Modification of primers and their switching characteristics
To improve the switching characteristics of specific primers, a single base of each primer was modified to create an artificial mismatch in the primer–DNA duplex in the 3′-terminal region of the primer. This modification of the primers improved the switching characteristics of primer extension according to the match or mismatch at the 3′-terminus. The switching characteristics in terms of primer extension reactions were evaluated with a template DNA containing a partial sequence of the human P53 gene (exon 8). The base type at the polymorphic site is A for wild-type DNA and T for mutant DNA. These are referred to as P53Wt and P53Mut, respectively. Four groups of primers were prepared. Each group contained four primers with the four possible terminal bases. Their 3′-termini are at the nucleotide position where a mutation can be observed. The first group had no modification in the terminal region. The other three groups contained a modification to cause an artificial mismatch in the primer–DNA duplexes at the second, third and fourth nucleotide positions from the 3′-termini of the primers, respectively.
Whether or not mismatch primer extension occurs depends on the DNA polymerase as well as on the annealing temperature used in the extension step. In general, higher temperatures are preferred, as they prevent mismatch primer extension. However, mismatch extension frequently occurs even at a high temperature such as 55°C. The artificial mismatch which we introduced at the third position from the 3′-terminus of the primer prevents mismatch primer extension. As shown in Figure , mismatch primer extension, which otherwise produced a signal as strong as several tenths of the matched signal, was decreased to a few percent of the matched signal by the artificial introduction of a mismatched base into the third position from the 3′-terminus of the primer. Only the matched primers produced large signals resulting from strand extension. The primer extension reactions can thus be controlled by the match or mismatch, at the 3′-terminus of the primer, with the template DNA. This switching characteristic in strand extension reactions is very accurate and is reliable enough to distinguish between wild-type and mutant DNAs. The ratios of mismatch extension products to match extension products were 0.66% for primer A, which matched with P53Mut, and 1.4% for primer T, which matched with P53Wt, as shown in Figure C. This technique is thus successful as an accurate way of determining the frequencies of alleles.
Figure 3 Comparison of primer extensions for wild-type and mutant targets. In (A) there is no artificial mismatch base in the primer; while in (B)–(D) an artificial mismatch base has been placed at the second, third and fourth positions from the 3′-terminus (more ...)
The data in Table show that this sharp switching characteristic is not dependent on the mutated base of the target. To confirm the switching characteristics of primers in DNA strand extension reactions, the results for all four possible bases at the polymorphic site were investigated using synthesized P53 gene fragments. We refer to these as P53-A, P53-C, P53-T and P53-G, respectively, according to the base in the allele. It was possible to perfectly control all primer extension reactions by the terminal base match or mismatch, as long as specific primers with an artificial mismatched base at the third position from the 3′-terminus were used. The results are listed in Table . As a small amount of strand extension from mismatched primers occurred, a calibration curve should be used to obtain an exact allele frequency. More recently it has been reported that extension from a mismatched primer can be minimized by adding single-stranded DNA-binding protein (SSB) to the extension solution (31
Relative abundances of extension products by match or mismatch at the 3′-termini of primers
Factors determining levels of background signals and the sensitivity of detection
If, in the present experiment, it were possible to use an ATP-degrading enzyme such as apyrase (as used in pyrosequencing), any endogenous PPi would be effectively degraded in the presence of APS and ATP sulfurylase, which would make highly sensitive detection of newly produced PPi possible. Unfortunately, this approach is not applicable to the present system because having the degradation of PPi proceeding in competition with the PPi assay reaction would affect quantitative SNP determination. However, a reduction in the amount of endogenous PPi remains very important in the realization of accurate allele frequency measurements. There are several possible sources of background signals. The reagents include a lot of PPi as a contaminant. Exogenous PPi is also produced by the thermal decomposition of dNTPs during the extension reactions. Furthermore, the APS used to convert PPi to ATP, and dATP, is a substrate of the luciferin–luciferase reaction. These background signals are produced through the following reactions.
APS + ATP sulfurylase luciferin + luciferase
PPi (reagent contaminant) ――――→ ATP ――――→ hv
luciferin + luciferase
APS ――――→ hv
Δ APS + ATP sulfurylase luciferin + luciferase
dNTP → dNMP + PPi ――――→ ATP ――――→ hv
luciferin + luciferase
dATP ――――→ hv
In pyrosequencing with a commercial pyrosequencer, ~1 pmol of sample is required. The present experiment consumed only 10 fmol, which is two orders of magnitude smaller than the quantity required for the current pyrosequencing method. When 10 fmol of template DNA was used to produce 55 base complementary strands, a signal intensity of ~100 mV was obtained in the present detection system. Background signals could be made very much smaller than this value by taking the following steps.
To analyze the components which affect bioluminometric measurement, various reagents were added to the bioluminometric detection system in a step-by-step fashion. The base solution contained luciferin, luciferase, template DNAs and polymerase. Even without adding other reagents such as ATP sulfurylase, the solution emitted a photo signal of 1.5 mV, which indicated that the reagents contained small amounts of endogenous ATP. By adding APS and ATP sulfurylase to the above solution, the background signal produced by endogenous PPi was evaluated as 146 mV, which included the contribution from the APS. As well as the contaminants in these reagents, solutions of dNTPs were found to contain a lot of endogenous PPi. As APS is an analog of ATP, it also reacts directly with luciferin to emit a photon, as has been reported previously (32
). Although dATP was a strong background source by reacting directly with luciferin to emit a photon, this was overcome by using dATPαS instead of dATP in BAMPER, a step that has previously been reported in work on pyrosequencing (13
In general, DNAs produced by a PCR process include a lot of PPi as well as unreacted dATP. This leads to a large background signal in a subsequent luminometric assay. PPi is produced by PCR and the thermal decomposition of dNTPs in the process of producing template DNA. This PPi and the dNTPs were removed by taking the following steps. The template DNA was initially purified using a QIAquick PCR purification kit. Single-stranded DNA was then prepared by a bead technology. Both endogenous PPi and residual dATP in PCR products were efficiently removed by the purification and bead washing procedures. As commercially available dNTP solutions contain large amounts of endogenous PPi, this PPi should be degraded using PPase, as described in Materials and Methods. Exogenous PPi is also produced by thermal decomposition of dNTPs. As such thermal decomposition will occur in the BAMPER process, we investigated this problem by heating a dNTP mixture to different temperatures for 2 min as shown in Figure . Little decomposition of the dNTPs was seen below 70°C, but this decomposition increased at higher temperatures. The use of a thermostable DNA polymerase requires a hot start where the reaction mixture is heated to 95°C. As decomposition of dNTPs is unavoidable in this case, reductions in the reaction time at high temperature and in the amounts of dNTPs present were both necessary as ways of reducing the amount of dNTP decomposition products and thus the levels of background signals. Therefore, a short reaction time of 30 s at 95°C and a reduced amount of dNTPs were used. To optimize this reduced amount of dNTPs, an experiment was carried out to determine the relationship between the amount of dNTPs and the bioluminescence signal intensity produced by DNA extension reactions with 50 nM P53Wt, as shown in Figure . This indicated that the amount of PPi produced with 50 nM template DNA was unchanged with a dNTP concentration >35 µM. So 50 µM of each of the dNTPs was a suitable concentration in this case. Since the amount of dNTPs was dependent on the amount of template used and the extension base length, we were able to adjust the amount of template on the basis of its extension base length. A concentration of 10–50 nM template P53Wt with an extension base length of 54 was appropriate for routine analysis, so 50 µM each dNTP with 50 nM template, P53Wt, was enough in most cases. This was about one-quarter of the amount of dNTPs used in the normal PCR protocol. The blank signal was decreased from 95 to 10 mV by this optimization. The background of the solution for detecting PPi was reduced to 6.5 mV by using PPase to degrade the PPi. This value was small enough to allow the detection of 10 fmol target DNA.
Signal intensity change from decomposition of dNTPs for 2 min at various temperatures. The concentration of each dNTP was 20 nM.
Figure 5 Signal intensity of 50 nM template at different concentrations of dNTPs. In the extension step the concentrations of template (P53Wt) and of primer were 50 and 100 nM, respectively. One microliter was injected for the determination. The concentration (more ...)
In BAMPER the addition of PPase to the reaction solution as an agent for degrading endogenous PPi is very important for reducing the background level. To prevent PPase degrading the PPi produced by the extension reaction, only a small amount of PPase was added. The amount of PPase was so small that the reaction solution had to be incubated for ~30 min to remove all of the endogenous PPi before adding APS to the solution. In this case it is not necessary to worry about the side-effects of PPase on PPi detection, because the PPi produced by the polymerase reaction was instantly and completely converted into ATP by ATP sulfurylase and APS before its degradation by the PPase present in the reaction mixture. This addition of PPase had no effect on the signal intensity produced by ATP, but significantly reduced the background level that arises from contaminant PPi in the reagents.
The background level produced by APS was linearly proportional to its concentration. Therefore, a small concentration of APS was preferable in terms of decreasing the level of the background signal. When a sufficient amount of luciferin was present in the reaction mixture, the signal intensity stayed constant at APS concentrations >1.5 µM. In the present experiment 2 µM APS was used in the SNP assay, and this produced a background signal level of 2.5 mV. With a fixed amount of APS, this background signal was stable during detection and it was thus easy to subtract it from the observed signals to obtain a reliable signal from the extension reaction.
The reduction in the amount of residual PPi and optimization of the reaction conditions allowed us to obtain a high degree of sensitivity of detection. For extension of a 54 base strand, the lower detection limit was 0.27 ± 0.02 fmol and the quantitative limit was 0.83 ± 0.05 fmol. This indicated that 10 fmol of a short strand of DNA, such as 200 bases, would be enough for routine SNP analysis. When a long DNA was used as template, the sensitivity of detection was improved. For example, for ssM13, with an extension length of 6289 bp, the lower detection limit was 14 amol. This is the most important advantage of BAMPER. It is impossible to detect 0.5 fmol ssM13 by pyrosequencing, while the signal produced with the same amount of ssM13 template in BAMPER was very strong. This is shown in Figure , where the same instrument was used for both forms of measurement. Although the intensity of the signal did not increase in linear proportion to base length, it did increase quite a lot. For a long strand of DNA the extension reaction might be incomplete, which is not good for quantitative determination. However, applying primer extension to a long strand of DNA is still useful in terms of highly sensitive detection of SNPs.
Figure 6 Comparison of signal intensities obtained with the prototype instrument in two different modes, pyrosequencing and BAMPER. The experimental conditions were the same, except that apyrase was not used in BAMPER. The sample was 0.5 fmol ssM13. BG indicates (more ...)
A simple instrument for BAMPER
The instrument for use in BAMPER may be very simple because it does not require iterative addition of four different dNTPs. While we have made a small device coupled to a photo-multiplier for BAMPER, a commercially available bioluminometer would also be suitable. Precise control of reagent injection is not necessary in BAMPER because a one-shot injection is enough to add the detection solution. The reaction volume was 10–40 µl for SNP typing, with both the prototype BAMPER device and a commercial bioluminometer. The amount of sample required for SNP typing can be decreased by reducing the reaction volume. For example, 10 fmol PPi, which corresponds to <100 amol target DNA (when, for example, the extension length is 100 bases), was successfully detected in a 500 nl solution with a good signal-to-noise ratio by BAMPER.
SNP typing and allele frequency analysis
SNP typing as well as allele frequency analysis were carried out with genomic DNA from our colleagues and with synthesized DNA. Typing one SNP for a homozygous or a heterozygous sample is the simplest case of allele frequency analysis. To start with, samples containing P53Wt, P53Mut and an equimolar mixture of P53Wt and P53Mut were analyzed, and the results are shown in Figure . The bioluminescence intensities obtained with the two specific primers were normalized to the total intensity. The results were almost as expected, which indicates that the method is applicable to SNP typing of a small amount of sample. In order to confirm the applicability of BAMPER to various SNPs, 16 polymorphic sites in human genomic DNA extracted from blood were investigated. As shown in Figure , all of the SNPs were accurately typed. The results are listed in Table and coincide with the sequencing results obtained by gel-based electrophoresis. Although some of the priming regions contained GC-rich sequences, these tests showed that BAMPER was effective for typing all the SNPs analyzed in this study. In some cases mismatch extension still occurred, but the relative proportion was <10% of the matched extensions, proving the usefulness of artificial introduction of a mismatched base into the primer. For example, the proportion of mismatch extensions for SNP16 was ~40% when such an artificial mismatched base was not introduced. The reproducibility of BAMPER analysis results was very good. For SNP-13 the whole SNP typing process, including PCR, preparation of single-stranded DNA, the bead washing process and BAMPER detection, was repeated eight times in parallel and the relative standard deviation was 3.8% (n = 8).
Figure 7 A bioluminescence assay with modified primer extension reactions for genotyping. Targets containing A (P53Wt) and T (P53Mut) at the polymorphic site were used. For homo-templates it was only possible to extend one primer. However, both primers extended (more ...)
Results of using BAMPER to type 16 SNPs. The intensity was normalized to the total intensity. The details of each SNP, including the corresponding gene name, mutation point and specific primers are given in Table .
SNPs used for the evaluation and typing analyses
As it would have been difficult to obtain real samples for an allele frequency study, synthetic wild-type and mutant P53 DNAs mixed in various ratios were used. Reactions with the two primers were carried out for each sample. The allele frequencies of P53Mut (T allele) in the artificially prepared samples were 0.02, 0.05, 0.1 and 0.7. The wild-type allele was A. As shown in Table , the correlation between the estimated allele frequencies and those observed for the artificially prepared DNA was very high, at r2
= 0.9999. The measurements were repeated four times and the results were highly reproducible. The largest uncertainty in frequency determination was estimated to be 0.9% for a frequency <10%. This is enough to distinguish differences between the allele frequencies of two groups in terms of medical characteristics. There may be some difficulty with accuracy in applying the method to determining allele frequencies of <5%. However, those frequencies that are <5% are likely to be of very minor importance and are usually not considered for further analysis, as has been discussed by Nigel Spurr (28
Comparison of calculated and observed allele frequencies for synthesized pooled DNA samples (four independent measurements were carried out to estimate the error rates, n = 4)
The complementary strand was also analyzed using two other specific primers to obtain a reliable frequency value. The observed allele frequencies in pooled DNA samples for both P53 strands were compared with estimated values, and are listed in Table . The observed allele frequencies were consistent for both strands, which indicated that the results obtained were reliable and that frequency analysis of one strand would be enough to determine the allele frequency in practical applications.
For allele frequency analysis the accuracy of pipetting operations as well as the number of samples in a pool must be carefully considered. The expected sampling error can be expressed as √[f
(1 – f
], where f
is the allele frequency of one of the two alleles and n
is the sample size (34
). The calculated errors due to sampling (sampling error) at different allele frequencies for n
= 100 and n
= 1000 are shown as the black and purple lines in Figure , respectively. It was found that when the sample size is smaller than 1000, the sampling error is greater than the standard deviation of the measurement (measurement error) at each allele frequency, as shown in Figure . On the other hand, the measurement error, which is small and independent of the number of samples, becomes dominant in allele frequency determination for large sample sizes such as n
> 1000. To obtain a reliable allele frequency, a large sample size (n
) is necessary.
Figure 9 Comparison of sampling errors (black and purple lines) and measurement errors (error bars) at different allele frequencies. Sample sizes were 100 for the black line and 1000 for the purple line, respectively. The green line was drawn by the regression (more ...)