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High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes 总被引:5,自引:0,他引:5
Shuber AP; Michalowsky LA; Nass GS; Skoletsky J; Hire LM; Kotsopoulos SK; Phipps MF; Barberio DM; Klinger KW 《Human molecular genetics》1997,6(3):337-347
As more mutations are identified in genes of known sequence, there is a
crucial need in the areas of medical genetics and genome analysis for
rapid, accurate and cost-effective methods of mutation detection. We have
developed a multiplex allele-specific diagnostic assay (MASDA) for analysis
of large numbers of samples (> 500) simultaneously for a large number of
known mutations (> 100) in a single assay. MASDA utilizes
oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA
samples are immobilized on a solid support and a single hybridization is
performed with a pool of allele-specific oligonucleotide (ASO) probes. Any
probes complementary to specific mutations present in a given sample are in
effect affinity purified from the pool by the target DNA. Sequence-specific
band patterns (fingerprints), generated by chemical or enzymatic sequencing
of the bound ASO(s), easily identify the specific mutation(s). Using this
design, in a single diagnostic assay, we tested samples for 66 cystic
fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell
anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations,
four mutations in Canavan disease, four mutations in Fanconi anemia, and
five mutations in BRCA1. Each mutation was correctly identified. Finally,
in a blinded study of 106 of these mutations in > 500 patients, all
mutations were properly identified. There were no false positives or false
negatives. The MASDA assay is capable of detecting point mutations as well
as small insertion or deletion mutations. This technology is amenable to
automation and is suitable for immediate utilization for high-throughput
genetic diagnostics in clinical and research laboratories.
相似文献
14.
Dou Q; Tarnuzzer RW; Williams RS; Schultz GS; Chegini N 《Molecular human reproduction》1997,3(11):1005-1014
15.
Rodgers KE; Girgis W; St Amand K; Campeau J; diZerega GS 《Human reproduction (Oxford, England)》1998,13(9):2443-2451
Adhesion formation is a major source of postoperative morbidity and
mortality. In this study, the ability of a variety of lazaroid formulations
[the antioxidant 21-aminosteroid PNU74006F (tirilazad) and the
non-steroidal 2-methylaminochroman derivative PNU83,836E] to reduce i.p.
adhesion formation in three rabbit models was examined. In initial studies,
PNU83836E was administered via Alzet miniosmotic pump to the site of
injury. In the sidewall and double uterine horn models, PNU83,836E was
administered via Alzet miniosmotic pump for the entire postoperative
interval. In the sidewall model, there was a dose- dependent reduction in
the area of the sidewall injury that was involved in adhesions. In the
double uterine horn model, PNU83,836E was administered via Alzet
miniosmotic pump to the area of injury for 1, 2, 3 or 7 days.
Administration for as little as 24 h after surgery significantly reduced
the extent of adhesion formation and the reduction was increased if it was
administered for longer. Further studies were conducted in which various
lazaroid formulations were administered as a bolus at the end of surgery.
In both the sidewall and double uterine horn models, administration of
either PNU83,386E (in citrate buffer) or PNU74006F (in cyclodextrin or
lipid emulsion vehicles) at the end of surgery reduced adhesion formation.
Administration of a bolus of PNU74006F 10 min prior to initiation of
surgery with or without additional treatment at the end of surgery further
increased its efficacy in the reduction of adhesion formation.
Administration of a minimum of 1.5 mg before and after surgery (3 mg total)
was required for maximal efficacy. These studies demonstrate that pre- and
postoperative administration of either a steroidal (PNU74006F) or
non-steroidal (PNU83,836E) lazaroid intraperitoneally reduced the formation
and reformation of postoperative adhesions in three animal models.
相似文献
16.
Renal transplantation has become a treatment of choice for patients with end stage renal disease. A successful transplant is the result of a combination of several factors acting synergistically, such as the degree of HLA compatibility between donor and the recipient, pretransplant blood transfusions, the recipient''s state of immunoreactivity and sensitization, immunosuppressive therapy given in post operative period etc. Donor selection appears to be the most critical factor for the long term success of the organ graft. In this brief review, some of the important parameters of donor selection in renal transplantation are highlighted.KEY WORDS: Histocompatibility (HLA) matching, Cross match, Sensitization 相似文献
17.
P G Pearson J G Slatter M S Rashed D H Han T A Baillie 《Chemical research in toxicology》1991,4(4):436-444
The reactivity toward peptides and proteins of S-(N-methylcarbamoyl)glutathione (SMG), the glutathione conjugate of methyl isocyanate, and the corresponding cysteine adduct, S-(N-methylcarbamoyl)cysteine (SMC), was investigated with the aid of in vitro model systems. Incubation of SMC or a trideuteriomethyl analogue of SMC with either the reduced or oxidized forms of oxytocin afforded similar mixtures of mono-, bis- and tris-N-methylcarbamoylated peptides. Structure elucidation of the mono and bis adducts by fast atom bombardment tandem mass spectrometry indicated that carbamoylation of oxytocin occurred preferentially at Cys-6 and that Cys-1 and/or Tyr-2 were secondary sites of modification. Upon incubation of S-[N-([14C]methyl)carbamoyl]glutathione (14C-SMG) with native bovine serum albumin (BSA), radioactivity became bound covalently to the protein in a time- and concentration-dependent fashion. "Blocking" of the lone Cys-34 thiol group of BSA in the form of a disulfide prior to exposure of the protein to 14C-SMG failed to decrease significantly the extent or time course of this covalent binding. It is concluded that carbamate thioester conjugates of MIC are reactive, carbamoylating entities which can donate the elements of MIC to nucleophilic functionalities on peptides and proteins. Free thiols appear to be preferred sites for such carbamoylation processes, a phenomenon that may have important toxicological consequences in the pathology of tissue lesions induced by MIC and related isocyanates. 相似文献
18.
We have demonstrated in our in vitro system that an extracorporeal lithotripter utilizing a movable ultrasound probe can fragment gallstones more effectively when the ultrasound probe is not partially blocking shock waves. Using a pressure transducer we measured the pressures in the focal volume of a Wolf Piezolith 2300 lithotripter with the ultrasound probe fully extended and fully retracted. We also chose 12 pairs of twin gallstones, each taken from the same gallbladder. One stone from each pair was subjected to shock waves while the ultrasound probe was fully extended and the other treated while the probe was fully retracted. Shock wave pressures (which are converted to a measurable voltage output by our transducer) were clearly lower when the ultrasound probe was extended (5.45 volts; SEM = 0.10 volts) as compared to when the ultrasound scanner was retracted (6.7 volts: SEM = 0.08 volts). Significantly more shock waves were required to completely fragment stones when the ultrasound scanner was extended than when it was retracted (p = 0.01 using the nonparametric Wilcoxon's signed rank test). These results show that, in the lithotripter tested, an extended in-line ultrasound scanner can partially block shock waves. Retraction of an extendible ultrasound probe may enhance stone fragmentation when operating at the highest shock wave intensity. 相似文献
19.
20.
Emergency and elective embolotherapy of various systemic arteries in 64 patients was carried out at a tertiary centre of Armed Forces. Specific indications were haemoptysis (n=43), preoperative (n=18), haematuria (n=1), epistaxis (n=1) and chemoembolization (n=1). The procedures were performed with gelfoam pellets (n=46), gelfoam pellets and absolute alcohol (n=1), polyvinyl alcohol particles (PVA) (n=14), steel coils (n=2) and Adriamycin-in-oil emulsion (n=1). Embolotherapy resulted in complete haemostasis in 37 (82.2%) out of 45 cases of haemorrhage. In eight cases (17.8%), it resulted in significant improvement. Complete haemostasis was achieved in both cases of haematuria and epistaxis. Pre-operative embolotherapy resulted in considerable reduction of peroperative blood loss in all the cases. Chemoembolization of Hepatocellular carcinoma resulted in partial regression of the tumour. The purpose of this study was to assess the efficacy, safety and reliability of vascular embolotherapy for control of life threatening haemorrhage and preoperative reduction of lesions.KEY WORDS: Embolization, Embolotherapy, Haemorrhage 相似文献