ABCMdb

PMID: 23503723

Zhou L, Palais RA, Ye F, Chen J, Montgomery JL, Wittwer CT
Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.
Clin Chem. 2013 Jul;59(7):1052-61. doi: 10.1373/clinchem.2013.202689. Epub 2013 Mar 15., [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCC7 p.Ile148Thr PRIMERS AND NOMENCLATURE Previously described CFTR primers (8) were modified with 5b18; tails to enable snapback genotyping of the 23 ACMG mutations (17), p.I148T, and 7 common benign variants. Login to comment 57 ABCC7 p.Arg75Gln c.869af9;11Cb0e;T, c.-8Gb0e;C, and p.R75Q) or synonymous variants. Login to comment 79 ABCC7 p.Ile1315Met We identified 2 previously unreported variants: p.I1315M (c.3945Ab0e;G) in exon 24 and c.4243-5Cb0e;T near exon 27. Login to comment 83 ABCC7 p.Arg553* In exon 12, two nearby variants (p.R551D and p.R553X) were genotyped with 1 snapback primer, because all alleles had unique stabilities (Fig. 2). Login to comment 86 ABCC7 p.Trp1282* Another method for genotyping separated loci is to design a bulge into the snapback hairpin, as demonstrated for exon 23 for variants p.W1282X and c.3870Ab0e;G (Fig. 4). Login to comment 126 ABCC7 p.Arg668Cys ABCC7 p.Ser686Tyr d One sample had 2 rare variants (R668C and S686Y) that required sequencing but were in the same amplicon. Login to comment 127 ABCC7 p.Trp1282* Another sample had 1 mutation (p.W1282X) and 1 benign variant (c.3870Ab0e;G) in cis, both within a snapback hairpin that required sequencing. Login to comment 134 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala For example, by directing snapback primers to genotype an additional 2 synonymous variants (p.Y1424Y and p.T966T) and a syntenic pair of variants (p.G576A and p.R668C) that constitute a complex allele (26, 27), the sequencing required for the patients we have analyzed could be reduced by an additional factor of 2 (see Table 5 in the online Data Supplement). Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Arg553* Scanning and genotyping of 2 mutations (p.G551D and p.R553X) in exon 12 of CFTR with 1 snapback primer. Login to comment 146 ABCC7 p.Gly551Asp ABCC7 p.Arg553* The wild-type Tm is at 72 &#b0;C, p.G551D is at 64 &#b0;C, p.R553X is at 60 &#b0;C, and a compound heterozygote in trans shows a broad peak with a Tm of 62 &#b0;C. Login to comment 166 ABCC7 p.Trp1282* Two variants (p.W1282X and c.3870Ab0e;G) separated by 23 bases were genotyped with 1 snapback primer by targeting its 5b18; tail to 2 discontinuous regions, thereby inducing a 10-bp secondary bulge in the hairpin. Login to comment 168 ABCC7 p.Trp1282* (B), Derivative melting curves of the hairpin after dilution and remelting to distinguish wild type (black lines), heterozygous p.W1282X (thick gray line), and heterozygous c.3870A/G (thin gray line). Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Arg553* Examples include p.G551D and p.R553X, which are separated by 4 bases (Fig. 2), and p.I507del and p.F508del (Fig. 3). Login to comment 174 ABCC7 p.Ser686Tyr Such was the case in the current study with p.S686Y in exon 14, which is 4 bases away from the ACMG CFTR mutation c.2052delA. Login to comment 178 ABCC7 p.Trp1282* Fig. 4 shows an example of a bulge-inducing snapback primer, in which ACMG mutation p.W1282X is separated by 23 bases from the common variant, c.3870Ab0e;G. Deleting 10 bp between the 2 variants in the complementary snapback tail produces a hairpin that induces a 10-bp bulge in the template. Login to comment