation of the tod transcripts have also been achieved. The level of tod transcription was determined to be 10⁷ transcripts/ 10 µg of total RNA, which is approximately 0.004% of the total RNA. Primer concentration, primer length, annealing temperature, template, dNTP, and MgCl₂, concentrations have also been optimized. The detection limit for differential display of the tod fragment was shown to be 0.015 ng of total RNA template.

Optimization of the RNA extraction buffer and fingerprinting of RNA obtained from soil samples inoculated with P. putida F1 have also been successful. The banding patterns of RNA from pure culture and soil samples are almost identical. Non-specific arbitrary primers have also been used to fingerprint P. putida F1 RNA to assess the effect of cDNA amplification. Subsequently a 543-bp todC2 gene fragment has been detected. Therefore, this study provides support that DDRT-PCR can be used to isolate the active genes from pure bacterial cultures and environmental soil bacteria.

Recommended Citation

Yao, Wen-Hsiang, "RNA fingerprinting of total bacterial RNA obtained by in situ extraction of soil microcosms inoculated with strain Pseudomonas putida F1. " Master's Thesis, University of Tennessee, 1996.
https://trace.tennessee.edu/utk_gradthes/11013