Analysis of t-AML by cDNA panhandle PCR.
(A) cDNA panhandle PCR analysis of total RNA from peripheral blood mononuclear cells at t-AML diagnosis. As shown by the smear in the second lane of gel (t-AML), a population of products of various sizes was obtained from reverse transcribing first-strand cDNAs with 5′-MLL-random hexamer-3′ oligonucleotides, generating second-strands by MLL primer extension and forming stem-loop templates and 2 sequential PCRs with primers, all fromMLL.9 dH2O control reactions with and without reverse transcriptase (RT) are in lanes 3 and 4. (B) Recombination PCR-generated subclones were screened with the sameMLL primers used in nested PCR.9 The 15 subclones contained inserts that ranged in size from 727 bp to 1653 bp. Subclone numbers above the lanes correspond with the sequences in panel C. Control lane is reagent control. (C) Three subclones contained an in-frame fusion of MLL exon 7 to GMPS at position 150 of the 2212-bp full-length cDNA (GenBank accession no. NM_003875)(top). Two subclones contained a fusion of MLLexon 8 to the same position of GMPS (top). Ten subclones suggested incompletely processed MLL-containing transcripts (bottom). (D) MLL exon 6/GMPS gene-specific primer set yielded 472-bp and 358-bp products, confirming transcripts fusing MLL exon 8 or MLL exon 7, respectively, toGMPS. Lane 2 shows purified product with MLL exon 8-GMPS fusion. Lane 3 shows purified product withMLL exon 7-GMPS fusion. Lane 4 shows a 223-bp product obtained with MLL exon 7/GMPSgene-specific primer set that confirmed MLL exon 7-GMPS fusion.
