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Articles |
1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler
1
Department of Clinical Chemistry, Georg-August-University, Robert Koch Strasse 40, 37075 Goettingen, Germany.
a Author for correspondence. Fax 49-551-39-2955; e-mail nahsen{at}gwdg.de
Background: 
1-Antitrypsin is the major plasma
serine protease inhibitor. Its deficiency is mainly associated with the
alleles PI*S and PI*Z and can
lead to obstructive lung disease in adults and to liver cirrhosis
during childhood.
Methods: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705.
Results: Unequivocal genotyping results were obtained for all investigated samples in an assay time of ~30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves.
Conclusions: To our knowledge, this is the first report of
simultaneous detection of two mutations in a single tube by PCR of
genomic DNA and the use of two different reporter dyes with the
LightCycler color compensation feature. This approach is a rapid,
convenient, and economic alternative to other methods described to date
for the detection of 1-antitrypsin deficiency
alleles.
The following articles in journals at HighWire Press have cited this article:
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  J. Prins, B. B. van der Meijden, R. J. Kraaijenhagen, and J. P.M. Wielders Inherited Chronic Obstructive Pulmonary Disease: New Selective-Sequencing Workup for {alpha}1-Antitrypsin Deficiency Identifies 2 Previously Unidentified Null Alleles Clin. Chem., January 1, 2008; 54(1): 101 - 107. [Abstract] [Full Text] [PDF]
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M. P. Kaczor, M. Sanak, and A. Szczeklik Rapid and Inexpensive Detection of {alpha}1-Antitrypsin Deficiency-Related Alleles S and Z by a Real-Time Polymerase Chain Reaction Suitable for a Large-Scale Population-Based Screening J. Mol. Diagn., February 1, 2007; 9(1): 99 - 104. [Abstract] [Full Text] [PDF]
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M. R. Snyder, J. A. Katzmann, M. L. Butz, P. Yang, D. B. Dawson, K. C. Halling, W. E. Highsmith, and S. N. Thibodeau Diagnosis of {alpha}-1-Antitrypsin Deficiency: An Algorithm of Quantification, Genotyping, and Phenotyping Clin. Chem., December 1, 2006; 52(12): 2236 - 2242. [Abstract] [Full Text] [PDF]
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 S. Andolfatto, F. Namour, A-L. Garnier, F. Chabot, J-L. Gueant, and I. Aimone-Gastin Genomic DNA extraction from small amounts of serum to be used for {alpha}1-antitrypsin genotype analysis Eur. Respir. J., February 1, 2003; 21(2): 215 - 219. [Abstract] [Full Text] [PDF]
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 F. Rodriguez, R. Jardi, X. Costa, M. Cotrina, R. Galimany, R. Vidal, and M. Miravitlles Rapid Screening for {alpha}1-Antitrypsin Deficiency in Patients with Chronic Obstructive Pulmonary Disease Using Dried Blood Specimens Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 814 - 817. [Abstract] [Full Text]
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P. S. Bernard and C. T. Wittwer Real-Time PCR Technology for Cancer Diagnostics Clin. Chem., August 1, 2002; 48(8): 1178 - 1185. [Abstract] [Full Text] [PDF]
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H. Millward, W. Samowitz, C. T. Wittwer, and P. S. Bernard Homogeneous Amplification and Mutation Scanning of the p53 Gene Using Fluorescent Melting Curves Clin. Chem., August 1, 2002; 48(8): 1321 - 1328. [Abstract] [Full Text] [PDF]
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 J. L. Sanchez and G. A. Storch Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA J. Clin. Microbiol., July 1, 2002; 40(7): 2381 - 2386. [Abstract] [Full Text] [PDF]
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A. Ruiz, G. Antinolo, I. Marcos, and S. Borrego Novel Technique for Scanning of Codon 634 of the RET Protooncogene with Fluorescence Resonance Energy Transfer and Real-Time PCR in Patients with Medullary Thyroid Carcinoma Clin. Chem., November 1, 2001; 47(11): 1939 - 1944. [Abstract] [Full Text] [PDF]
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N. von Ahsen, M. Richter, C. Grupp, B. Ringe, M. Oellerich, and V. W. Armstrong No Influence of the MDR-1 C3435T Polymorphism or a CYP3A4 Promoter Polymorphism (CYP3A4-V Allele) on Dose-adjusted Cyclosporin A Trough Concentrations or Rejection Incidence in Stable Renal Transplant Recipients Clin. Chem., June 1, 2001; 47(6): 1048 - 1052. [Abstract] [Full Text] [PDF]
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N. von Ahsen, M. Oellerich, and E. Schutz DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR Clin. Chem., December 1, 2000; 46(12): 1939 - 1945. [Abstract] [Full Text] [PDF]
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E. Schutz, N. von Ahsen, and M. Oellerich Genotyping of Eight Thiopurine Methyltransferase Mutations: Three-Color Multiplexing, ""Two-Color/Shared"" Anchor, and Fluorescence-quenching Hybridization Probe Assays Based on Thermodynamic Nearest-Neighbor Probe Design Clin. Chem., November 1, 2000; 46(11): 1728 - 1737. [Abstract] [Full Text] [PDF]
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F. A.J.T.M. van den Bergh, A. M. van Oeveren-Dybicz, and M. A.M. Bon Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection Clin. Chem., August 1, 2000; 46(8): 1191 - 1195. [Full Text] [PDF]
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P. S. Bernard and C. T. Wittwer Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes Clin. Chem., February 1, 2000; 46(2): 147 - 148. [Full Text] [PDF]
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