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S Bouma

Publications and source records attributed to S Bouma.

12 recordsLinked to original sources
PubMed1995

Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology.

The purpose of this study is to demonstrate the value of a multiplex amplification and readout system. The validation was done using as a model system the detection of deletions in nine possible dystrophin exons: 4, 8, 12, 17, 19, 44, 45, 48, and 51. The amplification system was gap ligase chain reaction, adapted to amplify selected regions of multiple exons simultaneously. The amplified products were read out with an immunochromatographic methodology, adapted from that used in the Abbott product line commercialized under the name Test Pack Plus. In each amplification, the beta-globin gene was incorporated and served as a procedural control. The complete process takes < 3 hr from DNA sample to result. The procedure is therefore rapid and simple, as well as being potentially very cost effective. The combination of these two technologies is shown to be a useful tool for the determination of deletions in the nine exons of the dystrophin gene. The results of a 100-patient sample study showed concordance with cDNA and PCR in current use. Equivalent performance at two sites was shown.

Base Sequence↗
PubMed1995

Simultaneous analysis of mutant and normal alleles for multiple cystic fibrosis mutations by the ligase chain reaction.

The ligase chain reaction (LCR) involves repetitive cycles of ligation of two adjacent pairs of oligonucleotides to form longer ligated products in a template-dependent manner. This study demonstrates the application of LCR for analysis of multiple small mutations. We adapted the technology for the simultaneous determination of the normal and mutant alleles in a competition format, as well as multiple mutations in a multiplex format. For these purposes, we used mutations causing cystic fibrosis, namely the delta F508, W1282X, and G551D mutations. Blunt ligation was compared to a strategy with a single base gap on one or both strands to be filled by thermostable polymerase prior to ligation. Blunt or gap strategies worked well for detection of the delta F508 mutation. Detection of the W1282X mutation worked well with a blunt strategy when high K+ concentration (180-220 mM) was used to reduce template-independent ligation. For reliable detection of the G551D mutation, we used mismatches in the oligonucleotides 2-5 bp away from the ligation site and hot start of the reaction to achieve allele specificity. Excellent discrimination of mutations was achieved using competitive LCR with six oligonucleotides (two common on one side of the mutation plus two wild type and two mutant on the opposite side with the mutation site at the end adjacent to the common oligonucleotides) and with multiplex-competitive LCR using 12 oligonucleotides to detect both alleles for two mutations in a single tube.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗