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RESEARCH PROFILES Dipstick for SNP analysis
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had been immobilized in the test zone of the strip. The capture generated a red band from the gold nanoparticles. As the buffer migrated further, the mixture passed through a “control zone” on the strip with immobilized
Control zone Test zone
Non-extended PEXT reaction product
Extended PEXT reaction product
Photograph of two dry-reagent dipsticks for the analysis of a SNP, one that lacks an extended product (left) and one that shows the presence of an extended product (right).
poly(dA) strands. Excess, unhybridized nanoparticle probes were captured in the control zone, forming a second red band and ensuring that the test worked properly. In their current paper, Ioannou and colleagues extended the assay to genotyping. The investigators looked at 6 SNPs in PCR-amplified products of the mannose-binding lectin gene (MBL2). Deficiencies in MBL2 gene have been shown to cause increased susceptibility to various infections and autoimmune disorders. Ioannou and colleagues performed PCR on the region of the MBL2 gene that carried the SNPs of interest. Next, they carried out quick primer extension (PEXT) reactions on the amplified DNA fragments. Each fragment was subjected to two PEXT reactions. One
A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 7
PENELOPE IOANNOU
Single-nucleotide polymorphisms (SNPs) are changes in single bases at specific positions in genes. Because SNPs can affect the function of a gene, clinical diagnostic laboratories regularly genotype a selected number of genes involved in diseases such as -thalassemia and familial hypercholesterolemia or genes that encode drug-metabolizing enzymes. But the current ways to analyze SNPs are relatively labor-intensive and time-consuming. To make the analysis quicker and easier, Penelope Ioannou and colleagues at Athens University, University of Patras, Foundation for Research and Technology Hellas, and Medical Genetics Athens University (all in Greece) describe in the January 15 issue of Analytical Chemistry (pp 395– 402) a dry-reagent dipstick test that can visualize SNPs in a gene within 10 min. “The idea for this method actually originates from the widely used pregnancy test. The pregnancy test is essentially a biosensor for human chorionic gonadotropin, with all required reagents deposited in a dry form on a suitable strip,” says Ioannou. “We thought to develop a method for the detection of DNA mutations that also provided a visual answer, without the need for sophisticated instruments or highly trained personnel.” The investigators had previously developed a dry-reagent dipstick format for the analysis of PCR-amplified products (Anal. Chem. 2003, 75, 4155–4160). Biotinylated PCR-amplified strands of DNA were hybridized to oligonucleotides with a poly(dA)-tail and applied to the dipstick. The strip was then immersed in an appropriate buffer. The strip contained gold nanoparticles with oligo(dT) strands attached to their surfaces. As the buffer migrated up, it rehydrated the gold nanoparticles. The nanoparticles linked themselves to the biotinylated DNA via poly(dA)/ (dT) hybridization. The DNA strands were then captured by streptavidin that
reaction was with a “normal” primer and the other was with a “mutant” primer. Both primers contained a poly(dA) tail at the 5´-end, but they differed in the final nucleotide at the 3´-end, which was at the position of the SNP. Both reactions were carried out in the presence of biotin-dUTP. Only the primer that had perfect complementarity to the target DNA was extended by a special DNA polymerase (which lacked exonuclease activity so it didn’t chew up the extended product) to result in a biotinylated product. Aliquots of 5 µL from each of the two PEXT reactions were applied to two dipsticks for rapid visualization. Only the biotinylated extension products were captured in the test zone by streptavidin. As before, the control zone was present to confirm that the assay worked. The two strips told the investigators whether a person was homozygous-normal, homozygous-mutant, or heterozygotic for a given SNP. “Of course, this method is only suitable for known mutations, not for unknown mutations,” points out Ioannou. The gold nanoparticles are stable for >1 year in the dry-reagent format and are amenable to storage. Ioannou also explains that the assay isn’t limited to the red color of gold nanoparticles. For instance, latex particles come in various colors. It could be possible to incorporate them into the assay to create a multicolored detection method for a number of SNPs in a single platform. The investigators are now looking for ways to improve their test. “The fact you have to perform two reactions per polymorphic site in this particular assay might be a limitation,” says Ioannou. “We’re looking for another kind of biosensor format in order to do both reactions in one tube. It would be nice if we could, let’s say, do two primer extension reactions in the same tube and then analyze the mixture with one dry reagent strip, not on two.” a —Rajendrani Mukhopadhyay
