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Technical Briefs |
1 Laboratory of Biochemistry and Biophysics, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China
aauthor for correspondence: fax 86-27-8788-2661, e-mail tanclswu{at}public.wh.hb.cn
PCR is an important technique for identifying specific nucleic acid targets; for example, sequences associated with diseases and pathogens in clinical, environmental, and food samples. Many techniques currently used for sequence-specific detection of PCR products either require manual processing or are limited in the speed or scale of analysis. The development of biosensors in recent years has provided promising techniques for efficient sequence-specific DNA analysis (1). Biosensors have been used to detect target sequences in PCR products. Most studies have been conducted with the commercially available biosensor BIAcore, which is based on surface plasmon resonance (SPR) technology. In those investigations, either sequence-specific oligonucleotide probe (2)(3)(4)(5)(6)(7)(8) or amplified products themselves (8)(9)(10)(11) were immobilized on the sensor chip, limiting their application to one sequence or to one particular sample. Such a strategy suffers several drawbacks: (a) it compromises the automation and high-throughput capability of such instruments when different targets are analyzed; (b) chip-to-chip variation makes it difficult to compare different measurements; and (c) probe immobilization and chip consumption make running costs substantial.
To overcome these drawbacks, we describe here a "one-chip-for-all" strategy that is capable of, in principle, detecting different target sequences by use of the same sensor chip. Target sequences are amplified by asymmetric PCR using a primer pair in which the low-concentration primer carries a common tag sequence that is identical to that of the oligonucleotide capture probe immobilized on the sensor chip. The PCR product is then injected and captured on the sensor chip, and its sequence identity can be further verified by use of a target-specific probe (see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue6/). We exemplify this application by analyzing three different genes: the cystic fibrosis transmembrane conductance regulator gene (CFTR), the mutation of which causes cystic fibrosis (12); the hTERT gene, which encodes the catalytic component of the human telomerase complex (13); and the human gene for the tumor suppressor p53 (Table 1
).
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Asymmetric PCRs were conducted with 100 ng of human genomic DNA from HeLa cells in a final volume of 50 µL, containing 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 1 µM high-concentration primer; 0.1 µM low-concentration primer, and 3 U/reaction of Taq polymerase (MBI). Thermal cycling was conducted on a Biometro thermal cycler with initial denaturation at 94 °C for 3 min, followed by 55 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s for CFTR and of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for hTERT and p53. Symmetric PCRs were conducted under the same conditions except that 0.1 µM each of both primers were used. PCR products were run in 12% polyacrylamide gels at 20 V/cm for 1 h in Tris-borate-EDTA buffer [90 mmol/L Tris-borate (pH 8.0), 2 mmol/L EDTA] and visualized by ethidium bromide staining.
Detection of PCR products by SPR was conducted at 30 °C on a BIAcore X biosensor with a streptavidin-coated SA5 sensor chip using HEPES-buffered saline containing, per liter, 10 mmol of HEPES (pH 7.4), 0.15 mol of NaCl, 3.4 mmol of EDTA, and 0.05 mL of the surfactant P20 as running buffer. The biotinylated probe was immobilized on the sensor chip surface by injection of 30 µL of probe at 6 ng/µL into one flow cell, giving a capture of 
300 response units (RU) of probe. The PCR sample was mixed with an equal volume of HEPES-buffered saline containing 1.6 mol/L NaCl, and a 20-µL aliquot of this mixture was injected at 2 µL/min followed by a flow of running buffer. In some experiments, 10 µL of 10 ng/µL sequence-specific or irrelevant oligonucleotide was subsequently injected at 2 µL/min, followed by a flow of running buffer. The sensorgram of a blank cell was simultaneously recorded as reference and subtracted from that of the sensing cell. The sensor chip was regenerated by a pulse of 5 µL of NaOH (20 mmol/L) in NaCl (1 mol/L).
Taking CFTR as an example, gel electrophoresis (Fig. 1A
) showed that the tag sequence in the low-concentration primer was introduced into the symmetric and asymmetric PCR products based on their molecular sizes. The asymmetric PCR produced two weak bands with slower migration in addition to the double-stranded product. Evidence that these two bands were single-stranded DNA was that they were sensitive to mung-bean nuclease, which specifically digests single-stranded DNA. When the sample was incubated with the 40mer probe complementary to the CFTR gene before electrophoresis, these bands disappeared and a new band appeared, indicating that they were both the single-stranded form of the expected products. Similar results were obtained for hTERT and p53.
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The BIAcore system monitors changes in mass of analyte at sensor chip surfaces by changes in refractive index (14). The tag sequence introduced to the single-stranded PCR product is complementary to and can hybridize with the capture probe immobilized on the sensor chip. When PCR sample is injected, the hybridization between the PCR product and the probe is reflected by an increase in RU. The response is proportional to the quantity of PCR product captured (15). As shown in Fig. 1B
, the amplified product hybridized to the capture probe, as demonstrated by the increase in RU. Little hybridization was detected when the tag sequence was not present in the low-concentration primer or the gene was omitted or amplified by symmetric PCR (data not shown).
The sequence identity of the captured PCR product was further verified by an injection of target-specific probe complementary to the CFTR sequence, which led to an additional increase of 143 RU, indicating that the captured product was indeed what was expected. Because the target-specific probe had 40 and the expected PCR product had 127 bases, an increment of 143 RU corresponded to 454 (143 x 127/40) RU of expected PCR product. The captured PCR product had a response of 500 RU before injection of the target-specific probe. This means that >90% of the captured DNA was the expected PCR product of CFTR. Note that the captured PCR product underwent a slow dissociation from the immobilized probe. Taking this into account, we estimated that >95% of the captured DNA was the expected PCR product. When noncomplementary probes were injected, no hybridization was detected. These results indicate that the detection is specific. Similar results were obtained when we used this same sensor chip to detect the other two targets, i.e., the hTERT and p53 genes (Fig. 2 in the online Data Supplement).
Because of mutations or single-nucleotide polymorphisms (16), target genes may not fully match the sequence-specific probes. Probes with one to three mismatches were injected after PCR product was captured on chip surface. The results shown in Fig. 1C
demonstrate that for a 40mer probe, mismatches of up to 3 bases had little effect on the hybridization. This indicates that target can be verified even when a few mismatches are present. On the other hand, this method should also be applicable to the detection of point mutations if a shorter probe ( 10 bases) is used for the second hybridization (11).
Optimization was conducted with respect to salt concentrations and temperatures (Fig. 3 in the online Data Supplement). Salt greatly enhanced the hybridization. This can be explained by the fact that both DNA and the carboxymethylated dextran coating at the sensor surface are negatively charged and salt will reduce the repulsion between them. Increasing the temperature also enhanced hybridization, although occasional increased noise signals might be produced at 35 °C. We therefore conducted our formal measurements at 30 °C in a high concentration (0.8 mol/L) of NaCl to obtain proper sensitivity and stability. For the BIAcore, background noise was within 1 RU (instrument specification). We obtained stable signals at 600 RU for 
50 ng and >500 RU for 10 ng of input genomic DNA (data not shown).
Flow-type biosensors such as the BIAcore biosensor are expected to be a valuable platform in diagnostics when automation and high-throughput capability are desired. Because there is no need to switch the chip for different targets or samples, the one-chip-for-all strategy described here should be especially suitable for automated, high-throughput analysis of large numbers of samples. The SPR technique avoids post-PCR processing, and the detection is highly specific, efficient, sensitive, and reproducible. The chip can be reused for hundreds of measurements in this particular case. After more than 260 measurements, the binding capacity of the chip we used decreased by <6%. Using one chip for different samples and targets should simplify operation, improve efficiency for routine analysis, and reduce the running costs associated with chip consumption and probe immobilization.
Acknowledgments
This work was supported by Grants 39925008 and 30270314 from the National Science Foundation of China and Grant G2000057001 from the Ministry of Science and Technology of China.
References
F508 (F508del) mutation of the cystic fibrosis gene by surface plasmon resonance and biosensor technology. Hum Mutat 1999;13:390-400.[CrossRef][Web of Science][Medline]
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