Figure 1
Figure 1. RT-PCR products on agarose gel. (A) Schematic scale of the coding region of VWF (exons 2-52) with the primer positions designed for amplification of the full-length VWF mRNA and corresponding amplicon segments. Agarose gel electrophoresis image shows the 14 overlapping RT-PCR products of VWF using total RNA from the IP’s blood as template, under thermocycling conditions with 2 minutes of extension time. The sequence chromatogram of segments 12 and 13 (both covering exon 44 carrying the silent mutation c.7464C>T) demonstrate single-peak manifestation of wt nucleotide C, c.7464C, indicating a fail in amplification of the mutant transcript. Lanes 13 and 14 were run in a separate gel but with similar running conditions. (B) RT-PCR products of segment 12 amplified with primers residing in exon 39 and exons 45/46 boundary, and with increased extension time (6 minutes) of thermocycling. RT-PCR products of RNA obtained from blood of the IP and her mother demonstrate a smaller product (920 bp) relevant to the normal transcript and an aberrant larger fragment (3130 bp) corresponding to the retained intron 44 in mRNA, whereas RT-PCRs using RNA from 5 healthy control subjects as template show only the smaller normal fragment (lanes 1-5). (C) RT-PCR amplification using allele-specific primers to confirm intron 44 retention. The primer combinations and expected amplicon sizes, if intron 44 is retained, are as follows: segment 1 (1437 bp), forward primer in exons 40/41 boundary and reverse primer targeted in intron 44, 861 nucleotides downstream of the exon 44 (lane 1); and segments 2, 3 and 4 (length 1873, 1683, and 896 bp, respectively), forward primers directed in intron 44 in 3 different positions (+787, +977, and +1764) and reverse primer in exons 48/49 boundary (lanes 2, 3, and 4). Sequence analysis of the cDNA segment 1 exhibited monoallelic presentation of the mutant variant T (c.7464T) in the sequence chromatogram, indicating that the aberrant transcript is derived solely from the mutant allele. Lane M represents the molecular weight marker (1-kb ladder).

RT-PCR products on agarose gel. (A) Schematic scale of the coding region of VWF (exons 2-52) with the primer positions designed for amplification of the full-length VWF mRNA and corresponding amplicon segments. Agarose gel electrophoresis image shows the 14 overlapping RT-PCR products of VWF using total RNA from the IP’s blood as template, under thermocycling conditions with 2 minutes of extension time. The sequence chromatogram of segments 12 and 13 (both covering exon 44 carrying the silent mutation c.7464C>T) demonstrate single-peak manifestation of wt nucleotide C, c.7464C, indicating a fail in amplification of the mutant transcript. Lanes 13 and 14 were run in a separate gel but with similar running conditions. (B) RT-PCR products of segment 12 amplified with primers residing in exon 39 and exons 45/46 boundary, and with increased extension time (6 minutes) of thermocycling. RT-PCR products of RNA obtained from blood of the IP and her mother demonstrate a smaller product (920 bp) relevant to the normal transcript and an aberrant larger fragment (3130 bp) corresponding to the retained intron 44 in mRNA, whereas RT-PCRs using RNA from 5 healthy control subjects as template show only the smaller normal fragment (lanes 1-5). (C) RT-PCR amplification using allele-specific primers to confirm intron 44 retention. The primer combinations and expected amplicon sizes, if intron 44 is retained, are as follows: segment 1 (1437 bp), forward primer in exons 40/41 boundary and reverse primer targeted in intron 44, 861 nucleotides downstream of the exon 44 (lane 1); and segments 2, 3 and 4 (length 1873, 1683, and 896 bp, respectively), forward primers directed in intron 44 in 3 different positions (+787, +977, and +1764) and reverse primer in exons 48/49 boundary (lanes 2, 3, and 4). Sequence analysis of the cDNA segment 1 exhibited monoallelic presentation of the mutant variant T (c.7464T) in the sequence chromatogram, indicating that the aberrant transcript is derived solely from the mutant allele. Lane M represents the molecular weight marker (1-kb ladder).

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