Optimized PCR labeling in mutational and microsatellite analysis

Department of Oncology and Neurosciences, Faculty of Medicine, University "Gabriele D'Annunzio", Via dei Vestini 1, 66013 Chieti, Italy.
^a Author for correspondence. Fax 39-871-3555-4110; e-mail Cama{at}unich.it.

	Abstract

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To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 µmol/L, with a peak at 1.8 µmol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [³⁵S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [³⁵S]dATP as the label.

	Introduction

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Radioactive PCR labeling is widely used in a number of applications, which include production of nucleic acid probes, single strand conformational polymorphism (SSCP)¹ analysis, and microsatellite typing (1)(2)(3)(4). The two most common methods used for PCR labeling are either end-labeling of primers before the reaction or incorporation of a labeled nucleotide during PCR amplification. Methods based on incorporation are more flexible and involve less experimental steps than those based on end-labeling. Although incorporation methods have been used for several years, there is a broad variation in the PCR-labeling conditions used by different laboratories (1)(2)(4)(5)(6)(7)(8)(9)(10). For instance, in SSCP analysis, many laboratories still use conditions identical to or comparable with the original PCR-labeling protocol described by Orita et al. (5)(6)(7)(8)(9). To improve the incorporation of the labeled nucleotide during PCR-SSCP, some of these protocols use unbalanced concentrations (up to 10-fold) of labeled vs unlabeled nucleotides (8)(9). Different concentrations of labeled and unlabeled nucleotides (with an imbalance of up to 100-fold) are also used for microsatellite analysis (4)(10). The use of disparate PCR-labeling conditions denotes a lack of standardization for these procedures. Therefore, studies investigating the influence of different labeling conditions should contribute to the selection of appropriate labeling protocols for specific PCR applications. In fact, suboptimal labeling conditions may lead to higher costs of the analysis, increased autoradiographic exposure time and/or reduced signals, which may in turn interfere with the interpretation of tests. Moreover, optimized PCR-labeling conditions could be advantageous for both radioactive and nonradioactive applications. In the attempt to optimize labeling and detection conditions for mutational and microsatellite analysis, we tested several PCR conditions. Standardized PCR-labeling conditions were also tested using S-labeled nucleotides in place of commonly used P-labeled nucleotides.

	Materials and Methods

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dna samples and sequences analyzed
DNAs used for PCR amplification, SSCP, and microsatellite analysis were obtained in previous studies from our laboratory concerning the following: non-insulin-dependent diabetes mellitus, familial adenomatous polyposis, and hereditary non-polyposis colorectal cancer (HNPCC) (11)(12)(13). In some cases, to test the effect of different variables on mutational analysis by SSCP, we selected samples known to harbor single nucleotide changes in the human insulin receptor (HIR) gene, in the adenomatous polyposis coli (APC) gene, and in the mismatch repair gene hMLH1 (11)(12)(13). Genomic DNA (gDNA) was isolated from whole peripheral blood, using standard procedures (14). Genomic DNA for microsatellite analysis was obtained by microdissection of paired tumor and nondiseased tissue samples from paraffin-embedded sections, as previously described (15).

To test the performance of PCR-labeling protocols, we selected DNA fragments of various lengths (expressed in basepairs, bp) derived from the following: exons 4 and 17 of the HIR gene (fragments of 149 bp and 304 bp, respectively), exon 15 of the APC gene (450 bp), the coding exon of the insulin receptor substrate-1 (IRS-1) gene (402 bp), exon 19 of the hMLH1 gene (169 bp), and exon 6 of the hMSH2 gene (233 bp). Dinucleotide repeat loci D3S1611 (134 bp) and D5S107 (165 bp) were selected for microsatellite analysis (16)(17). Oligonucleotide primers were generated using an Applied Biosystems DNA synthesizer model 392–05 (Applied Biosystems). The sequence and annealing temperatures are available upon request. Amplifications were carried out using a Perkin-Elmer 480 DNA thermal cycler (Perkin–Elmer).

pcr amplifications
PCRs were performed using gDNA as a template. For some applications (as specified below), to verify the amounts and the quality of templates used for labeling, we used a nested PCR protocol. In these cases, before PCR labeling, genomic DNA was amplified following an established PCR protocol; the templates were visualized on an ethidium bromide-stained agarose gel; and the amounts, as well as the quality, of the amplified products were evaluated. Primary unlabeled PCR reactions (10–25 µL), contained 100–250 ng of genomic DNA, oligonucleotide primers (1 µmol/L), 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl₂, Taq polymerase (25 000 U/L), and 200 µmol/L dNTP. Cycling conditions included an initial denaturation step at 94 °C for 5 min, followed by 28–32 temperature cycles at 94 °C for 60 s at an appropriate annealing temperature (based on the T_m of the primers) for 90 s, and at 72 °C for 90 s. The final extension step was at 72 °C for 10 min.

effects of dNTP concentration on pcr labeling and pcr yield
Amplified gDNAs (including a 149-bp and a 304-bp fragment of exons 4 and 17 in the HIR gene and a 450-bp fragment of exon 15 in the APC gene) were used as templates for the evaluation of PCR labeling and PCR yield. PCR-labeling reactions were performed in a final volume of 25 µL, containing 2.5 µL of diluted primary PCR products (final dilution 1:10 000), 25 pmol of each oligonucleotide primer, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl_2, 25 000 U/L of Taq polymerase, and a fixed amount (2.5 µCi) of [P]dATP (6 000 000 Ci/mol). Cold dATP at six different concentrations (i.e., 0.9, 1.8, 3.5, 7.0, 35, and 70 µmol/L) was used to adjust the specific activity of the labeled nucleotide. The concentrations of dCTP, dGTP, and dTTP were double those of the cold dATP except at the maximum concentration (70 µmol/L), in which all dNTPs were equimolar. The concentration of 70 µmol/L was selected because it was expected to produce an optimal DNA yield, on the basis of preliminary experiments and data from previous work (18). The concentration of dATP added as the label (0.016 µmol/L) was negligible, even compared with the lowest concentration of cold dATP added, and was not considered for the purpose of this experiment. Samples were denatured at 94 °C for 5 min, followed by 28 cycles of amplification, as described above. Eight microliters of amplified PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide. PCR labeling was evaluated on dried agarose gels, using a Bio-Rad Molecular Imager System (model GS-525, Bio-Rad). An estimate of PCR yield was obtained by analyzing the fluorescent signal from ethidium bromide-stained bands, using NIH Image software (Ver. 1.51).

effect of dNTP concentration and pcr dilution on sscp pattern
PCR-labeling reactions for SSCP analyses were performed in a final volume of 25 µL, containing 2.5 µL of primary PCR products diluted 1:1000 (final dilution 1:10 000), 25 pmol of each oligonucleotide primer, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl₂, 25 000 U/L of Taq polymerase, 2.5 µCi of [P]dATP (6 000 000 Ci/mol), and four different cold dATP concentrations (0.9, 1.8, 3.5, and 7.0 µmol/L). Samples were denatured at 94 °C for 5 min, followed by 28 PCR cycles. Samples were diluted 1:10 in 950 mL/L formamide buffer and denatured at 94 °C for 5 min, followed by rapid cooling on ice. For SSCP analysis, 2 µL aliquots were electrophoresed for 8 h through a nondenaturing 6% polyacrylamide gel. Two conditions were used for SSCP analysis: 4 °C in a buffer containing 45 mmol/L Tris-borate and 1 mmol/L EDTA, at 10 W constant power; and 24 °C in the same buffer plus 50 mL/L glycerol, at 7 W as previously described (19). To evaluate the effect of different dilutions on SSCP patterns, labeled PCR products obtained at a concentration of 7 µmol/L dATP were diluted 1:2.5, 1:5, and 1:10 in 950 mL/L formamide buffer, denatured by heating at 94 °C for 5 min, cooled on ice, and subjected to SSCP analysis.

effect of pcr cycles on sscp pattern
The effect of the number of PCR cycles on the SSCP pattern was evaluated by nested amplification of a 450-bp fragment of the APC gene. PCR labeling was performed in a final volume of 10 µL, containing unlabeled PCR product as a template (final dilution 1:10 000), 10 pmol of each primer, 7.0 µmol/L of dGTP, dCTP, and dTTP, 3.5 µmol/L of cold dATP, 1.5 µCi of [S]dATP (1 000 000 Ci/mol), 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl₂, and 25 000 U/L of Taq polymerase. Samples were subjected to a variable number of PCR cycles (10–28). Labeled PCR products, diluted 1:2.5 in formamide buffer, were denatured at 94 °C for 5 min and subjected to SSCP analysis, as described above. Gels were dried and autoradiographed for 1 day at room temperature. Parallel experiments to evaluate the effect of the number of cycles on PCR yield at reduced dNTP concentrations were conducted using unamplified gDNA as a template.

mutational analysis of IRS-1, hMSH2, and hMLH1 genes by sscp
Optimized labeling conditions described above were used in mutational screening of IRS-1, hMSH2, and hMLH1 genes. In these assays, we used 0.15 Ci/L of [S]dATP as the label, 3.5 µmol/L of cold dATP, and 7.0 µmol/L of the other nucleotides. PCR labeling was performed either by direct amplification of gDNA (IRS-1 gene), or by using a nested PCR protocol (hMSH2 and hMLH1 genes). Thirty-two cycles were used for direct labeling of gDNA (10 mg/L), whereas 16 cycles of secondary PCR (1:10 000, final dilution of primary PCR products) were used for nested amplification protocols. Amplified products were diluted 1:2.5 in formamide buffer and used for SSCP analysis.

microsatellite analysis
Because microdissected specimens constitute a rather unsteady source of DNA, we conducted microsatellite analysis using a nested protocol, consisting of a nonradioactive external PCR (30 cycles) followed by a radioactive secondary PCR (18–22 cycles), as we have previously described (15). Reactions were performed in 10 µL, containing 10 mmol/L Tris (pH 8.3), 1.5 mmol/L MgCl_2, 50 mmol/L KCl, 7.0 µmol/L dGTP, dTTP, and dCTP, 3.5 µmol/L dATP, 3 µCi of [S]dATP, (1 000 000 Ci/mol), 10 pmol of each primer, and 0.25 U of Taq polymerase. Radioactive PCR products were mixed with 1 volume of formamide buffer and subjected to denaturing electrophoresis in 5% polyacrylamide gels containing 8 mol/L urea. Gels were dried and autoradiographed for 1–4 days at room temperature.

	Result

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In this study, different variables, including the ratio of labeled vs unlabeled dNTPs, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols were tested to optimize the labeling and visualization of PCR products. Optimized PCR-labeling conditions were used for SSCP and microsatellite analyses.

effect of dNTP concentration on pcr labeling and pcr yield
To establish the optimal ratio of labeled dATP vs unlabeled dNTPs, we performed preliminary experiments using different dATP/dNTP ratios, ranging from 1:2 through 1:8. The 1:2 ratio of labeled dATP vs unlabeled dNTPs yielded an optimal labeling, whereas higher ratios caused a progressive inhibition of PCR amplification (data not shown). A 1:2 ratio was used in all subsequent PCR-labeling experiments, with the exception of tests performed at a concentration of 70 µmol/L dNTP, where a 1:1 ratio was used.

To study the kinetics of PCR labeling, we performed amplifications in the presence of a fixed amount of labeled dATP (see Materials and Methods). The specific activity was adjusted by adding cold dATP (ranging from 0.9 µmol/L through 70 µmol/L) in the reaction buffer. The kinetics of PCR labeling and DNA yield at varying dATP concentrations were tested by amplifying a 149-bp and a 304-bp fragment of the HIR gene, as well as a 450-bp fragment of the APC gene (Fig. 1 and data not shown). When a 149-bp fragment of the HIR gene was used, optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 µmol/L (Fig. 1 ). The PCR products reached the peak labeling intensity at a concentration of 1.8 µmol/L dATP, with a 9.3-fold increase compared with the labeling obtained using 70 µmol/L dATP (Fig. 1 ). On the other hand, the kinetics of PCR yield showed a gradual decrease in absolute amount of PCR product as a function of the decrease in dATP concentration from 70 to 0.9 µmol/L. Relative specific activities of 8.0, 4.2, 2.8, 2.1, 0.3, and 0.1 (average labeling/yield) were achieved by varying the dATP concentration from 0.9 to 70 µmol/L. In fact, at a dATP concentration corresponding to the maximum PCR labeling (1.8 µmol/L), the PCR yield decreased by 4.2-fold compared with the PCR yield obtained at 70 µmol/L dATP (Fig. 1 ). Comparable results were obtained using a 304-bp fragment of the HIR gene and a 450-bp fragment of the APC gene as templates (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of dNTP concentration on PCR labeling and PCR yield. A 149-bp segment of the HIR gene was amplified and labeled with [32P]dATP, as described in Materials and Methods. Shown are the kinetics of PCR labeling () and PCR yield () as a function of cold dATP concentration. Aliquots of amplified DNA (8 µL) were separated by electrophoresis on a 2% agarose gel containing ethidium bromide, and the amount of radiolabeled DNA in the corresponding bands was quantitated using a Bio-Rad Molecular Imager System. PCR yield was evaluated analyzing the fluorescent signals from ethidium bromide-stained bands by the NIH Image software. The data shown are the means of duplicate determinations from a representative of three independent experiments.

effect of dNTP concentration and pcr dilution on sscp pattern
The use of limiting dNTP concentrations may influence the fidelity of DNA replication by the Taq polymerase, thus affecting the reliability of mutational screening methods sensitive to single nucleotide changes. To verify whether limiting dNTP concentrations may affect the detection of nucleotide variants by SSCP, we amplified a 304-bp PCR-amplified fragment of the HIR gene, using two DNA specimens, one of which contained a single nucleotide change (Fig. 2 A). The SSCP patterns were similar, and the unique conformer of the sample bearing a nucleotide substitution was evident at all the different dATP concentrations within the range corresponding to optimal labeling. These findings were confirmed in independent experiments using PCR products of different sizes (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 2. Effect of dNTP concentration, PCR dilution, and PCR cycles on SSCP pattern. (A) To evaluate the effect of dNTP concentrations on SSCP patterns, we amplified a 304-bp fragment of the HIR gene in the presence of [32P]dATP and cold dATP (0.9–7.0 µmol/L). The samples analyzed in lanes 2, 4, 6, and 8 derive from a patient with a CACCAT silent polymorphism at codon 1058 of the HIR gene. The unique SSCP band corresponding to this polymorphism is indicated by an arrowhead. The experiment shown is representative of similar experiments performed using PCR fragments of different lengths (149 and 450 bp). (B) To study the effect of PCR dilution on SSCP pattern, the samples in lanes 1 and 2 of panel A were diluted 1:2.5 (lanes 1 and 2), 1:5 (lanes 3 and 4), and 1:10 (lanes 5 and 6) in a solution containing 950 mL/L deionized formamide, 1 g/L bromphenol blue, and 1 g/L xylene cyanol. The samples were denatured at 95 °C for 5 min and electrophoresed for 8 h through a nondenaturing 6% polyacrylamide gel at 10 W constant power. (C) To verify the effect of PCR cycles on SSCP pattern, we amplified a 450-bp fragment of the APC gene. Four amplified samples were diluted 1:10 0000 and subjected to PCR labeling for the indicated number of secondary PCR cycles (10–28), using [35S]dATP as the label. A unique SSCP conformer of the APC gene is evident in the second lane of each group (arrowheads). The SSCP conformer of the APC gene corresponded to a 5-bp frameshift deletion at codon 1309 of exon 15.

Dilution of labeled DNA in different volumes of denaturing solution may influence SSCP patterns and could affect the detection of nucleotide changes. Therefore, we tested different dilutions (1:10, 1:5, and 1:2.5) of the 304-bp amplified fragment of the HIR gene in formamide buffer (Fig. 2B ). As expected, the intensity of the SSCP bands was maximal at the lowest dilution (1:2.5). Although the SSCP pattern changed with increasing dilutions, the unique SSCP conformer corresponding to the nucleotide substitution (indicated by the arrow) was clearly identified at all dilution ratios.

effect of pcr cycles on sscp pattern
When a 1:10 000 dilution of PCR products in a nested PCR-labeling protocol is used, ~16 cycles of secondary PCR should be sufficient to reach amplification plateau. Furthermore, the use of limiting dNTP concentrations could decrease the number of cycles necessary to reach the plateau by reducing the PCR final yield. We tested the effect of varying the number (10–28) of secondary PCR cycles on SSCP patterns. In these experiments, PCR labeling of a 450-bp fragment of the APC gene was obtained, using a nested amplification protocol under limiting dATP concentrations (3.5 µmol/L). A relatively weak emitter, such as [S]dATP, was used as the label. The clearest SSCP patterns were obtained after 12–16 cycles of secondary PCR (Fig. 2C ). Increasing the number of PCR cycles did not enhance SSCP intensity, whereas the appearance of spurious bands reduced the definition of SSCP conformers. Therefore, low dNTP concentrations used in our PCR labeling protocols did not appear to decrease the number of cycles predicted to be necessary to reach a PCR plateau starting from a diluted PCR. We also compared the DNA yields obtained at reduced dNTP concentrations, using as a template either diluted PCR products (1:10 000, final dilution) or gDNA. In agreement with what observed in SSCP experiments, when amplified DNA was used as a template, PCR plateau was achieved with 16 cycles of secondary PCR. When unamplified gDNA was used, more cycles (30–34) were necessary to compensate for the lower number of target copies, both under limiting (3.5 µmol/L dATP) and under nonlimiting (70 µmol/L dATP) dNTP concentrations (data not shown).

mutational analysis of IRS-1, hMSH2, and hMLH1 genes by sscp
To validate the optimized PCR-labeling conditions in mutational analysis applications, we performed PCR-SSCP of the IRS-1, hMSH2, and hMLH1 genes, using reduced dNTP concentrations and [S]dATP as the label.

Analysis of the IRS-1 gene was performed using unamplified gDNA samples as templates. The PCR was run for 32 PCR cycles, and S-labeled products were subjected to SSCP analysis. A unique SSCP pattern (Fig. 3 A, lane 4) was evident after overnight film exposure and corresponded to a novel silent polymorphism of the gene. Analyses of hMSH2 and hMLH1 genes in HNPCC patients were performed using diluted PCR products as templates. Unique SSCP conformers were evident, after overnight film exposure, in the analyses of both the hMSH2 (Fig. 3B , lane 3) and the hMLH1 (Fig. 3C , lane 4) genes. The unique conformer of the hMSH2 gene corresponds to a novel nucleotide substitution.

View larger version (49K): [in this window] [in a new window]   Figure 3. Mutational analysis of IRS-1, hMSH2, and hMLH1 genes by SSCP. (A) SSCP pattern of the IRS-1 gene obtained using genomic DNA as the template for PCR labeling. The unique SSCP conformer in lane 4 (arrowhead) corresponds to a novel A C transversion at nucleotide 3489 of the IRS-1 gene (with no change of Pro 1163). (B and C) SSCP patterns of hMSH2 and hMLH1 genes, respectively, obtained using amplified DNA as template for PCR labeling. SSCP conformers are detected in lane 3 of (B) and in lane 4 of (C) (arrowheads). The unique SSCP conformers in lane 3 of (B) and in lane 4 of (C) corresponded, respectively, to a novel C T transition at nucleotide 1052 of the hMSH2 gene (with no change of Ala 328) and to a TTC deletion in the 3' untranslated region of the hMLH1 gene. All autoradiographic images shown in this figure were obtained after overnight exposure.

microsatellite analysis
Optimized PCR-labeling conditions were applied to microsatellite analysis. PCR labeling was performed using gDNA obtained from microdissected paraffin-embedded paired tumor and nondiseased tissue samples of HNPCC patients, using [S]dATP as the label. Microsatellite typing obtained using [S]dATP as the label (3 µCi) yielded results comparable with those obtained in an overnight exposure using a lower amount (0.5 µCi) of [P]dATP (Fig. 4 and data not shown). Examples of tumor-associated microsatellite alterations at loci D5S107 and D3S1611, corresponding to expansions/contractions of repeats (patients 1 and 2) and allelic loss (patient 3) are shown in Fig. 4 .

View larger version (32K): [in this window] [in a new window]   Figure 4. Microsatellite analysis of paired nondiseased and tumor samples from HNPCC patients. Examples of patterns obtained by microsatellite analysis of paired nondiseased (N) and tumor (T) samples derived from HNPCC patients. Genomic DNA was obtained by microdissection of paraffin-embedded tissues. The microsatellite markers D5S107 and D3S1611 were analyzed by PCR labeling, using [35S-]dATP as the label, and the amplified products were separated on 6% polyacrylamide denaturing gels. Autoradiographic films were exposed overnight to 4 days. Tumor samples in lanes 2 and 4 present replication errors consisting in expansion (lane 2) and contraction (lane 4) of microsatellite alleles (arrowheads). A banding pattern suggesting the loss of one microsatellite allele in the tumor sample (arrowhead) is shown in lane 6. No microsatellite alteration is observed in the tumor sample analyzed in lane 8.

	Discussion

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There is remarkable variability in the conditions of PCR labeling used by different laboratories (1)(2)(4)(5)(6)(7)(8)(9)(10). The use of disparate PCR-labeling protocols, despite the wide applications of this procedure, points out the need for standardized conditions. In the present study, we analyzed conditions that may affect the efficiency of PCR labeling and the detection of PCR-labeled products. These conditions were also validated in mutational analysis by SSCP and microsatellite typing applications.

Maximal PCR-labeling intensity was reached at a dATP concentration of 1.8 µmol/L. This concentration corresponded to an optimal ratio between an increase in specific activity and a decrease in DNA yield. When different DNA templates within the size range usually used for SSCP and microsatellite analysis (149–450 bp) were used, the optimal dNTP concentration for labeling was not affected by the length of the PCR fragment. In the range of dATP concentrations between 0.9 and 7.0 µmol/L, there was a modest reduction (~25%) in DNA labeling, compared with the peak obtained at 1.8 µmol/L. Considering that limiting dNTP concentrations may inhibit the amplification of critical PCR templates and that increasing the concentration of dATP up to 7 µmol/L has modest effects on the intensity of the signal, for most applications it may be advisable to use conditions less extreme than 1.8 µmol/L. We also considered nucleotide imbalance as a variable affecting PCR-labeling. Some protocols, in the attempt to increase the specific activity of PCR labeling without affecting PCR yield, limited only the concentration of the cold nucleotide corresponding to the one used for labeling (4)(8)(9)(10). In these protocols, the concentration of the other nucleotides was within the range that is expected to give optimal PCR yield with imbalance between labeled vs unlabeled nucleotides of up to 100-fold (4). However, it has been shown previously that excessive nucleotide imbalance may force nucleotide misincorporation and induce premature stoppage of the PCR reaction (20). In our hands, there was no increase in labeling intensity when nucleotide imbalances >1:2 were used. In fact, in agreement with the work by Innis et al. (20), greater unbalances appeared to inhibit PCR. Therefore, considering also the possibility that polymerase errors may interfere with mutational analysis, the use of excessive nucleotide imbalances does not appear to be advantageous and should be discouraged.

Mutational analysis by SSCP was used to validate optimized PCR-labeling protocols. We first evaluated whether limiting nucleotide concentrations could affect SSCP patterns, for example, by increasing the rate of Taq polymerase misincorporation. SSCP patterns obtained at four different dATP concentrations produced similar results, and single nucleotide changes were also evident at the lowest dNTP concentration used in our labeling protocols. Therefore, the reduced dNTP concentrations did not interfere with SSCP. Another variable considered was the influence of DNA templates. Clear SSCP patterns were obtained using DNA templates of different sizes derived from different genes, including APC, IRS-1, HIR, hMLH1, and hMSH2. In addition, SSCP patterns obtained using gDNA directly or amplified DNA as templates were equivalent and were visible after an overnight exposure. However, to obtain maximum DNA labeling, the number of PCR cycles had to be proportionally increased when gDNA rather than amplified products was used as a template. Of note is that the number of PCR cycles necessary to obtain clear SSCP patterns was influenced not only by the number of copies of template (i.e., gDNA vs diluted PCR) but also by the particular gene fragment amplified. In fact, some samples proved more difficult to amplify and required a greater number of cycles (up to 35 cycles in one-step labeling protocols and up to 28 cycles of secondary PCR in nested protocols) to obtain optimal signals. However, the number of PCR cycles should be kept as low as possible, because exceeding the optimal number reduced the definition of SSCP patterns.

With regard to the influence of the dilution of labeled PCR product on SSCP pattern, the original protocol by Orita et al. (5) used a 1:200 final dilution of labeled products in denaturing solution, which implied the use of large amounts of labeled nucleotide to obtain visible signals (~10-fold those used in our study). In our hands, dilutions of labeled products ranging between 1:10 and 1:2.5 had a considerable influence on SSCP patterns; however, SSCP bands as well as anomalous conformers were, if anything, more evident at lower dilutions. A low dilution ratio was particularly important to obtain readily visible signals with S-labeled reactions.

It is important to note that, using optimized conditions, we were able to visualize all the nucleotide changes that were already known to be present in our samples. In addition, the use of optimized PCR-labeling conditions was further validated by the detection of novel nucleotide variants in the IRS-1 and hMSH2 genes.

Microsatellite analysis is widely used in linkage studies (4), in forensic medicine investigations (21), and in the genotyping of tumors (22). Mononucleotide or dinucleotide repeats are often used in microsatellite analysis, and electrophoresis of these markers under denaturing condition results in the appearance of multiple bands for each of the polymorphic alleles (23). With currently used PCR-labeling protocols, the distribution of the signal in multiple bands requires the use of a strong emitter, such as P, to obtain visible signals in reasonable exposure times. We tested whether conditions that had been optimized for SSCP could be efficiently used in microsatellite typing. Under such conditions, we obtained clear typing of microsatellite alleles, which also allowed the detection of tumor-associated size shifts, which are typically observed in samples from HNPCC patients. In addition, the improved signal efficiency allowed the use of [S]dATP as the label. However, to compensate for the distribution of the signal in multiple bands, the concentrations of sulfur-radionucleotide were twice those used in the corresponding PCR-SSCP labeling protocol.

In conclusion, we analyzed some of the conditions that influence PCR labeling and detection in the attempt to optimize protocols for PCR labeling. Optimized conditions were validated in SSCP mutational screening and microsatellite analysis. This study should provide a useful reference for investigators who use PCR labeling. In fact, optimized conditions could contribute to cost reductions, decreased autoradiographic exposure times, improved quality of signals, and may reduce the amounts of radioactivity handled and/or lead to the use of relatively weak emitters. In this respect, the improved signal efficiency obtained with optimized protocols allowed the use of [S]dATP as the label in SSCP and microsatellite analyses in place of the commonly used P-labeled nucleotides. Although we used radioactive tracers, the optimized labeling conditions described in the present study should also prove useful for nonradioactive PCR-labeling procedures.

	Acknowledgments

 
This work was supported by Associazione Italiana Ricerca Cancro (AIRC) Special Project "Hereditary Colorectal Tumors"; by Consiglio Nazionale Ricerche (CNR) No. 96.00529.29.PF39; by Telethon Italy (No. E.295); and by Ministero Univesitá Ricerca Scientifica Technologica (40% and 60% grants to A.C. and P.B.).

	Footnotes

 
¹ Nonstandard abbreviations: SSCP, single strand conformational polymorphism; HNPCC, hereditary nonpolyposis colorectal cancer; HIR, human insulin receptor gene; APC, adenomatous polyposis coli gene; hMLH1, human MutL homolog 1 gene; IRS-1, insulin receptor substrate 1 gene; and hMSH2, human MutS homolog 2.

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