scholarly journals Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

1992 ◽  
Vol 2 (2) ◽  
pp. 157-162 ◽  
Author(s):  
A Iitia ◽  
E Hogdall ◽  
P Dahlen ◽  
P Hurskainen ◽  
J Vuust ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pcr Primers ◽  
Cystic Fibrosis Gene ◽  
Time Resolved ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Identification of Phytophthora alni subspecies in riparian stands in the Czech Republic

2013 ◽  
Vol 49 (Special Issue) ◽  
pp. S3-S10 ◽  
Author(s):  
P. Štěpánková ◽  
K. Černý ◽  
V. Strnadová ◽  
P. Hanáček ◽  
M. Tomšovský
Keyword(s):  
Czech Republic ◽  
Alnus Glutinosa ◽  
Pcr Primers ◽  
The Czech Republic ◽  
Specific Pcr ◽  
Related Taxon ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Subspecific Level

In the Czech Republic, Phytophthora alni was first confirmed in 2001 and the pathogen has been quickly spreading and occupying almost the whole area of the country. The pathogen attacks Alnus glutinosa or A. incana to a lesser extent and causes considerable losses of alder trees along hundreds of kilometres of riverbanks. The aim of our work was to perform the identification of P. alni isolates at the subspecific level using PCR and to determine the frequencies and distribution of particular subspecies. The allele-specific PCR primers focused on allele diversity of orthologs of ASF-like, TRP1, RAS-Ypt, and GPA1 genes were selected for identification. Eighty-eight per cent of the 59 analysed isolates belonged to P. alni ssp. alni while 12% were P. alni ssp. uniformis. P. alni ssp. multiformis has not been recorded in the country till now. The two subspecies differed in distribution. P. alni ssp. alni dominated in riparian stands along broader rivers in lowlands and the results confirmed the more effective spreading of P. alni ssp. alni based on its higher aggressiveness and ecological advantage. P. alni ssp. uniformis was acquired rather from riparian stands of small watercourses at higher altitudes. The insular distribution of P. alni ssp. uniformis may represent the remains of its former occurrence. Therefore, P. alni ssp. uniformis may be an indigenous subspecies suppressed by the more aggressive related taxon.

scholarly journals Improvement in Sensitivity of Allele-specific PCR Facilitates Reliable Noninvasive Prenatal Detection of Cystic Fibrosis

2004 ◽  
Vol 50 (4) ◽  
pp. 694-701 ◽  
Author(s):  
Ourania Nasis ◽  
Shanel Thompson ◽  
Tom Hong ◽  
Margaret Sherwood ◽  
Shawn Radcliffe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Alterations ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Specific Pcr ◽  
Wild Type Sequence ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Type Sequence

Abstract Background: Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. Methods: We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11–19.2 weeks), with mutation confirmation by chorionic villus sampling. Results: The method detected 5 copies of the CF D1152H mutant allele in the presence of up to ∼100 000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. Conclusions: This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.

scholarly journals Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus

1993 ◽  
Vol 295 (1) ◽  
pp. 313-315 ◽  
Author(s):  
A A Fryer ◽  
L Zhao ◽  
J Alldersea ◽  
W R Pearson ◽  
R C Strange
Keyword(s):  
Pcr Primers ◽  
Single Step ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Glutathione S Transferase ◽  
Specific Pcr ◽  
Target Dna ◽  
Allele Specific ◽  
Pcr Method ◽  
Allele Specific Pcr

We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms was confirmed by comparison with, firstly, an established PCR method that distinguishes GSTM1 *0 homozygotes from individuals with the other GSTM1 genotypes and, secondly, GSTM1 phenotypes determined using chromatofocusing.

Allele specific PCR with microfluorometry: application to the detection of del F508 mutation in cystic fibrosis

2002 ◽  
Vol 316 (1-2) ◽  
pp. 147-154 ◽  
Author(s):  
Akihiro Yamaguchi ◽  
Juan Alberto Nepote ◽  
Maliheh Kadivar ◽  
Yasuko Tagami ◽  
Masaru Fukushi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Molecular Analysis of Cystic Fibrosis Patients in Hungary – An Update to the Mutational Spectrum/Molekularna Analiza Obolelih Od Cistične Fibroze U Mađarskoj – Dopune Spektru Mutacija

2014 ◽  
Vol 34 (1) ◽  
pp. 46-51 ◽  
Author(s):  
Gergely Ivády ◽  
Katalin Koczok ◽  
Laszlo Madar ◽  
Eva Gombos ◽  
Izabella Toth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Newborn Screening ◽  
Sanger Sequencing ◽  
Mutation Detection ◽  
Detection Methods ◽  
Coding Region ◽  
Specific Pcr ◽  
Mutation Profile ◽  
Allele Specific ◽  
Allele Specific Pcr

Summary Background: In this study the authors present an update to the CFTR mutation profile in Hungary, utilizing data from a selected cohort of 45 cystic fibrosis (CF) patients from different regions of the country. Methods: Depending on the preceding analysis, four different mutation detection methods were used. A commercial assay targeting the most common CF-causing mutations was performed as the first test followed by an allele specific PCR for CFTRdele2,3(21kb), Sanger sequencing and MLPA analysis of the coding region of the CFTR gene. Results: In our recent study 27 different mutations were detected, including 2 novel ones (c.1037_1038insA and c.1394C>T). Besides F508del (c.1521_1523delCTT), the following mutations were found at a frequency of ≥ 4.0%: W1282X (c.3846G>A), N1303K (c.3909C>G), CFTRdele2,3(21kb) (c.54-5940_273+10250del21kb) and 2184insA (c.2052_2053insA). In addition, four mutations (G542X, Y1092X, 621+1G>T, and 2143delT) were found in more than one allele. Conclusions: The updated database of Hungarian mutations not only enables to increase the efficiency of the existing diagnostic approach, but also provides a further refined basis for the introduction of the molecular newborn screening (NBS) program in Hungary.

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