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. 2002 Jun 15;30(12):e55.
doi: 10.1093/nar/gnf054.

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes

Tony Jun Huang  1 , Minghsun Liu, Linda D Knight, Wayne W Grody, Jeff F Miller, Chih-Ming Ho
Affiliations

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes

Tony Jun Huang et al. Nucleic Acids Res. .

Abstract

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.

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Figures

Figure 1
Figure 1
(A) Basic EDEMNA scheme. See text for details. (B) Adaptation of CARD to EDEMNA. Following the addition of HRP-conjugated anti-fluorescein monoclonal antibodies, instead of adding the HRP substrates, fluorescein-labeled tyramide molecules are added to the mixture. The HRP-mediated reactions deposit the additional fluorescein labels around the enzymes, creating additional targets for the HRP-conjugated antibodies to bind and resulting in higher output signals.
Figure 2
Figure 2
Comparing the ability of linear detector probe and hairpin-forming detector probes to detect the single nucleotide difference between the NDH-PM (Perfect Match) and mismatched (1 bp Mismatch) targets. (A) Using linear detector probe. (B) Using hairpin-forming detector probe.
Figure 3
Figure 3
Effect of incorporating competitor probes into EDEMNA. With increasing competitor probe concentrations (100 nM–1 mM), signals generated by the mismatched target (1 bp Mismatch) were progressively reduced to levels comparable with the background with no significant effect on the signals generated by NDH-PM (Perfect Match).
Figure 4
Figure 4
Signal amplification using CARD. Using NDH-PM as the target, without CARD, the output signals became indistinguishable from the background at the target concentration of 1 pM. With CARD, the output signals became indistinguishable from the background at 0.01 pM.
Figure 5
Figure 5
Screening for the factor V Leiden mutation using EDEMNA. (A) Homozygous wild-type. (B) Heterozygous carriers.
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