Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: identification of cytokine polymorphism by pyrosequencing analysis.
BACKGROUND
DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping.


METHODS
Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed.


RESULTS
IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B -511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B -31; 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B +3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples.


CONCLUSIONS
We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples.
Sun
Yi-Qian
YQ
Department of Biomedicine and Surgery, University Hospital of Linköping, Sweden.
Monstein
Hans-Jürg
HJ
Ryberg
Anna
A
Borch
Kurt
K
D001483:Base Sequence; D001706:Biopsy; D001782:Blood Donors; D016207:Cytokines / Q000097:blood* / Q000235:genetics*; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D016480:Helicobacter pylori / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic / Q000235:genetics*; D013997:Time Factors
D016207:Cytokines; D004247:DNA