Abstract
The feasibility of direct mRNA sequencing for the purpose of characterizing mutations of relatively low abundance messages was examined. A 906 base pair PstI fragment corresponding to the coding region of hypoxanthine phosphoribosyltransferase (HPRT) cDNA was inserted into a pGEM plasmid and HPRT RNA was generated in quantity. Varied amounts of HPRT RNA were diluted with 12 ug of human lymphoblast poly A+ RNA. A 20-base oligonucleotide primer, complementary to HPRT mRNA, and RNA were heated to 80° C for 3 min, hybridized for 45 min at 54° C and subjected to Sanger dideoxy sequencing reactions. dNTP-labelled sequencing produced reliable results at a message abundance of greater than 0.1%, but was marred by anomalous banding and high background at lower abundances. Primer-labelled sequencing produced reliable sequence at a message abundance of 0.05%. The minimal message requirement for the primer-labelled sequencing method was determined by using between 3 and 50 ug of poly A+ RNA in the sequencing of HPRT mRNA (∼ 0.01% of poly A+ mRNA) and phosphoglycerate kinase (PGK) mRNA (∼ 0.05% of poly A+ mRNA). Reliable sequence was obtained from 0.01 pmole of either PGK or HPRT message. The region of interest in a PGK variant previously characterized as a single amino acid substitution was sequenced. Direct sequencing is indicated to be useful for messages whose abundance is equal or greater than 0.05%. Supported by the Medical Research Council grant MT-8665.
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Joshi, R., Snyder, F. 147 CONDITIONS AND LIMITATIONS FOR DIRECT mRNA SEQUENCING USING OLIGONUCLEOTIDE PRIMERS. Pediatr Res 24, 135 (1988). https://doi.org/10.1203/00006450-198807000-00171
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DOI: https://doi.org/10.1203/00006450-198807000-00171