Comparative Mutation Detection Screening of the Type VII Collagen Gene (COL7A1) Using the Protein Truncation Test, Fluorescent Chemical Cleavage of Mismatch,

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Comparative Mutation Detection Screening of the Type VII Collagen Gene (COL7A1) Using the Protein Truncation Test, Fluorescent Chemical Cleavage of Mismatch, and Conformation Sensitive Gel Electrophoresis Neil V. Whittock, Gabrielle H.S. Ashton, Rafik Mohammedi, Jemima E. Mellerio, Christopher G. Mathew, Stephen J. Abbs, Robin A.J. Eady, John A. McGrath Journal of Investigative Dermatology Volume 113, Issue 4, Pages 673-686 (October 1999) DOI: 10.1046/j.1523-1747.1999.00732.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic representation of the principles of the COL7A1 nested RT-PCR protein truncation test. Normal and mutant COL7A1 transcripts are represented. The point mutations in the mutant transcript are represented by X and Y. Total cellular RNA isolated from blood lymphocytes is subjected to four separate RT reactions using COL7A1 specific primers, followed by four separate nested PCR denoted F1–F4. In addition to the COL7A1 specific sequence, each 5′ secondary PCR primer contains consensus sequences for the T7 bacteriophage RNA promoter and a eukaryote initiation codon (ATG). Unpurified PCR products for fragments 1–4 are transcribed and translated using a T7 coupled rabbit reticulocyte lysate system and analyzed using discontinuous SDS-PAGE and autoradiography. M, protein markers. Lanes 1–4 denote the four protein bands F1–F4 transcribed and translated from the normal transcript, and lanes 6–9 denote the four protein bands for the mutant transcript. Aberrant protein bands can be seen for the mutant transcript in lane 6 (F1) and lane 9 (F4) corresponding to the heterozygotes R578X(X) and 7786delG(Y), respectively. Journal of Investigative Dermatology 1999 113, 673-686DOI: (10.1046/j.1523-1747.1999.00732.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Schematic representation of the principles of the COL7A1 solid phase fluorescent chemical cleavage of mismatch strategy. One of the fragments for patient and control is represented. The sequence change in the patient fragment is represented by X. Genomic DNA is isolated from blood lymphocytes and subjected to 21 separate fluorescent PCR ranging in size from 620 bp to 1604 bp. The control PCR utilizes biotin-labeled primers as indicated. Patient and control DNA (probe) is hybridized to form two heteroduplexes (stage 1), which are then bound along with any probe homoduplex to streptavidin beads via the biotin molecules in order to remove any patient homoduplexes formed. Any mismatches are modified (stage 2) using either hydroxylamine or osmium tetroxide, and the DNA is cleaved (stage 3) at the mismatch site by the use of piperidine. Cleavage products are eluted (stage 4) from the beads and electrophoresed through a 6% acrylamide gel (stage 5) on an ABI 377 genetic analyzer and analyzed using Genescan software. Journal of Investigative Dermatology 1999 113, 673-686DOI: (10.1046/j.1523-1747.1999.00732.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Fluorescent CCM analysis of COL7A1 demonstrating the mutation 7786delG. Electropherogram generated by the Genescan software of fragment 19 (exons 103–108) for two patients heterozygous for the common British mutation 7786delG (lanes 1 and 2) and a negative control (lane 3). Lane 4 contains the ROX2500 DNA size standards. Cleavage peaks can be seen for the 7786delG mutation (458 bp + 1082 bp) in lanes 1 and 2, but are absent for the negative control. The full-size uncleaved DNA peak is 1540 bp. Journal of Investigative Dermatology 1999 113, 673-686DOI: (10.1046/j.1523-1747.1999.00732.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions