ABCMdb

PMID: 15890513

Eaker S, Johnson M, Jenkins J, Bauer M, Little S
Detection of CFTR mutations using ARMS and low-density microarrays.
Biosens Bioelectron. 2005 Dec 15;21(6):933-9., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene ( F508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Login to comment 88 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Each mutation on the panel ( F508, 1717-1G>A, G542X, 621+1G>T, and N1303K) was tested individually (Fig. 1), and in a multiplex reaction containing all five primer pairs (Fig. 1B, lanes 8 and 9). Login to comment 97 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* PCR reactions containing various DNA templates to evaluate the performance of the ARMS reaction to detect the specific mutations are listed as follows: (A2) wild-type (wt); (A3) N1303K; (A4) wt; (A5) 621+1G>T; (A7) wt; (A8) G542X; (B1) wt; (B2) F508; (B3) wt; (B4) W1282X; (B6) wt; (B7) 1717-1G>A; (B8) wt; (B9) multiplex F508 and W1282X (using F508/W1282X compound heterozygous DNA template). Login to comment 98 ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Table 1 Probe sequences specific for each mutation, 5 -amine-modified with C6 spacers Mutant probe Sequence 621+1G>T TTT GAT TTA TAA GAA GTT AAT ACT TCC TTG CAC AGG F508 GGC ACC ATT AAA GAA AAT ATC ATT GGT GTT TCC TA 1717-1G>A CTA TTT TTG GTA ATA AGA CAT CTC CAA GTT TGC AG G542X ATA GTT CTT TGA GAA GGT GGA ATC ACA CTG N1303K TAG AAA AAA GTT GGA TCC CTA TGA ACA GTG G W1282X TTT GCA ACA GTG AAG GAA AGC CTT T To each array/glass slide, Cy5-labeled PCR products were hybridized to each array/glass slide, and the fluorescence measured by both a commercial scanner and an inexpensive detection device designed in-house (Fig. 2). Login to comment 101 ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Each spot is 1 mm in diameter and contains: (1) F508; (2) 1717-1G>A; (3) N1303K; (4) 621+1G>T; (5) W1282X; (6) G542X; and (R) reference spot containing amino-linked 15-mer with a terminal Cy5 label (seen as a smear in B and E due to bleaching of the scanner. Login to comment 102 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* Each box represents a separate hybridization with the PCR products from ARMS reactions containing known DNA: (A) wt; (B) 1717-1G>A; (C) N1303K; (D) 621+1G>T; (E) F508; (F) G542X. Login to comment 105 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* After establishing a baseline fluorescence for each slide, ARMS-products from F508, 1717-1G>A, G542X, 621+1G>T, and N1303K DNA templates were hybridized, and detection on each corresponding spot was achieved, leaving the other mutant spots unbound and unlabeled. Login to comment 106 ABCC7 p.Asn1303Lys This was seen for the 1717-1G>A, N1303K, 621+1G>T, and weakly for F508. Login to comment 107 ABCC7 p.Gly542* ABCC7 p.Gly542* Fig. 2F shows that the ARMS product from G542X DNA bound both to the G542X probe as well as the 1717-1G>A probe. Login to comment 108 ABCC7 p.Gly542* This arose due to the fact that the G542X ARMS primers overlap the 1717-1G>A region. Login to comment 109 ABCC7 p.Gly542* ABCC7 p.Gly542* However, the 1717-1G>A ARMS primers do not overlap the G542X sequence, and does not produce a product which binds to the G542X probe (Fig. 2B). Login to comment 121 ABCC7 p.Asn1303Lys ABCC7 p.Gly542* (A) and (B) show the readings (screen shots) using slides from Fig. 2(A) N1303K and (B), G542X. Login to comment 125 ABCC7 p.Asn1303Lys The yellow bar represents a positive fluorescence reading for the N1303K spot (detecting hybridization). Login to comment 140 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Fig. 4A shows a screen-shot using the N1303K slide from Fig. 2C, displaying the reference spot (yellow bar) and the single N1303K spot (green bar). Login to comment 141 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Fig. 4A shows a screen-shot using the N1303K slide from Fig. 2C, displaying the reference spot (yellow bar) and the single N1303K spot (green bar). Login to comment 142 ABCC7 p.Gly542* ABCC7 p.Gly542* Multiple spots can be displayed as well, seen in Fig. 4B using the G542X slide from Fig. 2F (reference, blue bar; 1717-1G>A, green bar; G542X, yellow bar). Login to comment 143 ABCC7 p.Gly542* ABCC7 p.Gly542* Multiple spots can be displayed as well, seen in Fig. 4B using the G542X slide from Fig. 2F (reference, blue bar; 1717-1G>A, green bar; G542X, yellow bar). Login to comment