Fig 1.
Principle of PNA-LNA mediated LAMP.
When the target gene is wild-type, the clamping PNA probe forms a stable duplex with the dumbbell structure, and interferes with the annealing and extension of the LNA primer. On the other hand, when the target gene is mutated, the clamping PNA probe does not anneal with the cDNA because of the single-base mismatch, and the LNA primer breaks its internal interaction to bind the target, and the extension reaction proceeds.
Fig 2.
Set of PNA-LNA mediated LAMP primers used.
(a) The figure shows the DNA sequence of codon 12 of the KRAS gene. The wild and mutated nucleotides in KRAS codon 12 are shown in bold blue type (G for the wild type, A for G12D and T for G12V.). (b) The figure indicates the design of the set of LAMP primers and the PNA probe. The italic faces indicate modification site of PNA and the bold faces indicate modification site of LNA.
Fig 3.
Amplification products of KRAS by PNA-LNA mediated LAMP.
(a-d) The graphs indicate the fluorescence intensity of the LAMP products as measured using real-time PCR equipment. (e) The figure shows the detection by agarose gel electrophoresis. (f) The fluorescence emitted from LAMP products in microtubes under UV light was visually assessed. LNA primer complementary to the G12D mutant gene was used for Panc-1 and Bx cDNA samples, and the LNA primer complementary to the G12V mutant gene was used for QGP-1 and HS766T cDNA samples. M: size maker, lane 1–8: LAMP products from Panc-1 with PNA, Panc-1 without PNA, QGP-1 with PNA, QGP-1 without PNA, Bx with PNA, Bx without PNA, HS766T with PNA and HS766T without PNA, lane 9: negative control without cDNA.
Fig 4.
The LOD of PNA-LNA mediated LAMP.
Mutant type Panc-1 and QGP-1 samples were serially diluted with wild type Bx and HS766T samples (100% Bx and 100% HS766T) to give Panc-1 and QGP-1 cDNA samples concentrations of 100, 10, 1, 0.5, 0.1 and 0%, which were then analyzed using PNA-LNA mediated LAMP. As a negative control, we use 100% Bx and 100% HS766T cDNA samples. (a, c) The graph shows the fluorescence intensity of LAMP products measured using real-time PCR equipment. (b, d) The fluorescence emitted by LAMP products in microtubes under UV light was visually assessed. (e) The graph shows the respective graph of TT vs %Mutant/ WT for both mutations. The TT value was defined as the time it took for fluorescence intensity to reach a threshold value of 100 after baseline subtraction.
Fig 5.
Detection of KRAS mutation subtypes using PNA-LNA mediated LAMP.
(a) By using an LNA primer with a sequence complementary to that encoding the G12D mutation, cDNA from the Panc-1 cell line that expresses the KRAS G12D mutant was amplified more rapidly than cDNA from the QGP-1 cell line that expresses the KRAS G12V mutant (P<0.01). (b) Conversely, by using an LNA primer with a sequence complementary to that encoding the KRAS G12V mutation, cDNA from the QGP-1 cell line that expresses the KRAS G12V mutant was amplified more rapidly than cDNA from the Panc-1 cell line that expresses the KRAS G12D mutant (P<0.01). ΔTT is defined in the method.