Mesoplasma florum:Primer extension

Goal: identify transcription start sites of the entire genome

• Isolate total RNA
• Reverse transcribe the RNA using a 5' fluorescently labeled primer
• Isolate ssDNA
• Mix ssDNA into a standard sequencing reaction
• Sequence the mixed product

Fluorescent labels

• LIZ standard
  • VIC or HEX
  • NED or TAM
  • FAM
  • PET
• ROX standard
  • VIC or HEX
  • NED or TAM
  • FAM
• FAM is cheap and suitable

Custom Genescan markers

• Bioventures, ROX, TAM labeled

References

The original reference is Altermann99; Lledo05 is closest to our intended approach, mixing primer extension products with a sequencing reaction.

1. Altermann E, Klein JR, and Henrich B. Synthesis and automated detection of fluorescently labeled primer extension products. Biotechniques. 1999 Jan;26(1):96-8, 101. DOI:10.2144/99261st03 |
2. Fekete RA, Miller MJ, and Chattoraj DK. Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension. Biotechniques. 2003 Jul;35(1):90-4, 97-8. DOI:10.2144/03351rr01 |
3. Gould PS, Bird H, and Easton AJ. Translation toeprinting assays using fluorescently labeled primers and capillary electrophoresis. Biotechniques. 2005 Mar;38(3):397-400. DOI:10.2144/05383ST02 |
4. Lledó B, Marhuenda-Egea FC, Martínez-Espinosa RM, and Bonete MJ. Identification and transcriptional analysis of nitrate assimilation genes in the halophilic archaeon Haloferax mediterranei. Gene. 2005 Nov 21;361:80-8. DOI:10.1016/j.gene.2005.07.011 |
5. Lloyd AL, Marshall BJ, and Mee BJ. Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis. J Microbiol Methods. 2005 Mar;60(3):291-8. DOI:10.1016/j.mimet.2004.10.009 |
6. Mathews SA and Timms P. Identification and mapping of sigma-54 promoters in Chlamydia trachomatis. J Bacteriol. 2000 Nov;182(21):6239-42. DOI:10.1128/JB.182.21.6239-6242.2000 |
7. Nesper J, Blass J, Fountoulakis M, and Reidl J. Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion, and integration. J Bacteriol. 1999 May;181(9):2902-13. DOI:10.1128/JB.181.9.2902-2913.1999 |
8. Thorell HD, Karlsson J, Portelius E, and Nilsson T. Cloning, characterisation, and expression of a novel gene encoding chlorite dismutase from Ideonella dechloratans. Biochim Biophys Acta. 2002 Sep 27;1577(3):445-51. DOI:10.1016/s0167-4781(02)00446-3 |