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Sequencing of abnormal bands was used to identify mutations.
Samples showing abnormal bands by SSCP were subjected to DNA sequencing analysis.
Abnormal bands were observed in sample with BamHI or HindIII digestion, confirming that FGFR1 was rearranged by the translocation.
Sequencing of one of these abnormal bands (T2) revealed a G → T transversion in exon 5, and sequencing of another such band (T3) revealed a 21 bp deletion of the 3′ end of exon 5. Analysis of normal tissue from the patient showed only the normal sequence, demonstrating that these variants occurred somatically.
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Diagnosing BSE via western blotting is a well characterized method; however, samples with very low PrPsc concentrations show abnormal banding profiles that do not coincide with the typical criteria required for a positive diagnosis.
By PCR-SSCP for all 16 exons, abnormal band shifts were not found in all cases.
Any samples showing abnormal band shifts were re-analysed by repeating both first- and second-round SSCP PCR reactions.
Additional PCRs around the mutation were performed between exons 12 and 16 (data not shown); yet no abnormal band appeared.
In hOGG1 gene, 4 lines (SNU-308, SNU-478, SNU-869 and SNU-1079) were found to harbour abnormal band shift bands in exon 5.
There were no abnormal band shift bands in the β -catenin, DPC4, STK11, and TGF-β RII genes by PCR-SSCP analysis.
Tumor samples exhibiting abnormal band migration by SSCP were sequenced on both the forward and reverse DNA strands by using P-labeled cycle sequencing methods.
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