ABCMdb

PMID: 14764639

Nasis O, Thompson S, Hong T, Sherwood M, Radcliffe S, Jackson L, Otevrel T
Improvement in sensitivity of allele-specific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis.
Clin Chem. 2004 Apr;50(4):694-701. Epub 2004 Feb 5., [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCC7 p.Asp1152His ABCC7 p.Asp1152His CF mutation D1152H consists of a single nucleotide substitution (G to C) at nucleotide 3586, changing Asp to His at amino acid position 1152 (27). Login to comment 40 ABCC7 p.Asp1152His Patient 6 was carrying a fetus known to be heterozygous for the CF D1152H mutation (determined by a commercial laboratory by use of invasive prenatal diagnosis). Login to comment 41 ABCC7 p.Asp1152His This patient was a carrier for the CF ⌬F508 mutation, and the father of the fetus was a carrier for D1152H. Login to comment 42 ABCC7 p.Asp1152His To obtain DNA containing the D1152H mutation for use in the model experiment, we collected ϳ10 mL of blood from the father. Login to comment 52 ABCC7 p.Asp1152His Patient Maternal age, years Gestational age, weeks Time between collection and processing of blood, days Fetal D1152H mutation status DNA in PCR tubes,a ng 1 32 19.2 3 -/- 53.3 2 33 15.5 6 -/- 307.7 3 35 11.2 1 -/- 2.0 4 37 15.1 3 -/- 4.3 5 29 18 5 -/- 318.5 6 30 11 0 ϩ/- 0.3 B. Login to comment 61 ABCC7 p.Asp1152His dna used in the model pcr experiment Genomic DNA containing CF mutation D1152H was isolated from 800 ␮L of whole blood from a heterozygous male carrier (father of the fetus carried by patient 6) by use of the DNA Blood Mini Kit according to the manufacturer`s protocol. Login to comment 62 ABCC7 p.Asp1152His DNA samples with various ratios of mutant to wild-type alleles were prepared in the PCR reaction tubes by combining 30 pg of D1152H heterozygous carrier DNA with 3, 30, and 300 ng of wild-type female DNA obtained from Sigma. Login to comment 63 ABCC7 p.Asp1152His oligonucleotides The sequences of oligonucleotide primers 1152F (5Ј- GATAAGACTTACCAAGCTATCCACATG-3Ј) and 1152R (5Ј-GAGTTGGTATTATCCTGACTTTAGCCA-3Ј), used for amplification of DNA carrying the D1152H mutation, were designed with Primer Express 1.5 software (Applied Biosystems) and synthesized by Proligo. Login to comment 64 ABCC7 p.Asp1152His Forward primer 1152F is specific for the D1152H mutation and exhibits a perfect match to the mutated sequence. Login to comment 67 ABCC7 p.Asp1152His This oligonucleotide was used in the initial experiments and represents a destabilized D1152H mutation-specific primer. Login to comment 71 ABCC7 p.Asp1152His conventional pcr All ASPCR amplifications for detection of the D1152H mutation on the CFTR gene were performed on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). Login to comment 95 ABCC7 p.Asp1152His However, because of frequent inconsistencies in PCR amplification when working with quantities of target lower than five copies-probably caused by an uneven distribution of intact DNA target molecules among the samples-we decided to include five copies of the mutated allele in all aliquots of D1152H-positive artificial specimens used in PCRs. Login to comment 96 ABCC7 p.Asp1152His These artificial specimens were used to develop the D1152H mutation detection assay. Login to comment 97 ABCC7 p.Asp1152His Considering that the diploid DNA content of one cell is 6.6 pg (28), five copies of the mutated allele should be present in ϳ30 pg of DNA isolated from a heterozygous D1152H carrier. Login to comment 99 ABCC7 p.Asp1152His ABCC7 p.Asp1152His Thus, to make model D1152H-positive samples that resemble conditions in maternal plasma- and to allow for biological variation and additionally to test the limits of our procedure-we prepared several PCR samples by mixing 30 pg of D1152H heterozygous carrier DNA (isolated from the father of the fetus carried by patient 6) with 3, 30, and 300 ng of wild-type female DNA (Sigma). Login to comment 101 ABCC7 p.Asp1152His However, our initial experiments, using mutation-specific primer 1152F, reverse primer 1152R, Accuprime Taq polymerase without 3Ј-exonuclease activity (Invitrogen), dNTPs and reaction buffer providing a final magnesium chloride concentration of 1.5 mM (Roche), failed to differentiate between the D1152H mutated and wild-type CF alleles (Fig. 1A). Login to comment 110 ABCC7 p.Asp1152His Attempts to differentiate between D1152H mutant and wild-type CF alleles with regular ASPCR conditions. Login to comment 112 ABCC7 p.Asp1152His ABCC7 p.Asp1152His ABCC7 p.Asp1152His The CF D1152H-specific bands (lanes 2-4) were produced in PCRs containing 30 pg of CF DNA with 0, 6, and 3 ng of female control DNA, respectively. Lanes 5 and 6 correspond to PCR tubes containing 6 and 3 ng of female DNA, respectively, without the presence of any mutated D1152H DNA and show amplification of the CF D1152H-specific bands. Login to comment 116 ABCC7 p.Asp1152His The CF D1152H-specific bands (lanes 2-4) were produced in PCRs containing 30 pg of CF DNA with 0, 6, and 3 ng of female control DNA, respectively. Login to comment 117 ABCC7 p.Asp1152His Lane 5 corresponds to the PCR tube with 6 ng of female DNA and shows amplification of the CF D1152H-specific bands. Login to comment 124 ABCC7 p.Asp1152His By contrast, the combination of a primer pair containing perfectly matched D1152 mutation-specific forward primer 1152F and reverse primer 1152R, Pfx PCR buffer from the Accuprime Pfx DNA Polymerase Kit (Invitrogen), Accuprime Taq polymerase (without proofreading activity; from the Accuprime Taq DNA Polymerase System), and TaqMaster PCR Enhancer (Eppendorf) allowed consistent detection of 5 copies of the CF D1152H mutant allele in the presence of up to ϳ100 000 copies of the wild-type allele without interference from the wild-type sequence. Login to comment 127 ABCC7 p.Asp1152His ABCC7 p.Asp1152His Approximately 5 copies of the D1152H mutated allele contained in 30 pg of D1152H heterozygous genomic DNA was amplified in the presence of 0-300 ng of wild-type female DNA (i.e., up to ϳ100 000 copies of the wild-type allele). Login to comment 128 ABCC7 p.Asp1152His No amplification was observed with 0-300 ng of female DNA in the absence of the D1152H mutated allele. Login to comment 129 ABCC7 p.Asp1152His analysis of dna samples isolated from maternal plasma Testing for the presence of the D1152H mutation. Login to comment 130 ABCC7 p.Asp1152His We studied DNA from the plasma of a woman known to be carrying a fetus with the CF D1152H mutant gene inherited from the father (Table 1A, patient 6) and five controls. Login to comment 131 ABCC7 p.Asp1152His The sample from patient 6 (Fig. 3A, lanes 2-4) showed amplification of the mutant D1152H allele in all three reactions. Login to comment 133 ABCC7 p.Asp1152His Samples from four of five control patients (Fig. 3, B and C) showed no amplification of the mutant D1152H allele, but patient 2 (Fig. 3C, lanes 5-7) showed a band in two of three amplifications. Login to comment 134 ABCC7 p.Asp1152His By contrast, the results from the CVS of patient 2 and a blood test performed on the father of the pregnancy were both negative for the D1152H mutation. Login to comment 145 ABCC7 p.Asp1152His PCR results obtained with experimental models containing CF D1152H heterozygous carrier DNA and wild-type female DNA. Login to comment 146 ABCC7 p.Asp1152His The CF D1152H-specific bands (lanes 2-5) were produced in PCRs containing 30 pg of CF DNA with 0, 300, 30, and 3 ng of female DNA, respectively. Lanes 6-9, which correspond to PCR tubes with 300, 30, 3, and 0 ng of female DNA, respectively, show no amplification product. Login to comment 149 ABCC7 p.Asp1152His Testing for D1152H mutation in DNA samples isolated from blood plasma of pregnant women. Login to comment 150 ABCC7 p.Asp1152His (A), CF D1152H mutation-specific band is present in all lanes containing samples from patient 6 (lanes 2-4). Login to comment 168 ABCC7 p.Asp1152His testing of an aspcr-based d1152h mutation assay on appropriately handled maternal blood samples obtained from d1152h-negative control pregnancies To demonstrate the reliability of the assay in terms of suppression of nonspecific amplification interference from the wild-type CFTR allele, we isolated new negative DNA control samples from appropriately processed maternal blood specimens corresponding to pregnancies with no D1152H mutation (Table 1B, patients 7-11). Login to comment 185 ABCC7 p.Asp1152His Testing for D1152H mutation in negative-control DNA samples isolated from appropriately processed blood plasma of pregnant women. Login to comment 186 ABCC7 p.Asp1152His (A), no CF D1152H mutation-specific bands are present in lanes containing samples from patients 7 (lanes 2-4), 8 (lanes 5-7), and 9 (lanes 8-10). Login to comment 188 ABCC7 p.Asp1152His The band in lane 9 represents the positive control: 30 pg of genomic DNA heterozygous for the D1152H mutation. Login to comment