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A novel method of detecting alpha-1 antitrypsin deficiency of Z mutant (GAG(342)AAG) in a single PCR reaction using base-quenched probe

Qin, Li; Luo, Guanghua; Zhang, Jun and Xu, Ning LU (2014) In Clinica Chimica Acta 427. p.29-33
Abstract
Background: Alpha-1 antitrypsin (A1AT) is a protease inhibitor that protects the tissues from degradation by neutrophil elastase under certain pathological process. Alpha-1 antitrypsin deficiency (A1ATD) could associate with both lung and liver pathogenicities. Of all the deficiency alleles, Z mutant is the most common variant and causes severe complications. Here, we described a novel and quick method to detect Z mutant using the base-quenched probe technique in only one single PCR reaction. Methods: Primers and probe were designed based on the base-quenched probe technique. Two vectors, representing the two genotypes, were constructed as amplification templates for validating the method. The Z mutant (GAG(342)AAG) was analyzed according... (More)
Background: Alpha-1 antitrypsin (A1AT) is a protease inhibitor that protects the tissues from degradation by neutrophil elastase under certain pathological process. Alpha-1 antitrypsin deficiency (A1ATD) could associate with both lung and liver pathogenicities. Of all the deficiency alleles, Z mutant is the most common variant and causes severe complications. Here, we described a novel and quick method to detect Z mutant using the base-quenched probe technique in only one single PCR reaction. Methods: Primers and probe were designed based on the base-quenched probe technique. Two vectors, representing the two genotypes, were constructed as amplification templates for validating the method. The Z mutant (GAG(342)AAG) was analyzed according to the melting curve. Finally, the accuracy was confirmed by direct sequencing. Results: Z mutant could be accurately distinguished from the wild type. The wild type resulted in high melting temperature (TM) (48.64 +/- 133 degrees C), while when the Z mutation was present, the TM was shifted to an obvious low TM (4138 +/- 0.9017 degrees C). The sensitivity reached a low of 10(3) copies of template DNA with a clear melting valley and a complete concordance occurred between this method and the direct DNA sequencing. Conclusion: The present described method is simple, quick and economic as well as suitable for large-scale genotyping studies and clinical testing of Z mutant in patients with emphysema and cirrhosis. (C) 2013 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Single nucleotide polymorphism, Alphal-antitrypsin deficiency, PIZ, mutant
in
Clinica Chimica Acta
volume
427
pages
29 - 33
publisher
Elsevier
external identifiers
  • wos:000330819200008
  • scopus:84886030729
ISSN
0009-8981
DOI
10.1016/j.cca.2013.09.042
language
English
LU publication?
yes
id
e1c9ecb4-4b37-4b8f-87cd-c2f1a4718f36 (old id 4376544)
date added to LUP
2014-04-07 09:20:51
date last changed
2017-01-01 03:28:24
@article{e1c9ecb4-4b37-4b8f-87cd-c2f1a4718f36,
  abstract     = {Background: Alpha-1 antitrypsin (A1AT) is a protease inhibitor that protects the tissues from degradation by neutrophil elastase under certain pathological process. Alpha-1 antitrypsin deficiency (A1ATD) could associate with both lung and liver pathogenicities. Of all the deficiency alleles, Z mutant is the most common variant and causes severe complications. Here, we described a novel and quick method to detect Z mutant using the base-quenched probe technique in only one single PCR reaction. Methods: Primers and probe were designed based on the base-quenched probe technique. Two vectors, representing the two genotypes, were constructed as amplification templates for validating the method. The Z mutant (GAG(342)AAG) was analyzed according to the melting curve. Finally, the accuracy was confirmed by direct sequencing. Results: Z mutant could be accurately distinguished from the wild type. The wild type resulted in high melting temperature (TM) (48.64 +/- 133 degrees C), while when the Z mutation was present, the TM was shifted to an obvious low TM (4138 +/- 0.9017 degrees C). The sensitivity reached a low of 10(3) copies of template DNA with a clear melting valley and a complete concordance occurred between this method and the direct DNA sequencing. Conclusion: The present described method is simple, quick and economic as well as suitable for large-scale genotyping studies and clinical testing of Z mutant in patients with emphysema and cirrhosis. (C) 2013 Elsevier B.V. All rights reserved.},
  author       = {Qin, Li and Luo, Guanghua and Zhang, Jun and Xu, Ning},
  issn         = {0009-8981},
  keyword      = {Single nucleotide polymorphism,Alphal-antitrypsin deficiency,PIZ,mutant},
  language     = {eng},
  pages        = {29--33},
  publisher    = {Elsevier},
  series       = {Clinica Chimica Acta},
  title        = {A novel method of detecting alpha-1 antitrypsin deficiency of Z mutant (GAG(342)AAG) in a single PCR reaction using base-quenched probe},
  url          = {http://dx.doi.org/10.1016/j.cca.2013.09.042},
  volume       = {427},
  year         = {2014},
}