Abstract
Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3′-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples.
Highlights
Nucleic acids are typically amplified using the polymerase chain reaction (PCR), which requires thermal cycling, and while portable microsystems capable of carrying out PCR have been developed, a more attractive solution is the use of isothermal amplification, including nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA), as well as recombinase polymerase amplification (RPA)
Four thiolated reverse primers were immobilized on the surface of the wells of a maleimide microtiter plate, where the primers were designed to be identical and complementary to the target up to the base adjacent to the polymorphic site located at the 3′ end, where each primer had a different terminal base
Isothermal recombinase polymerase amplification based solidphase primer extension was used for the detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample
Summary
A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The displaced DNA strand is stabilized by single-stranded binding proteins, and the recombinase disassembles and a strand displacing DNA polymerase binds to the 3′ end of the primer and elongation is initiated.[1,6,7] Solid-phase RPA has been reported, where one of the two primers is immobilized on a substrate and the other is maintained in the solution phase, resulting in surfacetethered amplified DNA targets.[8−10] The amplified DNA can be detected using a variety of different transduction methodologies, including electrochemical and chemiluminescence detection.[11,12] Solid-phase RPA is attractive due to its compatibility with multiplexed. Different approaches have been developed for the detection of solid phase amplification products, including the use of biotin labeled solution phase primer, followed by the addition of enzyme labeled streptavidin,[15] or alternatively the use of enzyme labeled primer[15,16] or redox decorated nanoparticle labeled primer.[17]
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