ABCMdb

PMID: 9662393

Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC
Mutation detection and single-molecule counting using isothermal rolling-circle amplification.
Nat Genet. 1998 Jul;19(3):225-32., [PubMed]

Sentences

No. Mutations Sentence Comment

23 ABCC7 p.Gly542* We designed an allele-discriminating probe for a 46-nt target sequence in the CFTR G542X gene locus (Fig. 2a). Login to comment 35 ABCC7 p.Gly542* Allele discrimination using HRCA and genomic DNA An alternative probe design for a 46-nt target sequence in the CFTR G542X gene locus (Fig. 2c) was used in this experiment. Login to comment 51 ABCC7 p.Gly542* a, Sequence of the hybridizing arms of a circularizable probe, and two alternative 8-base gap probes designed for the CFTR G542X locus. Login to comment 64 ABCC7 p.Gly542* c, Sequence of the hybridizing arms of a circularizable gap-fill probe designed for the CFTR G542X mutation locus. Login to comment 80 ABCC7 p.Gly542* Human lymphocytes that were either wild-type, homozygous or heterozygous for the G542X locus were used as a DNA source. Login to comment 81 ABCC7 p.Gly542* An assay was performed where G542X probes of the gap-fill design (Fig. 2c) were extended and ligated using genomic DNA targets, and amplified using HRCA. Login to comment 91 ABCC7 p.Gly542* We prepared slides containing an oligonucleotide probe (P1) specific for a 39-base sequence adjacent to the G542X locus of the CFTR gene. Login to comment 118 ABCC7 p.Gly542* We performed an assay to measure the ratio of mutant to wild type strands at the G542X locus in genomic DNA samples that had been constructed to simulate the presence of rare somatic mutations. Login to comment 122 ABCC7 p.Gly542* a, Design of primers used to amplify an 89-base probe that had been extended by DNA polymerase copying of 7 nt (shown in small type) from the G542X target region, and circularized by DNA ligase. Login to comment 126 ABCC7 p.Gly542* Lanes 1-7 were seeded, respectively, with 2×105, 2×104, 2×103, 2×102, 2×101, 2×101 (repeat), or zero molecules of G542X circularized mutant probe. Login to comment 128 ABCC7 p.Gly542* c, Detection of the G542X point mutation. Login to comment 129 ABCC7 p.Gly542* DNA samples from homozygous wild type, heterozygous or homozygous mutant cultured lymphocytes were denatured by heating for 4 min at 96 °C, and mixed with the gap-fill open circle probe for the G542X locus shown in 6A. Login to comment 142 ABCC7 p.Gly542* P1 is designed to form 39 bp with the G542X target, and the 5´ terminus of P1 contains a 5´-phosphate to permit ligation. Login to comment 163 ABCC7 p.Gly542* To test the feasibility of padlock probe detection on deproteinized DNA, we ligated the G542X padlock probe (Fig. 2a) on a sample of wild-type human genomic DNA which had been immobilized and denatured on a polylysine coated glass slide, at a density of approximately 480 haploid genomes per square mm. Login to comment 167 ABCC7 p.Gly542* The same padlock probe, with a gap oligonucleotide specific for the wild-type G542X locus, was ligated on salt-extracted nuclei prepared by the 'halo` method11,12. Login to comment 179 ABCC7 p.Gly542* a, Observation of non-condensed rolling circle DNA product from a padlock probe specific for the CFTR G542X wt locus, labelled with BUDR as a hapten, lying on the surface of a polylysine-coated slide. Login to comment 180 ABCC7 p.Gly542* b,c, Observation of partly condensed (b) and fully condensed (c) rolling-circle amplification signals generated by CFTR G542X wt padlock probes that had been hybridized to nuclear 'halo` cytological preparations (see text for details). Login to comment 182 ABCC7 p.Gly542* The frequency of nuclei displaying one or two RCA signals in halo preparations of wild-type cells was 197/2182 for the G542X locus wt probes, 1936/2573 for deltaF508 locus wt probes. Login to comment 236 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* The sequences are: CFTR G542X phosphorylated open circle probe (85 bases) for the gap oligo method, 5´-AAGAACTATATTGTCTTTCATTCTTGCATG- GTCACACGTCGTTCTAGTACGCTTCTAGTACGCTTTTCCACTCAGTG- TGATTCCA-3´; G542X wild type phosphorylated gap probe, 5´- CCTTCTCC-3´; G542X mutant phosphorylated gap probe, 5´- CCTTCTCA-3´; primer for rolling circle reaction, 542X.P1.18, 5´- ACGACGTGTGACCATGCA-3´. Login to comment 238 ABCC7 p.Gly542* ABCC7 p.Gly542* Primers for the HRCA reactions performed with gap-fill probe were: first primer, 542X.P1-23, 5´- CTAAAGCTGAGACATGACGAGTC-3´; alternative second primers for allele discrimination, 542X.P2-23-C, 5´-CTCAGTGTGATTCCACCTTC-3´ or 542X.P2-23-A, 5´-CTCAGTGTGATTCCACCTTCACA-3´; G542X artificial wild type target, 5´-TTGCAGAGAAAGACAATATAGTTCTTGGAGAAG- GTGGAATCACACTGAGTGGA-3´; G542X artificial mutant target, 5´- TTGCAGAGAAAGACAATATAGTTCTTTGAGAAGGTGGAATCACACTG- AGTGGA-3´. Login to comment 242 ABCC7 p.Gly542* Oligonucleotides for the RCA-CACHET method, carbon spacers in parenthesis: phosphorylated P1-G542X, 5´-GAGAAGGTGGAATCACACTGAGTG- GAGGTCAACGAGCAATTTTTTTTTTT-(C7-NH2)-3´; P2wt, 3´-GTT- CTTGATATAACAGAAAGTTTT-(C18)-TTTTTATGATCACAGCTGAGGA- TAGGACATGCGA-3´; P2mu, 3´-TTTCTTGATATAACAGAAAGTTTT- (C18)-TTTTTACGTCGTCCGTGCTAGAAGGAAACACGCA-3´; pre-made amplification circles with cognate detector tags, Cwt, 5´-CGCATGTCCTAT- CCTCAGCTGTGATCATCAGAACTCACCTGTTAGACGCCACCAGCTCC- AACTGTGAAGATCGCTTAT-3´; detector tag Fl-det1c-dinitrophenol (DNP), FITC-TCAGAACTCACCTGTTAG-3´-DNP; detector tag Fl-det1d- DNP, FITC-ACTGTGAAGATCGCTTAT-3´-DNP; Cmu, 5´-GCGTGTTTC- CTTCTAGCACGGACGACGTATATGATGGTACCGCAGCCAGCATCACC- AGACTGAGTATCTCCTATCACT-3´; detector tag Cy3-det2b-DNP, Cy3- TATATGATGGTACCGCAG-3´-DNP; detector tag Cy3-det2c-DNP, Cy3- TGAGTATCTCCTATCACT-3´-DNP. Login to comment 249 ABCC7 p.Gly542* Cell lines of known genotypes for the G542X locus (GM07828[+/+], GM11497B[+/-], GM11496[-/-]) were purchased from Corriell Cell Repositories. Login to comment 255 ABCC7 p.Gly542* The circularizable G542X probe and an equimolar mixture of the two allele-specific gap oligonucleotides were ligated in the presence of the mutant sequence artificial DNA target. Login to comment 256 ABCC7 p.Gly542* Circular DNA resulting from the ligation of G542X gapped probes were amplified with ø29 DNA polymerase, and the amplified ssDNA was cloned and sequenced. Login to comment 257 ABCC7 p.Gly542* The resulting sequence contained only T at the position of the G542X mutation in 20 independent clones, indicating that only the mutant gap oligonucleotide had been ligated and amplified. Login to comment 258 ABCC7 p.Gly542* With the wild-type template, the RCA product contained only G at the G542X site in 20 clones (data not shown). Login to comment 272 ABCC7 p.Gly542* The oligonucleotide P1-G542X was immobilized over an area of 1 mm in diameter on the surface of a glass slide activated with reactive groups. Login to comment 274 ABCC7 p.Gly542* Covalent coupling was obtained by reaction of a primary amine attached to the 3´ end of the P1-G542X oligonucleotide. Login to comment 275 ABCC7 p.Gly542* Genomic DNA preparations obtained from human cell lines that were either wild type or homozygous mutant at the CFTR-G542X locus were mixed in different pre-determined ratios (mutant/wild type equal to 1:0, 1:1, 1:25 and 1:100) and then amplified by PCR as described17. Login to comment 305 ABCC7 p.Gly542* ABCC7 p.Gly542* Padlock probe ligationin situ was performed for 2 h at 52 °C by incubating 18 µl of reaction mixture under a cover slip sealed with rubber cement as follows: 150 nM phosphorylated G542X 89-mer probe, 1200 nM wild type G542X gap probe, in 20 mM Tris•HCl (pH 8.3), 50 mM KCl, 0.5 mM NAD, 10 mM MgCl2, 150 µg/ml acetylated BSA, 0.01% Triton X-100 and 0.7 U/µl Ampligase DNA ligase. Login to comment