A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis

Abstract

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5'-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

Figure 1. Detection of the specificity of UP-M-PCR.

(A) Comparison of specificity between specific primers and compound specific primers. Lanes 1∼15, amplicon fragments of specific primer; lanes 1′∼15′, amplicon fragments of UP and compound specific primer; lane 0/0′, negative control without template; lane M, 100 bp DNA Marker. (B) Lanes 1∼15, amplicon fragments of one specific primer in singlet PCR; lanes 1′∼15′, amplicon fragments of fifteen-plex PCR with only one certain template DNA; lane M, 100 bp DNA Marker.

Figure 2. Impact of concentration of universal primer on singlet PCR.

Lane 0, negative control without template; lanes 1, 2, 3, 4, amplicon fragments by compound specific primer pair Lec-118-F/R at the concentration of 500 nmol L⁻¹, 50 nmol L⁻¹, 25 nmol L⁻¹, 5 nmol L⁻¹ respectively; lanes 5, 6, 7, 8, amplicon fragments by UP (500 nmol L⁻¹) and compound specific primer Lec-118-F/R at a series concentrations of 500 nmol L⁻¹, 50 nmol L⁻¹, 25 nmol L⁻¹,5 nmol L⁻¹; lane M, 100 bp DNA Marker.

Figure 3. Optimization of the UP-M-PCR.

Lane A, B, C, D, E, amplicon fragments by UP (500 nmol L⁻¹) and compound specific primer hpt-839, nptII-508, pat-262, bar-226 and sps-110 at a series concentrations of 500 nmol L⁻¹, 50 nmol L⁻¹, 25 nmol L⁻¹, 5 nmol L⁻¹, 0.5 nmol L⁻¹; lane F1, amplicon fragments by UP at 500 nmol L⁻¹ and all compound specific primers at 25 nmol L⁻¹; lane F2, amplicon fragments by UP at 500 nmol L⁻¹ and all compound specific primers at the optimized concentration; lane G1,G2,G3, amplicon fragments by all primers at the optimized concentration with TaKaRa Taq™, Phire™ Hot Start DNA polymerase, iProof™ High-Fidelity DNA polymerase; lane H1, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the common amplification conditions; lane H2, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the optimized amplification conditions; lane M, 100 bp DNA Marker.

Figure 4. Multiplex PCR assay for testing of primer interference.

Using equivalent DNA mix of six different GM events including Bt11 of maize, MON 15985 of cotton, GM rice with bar gene, Huanong No. 1 of papaya, RRS of soybean and GM canola with hpt gene. Lanes 1∼15, PCR runs starting with the largest amplicon (by hpt gene-specific primer, amplicon size 879 bp), followed by the addition of a second primer pair, until the fifteenth primer pair; lanes1′∼15′, PCR runs starting with the fifteen-plex amplicon, followed by the elimination of the largest amplicon primer pair (by hpt gene-specific primer, amplicon size 879 bp), until only the smallest amplicon (by FatA gene-specific primer, amplicon size 116 bp) remained; lane M, 100 bp DNA Marker.

Figure 5. Sensitivity detection of single template by UP-M-PCR.

a, MON 15985 of GM cotton; b, Huanong No. 1 of GM papaya; c, Bt11 of GM maize; d, GM rice with bar gene; e, GM canola with hpt gene; f, RRS of GM soybean. Lanes 1∼5, template concentration from 50, 5, 0.5, 0.1 ng to 0.05 ng; lane M, 100 bp DNA Marker.

Figure 6. Application of UP-M-PCR.

Lane 1,2, GM/non-GM cotton, with endogenous genes sad1 (amplicon size 131 bp); Lane 3,4, GM/non-GM papaya, with endogenous genes Pa (amplicon size 403 bp); Lane 5,6, GM/non-GM maize, with endogenous genes Ivr (amplicon size 266 bp); Lane 7,8, GM/non-GM rice, with endogenous genes sps (amplicon size 150 bp); Lane 9,10, GM/non-GM canola, with endogenous genes FatA (amplicon size 116 bp); Lane 11,12, GM/non-GM soybean, with endogenous genes Lec (amplicon size 158 bp); Lane 0, negative control without template; lane M, 100 bp DNA Marker.

Figure 7. Amplification routine of UP-M-PCR.

Each compound specific primer contained a universal sequence at the 5′-end (red) and the specific primer at the 3′-end (blue). The amplified fragments with the primer pairs of different targets are individually marked in different colors. The amplified fragments only by the universal primer are marked in red.