Fig 1.
Overall flow chart comparing conventional and single-tube methods.
We introduce the use of perfluorocarbon (PFC) for cell lysis and magnetic particles for nucleic acid purification in single-tube reaction. Compared with the conventional methods, using only one tube greatly simplify the procedures and reduce the risk of cross-contamination. No incubation or enzyme digesting time is needed.
Table 1.
Single-tube nucleic acid amplification using various sample volumes.
Fig 2.
Summary of single-tube reactions.
(A) Single-tube nucleic acid purification. All reagents (including lysis buffer containing PFC and wash buffer) can be added in one step, greatly simplifying the procedure. After a brief and intense vortex a homogenization state is achieved (see discussion). Nucleic acids are then purified by 1 μm, dextran-coated and positively charged magnetic beads. (B) Single-tube PCR. After isolation, reagents for use in nucleic acid amplification can be added directly into the same tube without further treatment, or, all the reagents required for nucleic acid isolation and amplification (including lysis buffer, washing buffer and PCR reagents) can be added in one step in the very beginning of the experiment. PCR is then conducted in PFC micelles. The amplified products are collected and purified by coated magnetic beads. (C) Single-tube emulsion PCR. After cell lysis and magnetic bead purification of nucleic acids, reagents for emulsion PCR can be added directly into the same tube after discarding the supernatant and the cellular debris. Tests can be performed in microemulsions (water-in-oil droplets) containing sequence-specific captures and probes for further high-throughput detection. The tube sizes in each column are not to scale.
Fig 3.
RNA isolation/purification from frozen mouse liver tissue, ITRI (I, duplicate), and Qiagen (Q) samples analyzed by Agilent 2100® gel electrophoresis.
Compared with the conventional methods, using single-tube reaction greatly simplified the procedures (see above). Analysis by Agilent 2100® bioanalyzer revealed that the sample qualities were comparable. This demonstrates that by using single-tube reaction the experiment time can be greatly reduced while the nucleic acid quality is maintained.
Table 2.
The influences of different perfluorohexane ratios on RNA yields and qualities.
Fig 4.
cDNA yields and qualities of single-tube reaction samples.
(A) PCR amplification of the GAPDH gene, performed in one tube and in one step with the nucleic acid isolation. cDNA yields and qualities of samples amplified using single-tube reactions were comparable to those amplified using commercialized kits (Fermentas Maxima®) in two steps after nucleic acid isolation. (B) Standard dengue virus sera samples were tested by RT-PCR to see if trace amounts of specific sequences could be detected. Amplified products from single-tube reaction and commercialized kits were comparable.
Fig 5.
Single-tube multiplex PCR and single-tube PCR detection of selected plant genes.
(A) Multiplex PCR reactions were successfully conducted in a single tube (S1.2.3: Sample 1.2.3. N.C.: Negative control). (B) Single-tube amplification for various plant tissues, demonstrating the cell lysis power of PFC in treating plant tissues that contained rigid cell walls.
Fig 6.
(A)(B)(C) Target 1 represents the DNA-probe-Cy3 in which the probe 1-Cy3 was labeled. Target 2 represents the DNA-probe-Cy5 in which the probe 2-Cy5 was labeled. WL represents the condition under white light. The results indicate that the biosample treated with a single-tube reaction was in complete oil-ball shapes after being amplified with emulsion PCR, which was beneficial for the next optical analyses. (D)(E) The formation of droplets is shown, which could be placed in arrays. The bright spots inside the droplets are fluorescent emissions from specifically labeled probes. N.C. represents negative control.