Arginine-based PNA microarrays for APOE genotyping.
Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimer's disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.
Calabretta
Alessandro
A
Department of Organic and Industrial Chemistry, University of Parma, Viale G.P. Usberti 17a, I-43100, Parma, Italy.
Tedeschi
Tullia
T
Di Cola
Gabriella
G
Corradini
Roberto
R
Sforza
Stefano
S
Marchelli
Rosangela
R
D001057:Apolipoproteins E / Q000235:genetics*; D001120:Arginine / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D015394:Molecular Structure; D020135:Peptide Nucleic Acids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D044366:Transition Temperature
D001057:Apolipoproteins E; D020135:Peptide Nucleic Acids; D001120:Arginine