Rapid DNA Extraction Protocol for Detection of Alpha-1 Antitrypsin Deficiency from Dried Blood Spots by Real-Time PCR

  • R. Struniawski
  • A. Szpechcinski
  • B. Poplawska
  • M. Skronski
  • J. Chorostowska-Wynimko
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 756)


The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.


Alpha1-antitrypsin deficiency Dried-blood spot Genotyping Real time PCR Screening 



Supported by National Institute of Tuberculosis and Lung Diseases in Warsaw (project 5/8) and by Ministry of Science and Higher Education, Republic of Poland (grant N404081539).

Conflicts of interest: The authors declare no conflicts of interest in relation to this article.


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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • R. Struniawski
    • 1
  • A. Szpechcinski
    • 1
  • B. Poplawska
    • 1
  • M. Skronski
    • 1
  • J. Chorostowska-Wynimko
    • 1
  1. 1.Laboratory of Molecular Diagnostics and ImmunologyNational Institute of Tuberculosis and Lung DiseasesWarsawPoland

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