Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently, peptide nucleic acid PNA clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA. To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K- ras gene based on a high-fidelity DNA polymerase. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity DNA polymerases. Detection of microscopic spread of tumor cells is of interest in several areas of cancer research.
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Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Recently, peptide nucleic acid PNA clamp real-time PCR has provided a time-sparing and sensitive method for the detection of mutations in the presence of a large excess of wild-type DNA.
To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K- ras gene based on a high-fidelity DNA polymerase. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity DNA polymerases.
Detection of microscopic spread of tumor cells is of interest in several areas of cancer research. Common strategies to distinguish tumor cells from normal cells are based on different histological properties, specific protein and mRNA expressions, and tumor-specific mutations.
Several strategies have been used to detect tumor-specific mutations sensitively. Subsequently, several studies using similar assays to detect microscopic cancer dissemination have been reported. DNA concentrations were determined by UV nm spectrophotometry. Platinum Taq is a recombinant Thermus aquaticus Taq DNA polymerase with a thermolabile inhibitor to achieve hot-start capabilities according to the manufacturer.
HotGoldStar polymerase is a modified recombinant Taq DNA polymerase with hot-start capabilities according to the manufacturer. The purity of all PCR products was monitored by melting curve analysis. Mutant mut. The identity of the PCR products was initially also confirmed by sequencing.
PCR setup and analysis were performed in separate rooms to avoid contamination. Each sample was analyzed in triplicate both with and without PNA. Peripheral blood lymphocytes were isolated from blood samples of healthy volunteers by Lymphoprep density centrifugation as recommended by the manufacturer Axis-Shield, Dundee, Scotland, UK. The colon carcinoma cell line LST was cultured as described above, grown to near confluence, and trypsinated as suggested by the European Collection of Cell Cultures.
Cell densities were determined using a hemocytometer. The cell suspensions were repeatedly vortexed at slow speed to keep the suspensions homogeneous. DNA was isolated from the cell suspensions as described above. Sequencing was performed using the BigDye Terminator v1. Confidence intervals were computed using the t distribution. We aimed to develop a PNA-clamped real-time PCR assay for sensitive detection of all possible carcinoma-associated mutations in K- ras codons 12 and 13 in lymph nodes wt K- ras from colorectal cancer patients.
To distinguish all possible codon 12 and 13 mutant PCR products from wild-type PCR products by melting curve analysis, we altered the probe sequence to be wild-type specific. Thus, the PNA clamping seemed to be incomplete. This led us to hypothesize that the mutations were introduced when Taq polymerase made replication errors and that any error leading to a mismatch between the PNA and DNA would be enriched during the PCR because of weaker PNA-clamping compared with wild-type template.
If so, the utilization of high-fidelity DNA polymerase would reduce the frequency of new mutations and thus increase the sensitivity of the assay. Further, the use of mutation-specific hybridization probes would not enhance the assay as it would be impossible to distinguish the mutations arising during the PCR from the mutations present in the sample to be analyzed. Schematic pictorial of our PNA clamp assay.
In our design, the primer and the PNA bind competitively to part of the same sequence. To explore the sensitivity of our assay in a cellular context, we analyzed dilution series of 10 2 , 10 3 , 10 4 , and 10 5 LST cells in 10 7 normal lymphocytes. We found several different mutations in the PNA-binding site Figure 5 , confirming our hypothesis. The DNA sequences are shown above the chromatograms, with codons 12 and 13 underlined.
Only the introduced mutations are shown explicitly. PCR-induced errors may cause cloning errors and overestimation of genetic diversity. Selective amplification of mutant templates by PNA clamping of the wild-type template also favored amplification of PCR products with polymerase-induced errors within the PNA-binding site.
During the next cycles, the PNA binding to the newly synthesized strands containing errors in the PNA-binding site would be reduced or completely inhibited, depending on the kind of mutation, and exponential amplification could occur. Thus, polymerization errors seem to be a plausible explanation for the mutations we observed when analyzing wild-type template with PNA present. Interestingly, there is evidence indicating that guanine substitutions destabilize PNA binding more than other substitutions.
Although the peaks corresponding to introduced mutants in the sequencing chromatograms were quite convincing, the wild-type peaks in the same position were usually higher Figure 5A. If all mutant peaks correspond to one type of mutant PCR product, this could indicate that the main reason for exponential amplification from wild-type template is incomplete PNA-binding and not polymerase errors. However, this model would not explain why we achieved substantially higher sensitivity when applying a high-fidelity DNA polymerase.
A potentially better explanation is that multiple mutants are introduced separately in the first part of the PCR and afterward compete during the amplification.
Possibly, all kinds of mutations are introduced in the PNA-target, but only the most destabilizing mutants become sufficiently amplified to be observed by sequencing.
Considering the fact that the cell line used to determine the sensitivity LST was heterozygous for the K- ras mutation, the sensitivity was even higher , The sensitivity was determined by diluting colon carcinoma cells in a large excess of peripheral blood cells unclear whether nucleated or not.
The apparent increase in sensitivity seems to be achieved by the use of a mutation-specific hybridization probe. With that approach, melting curve analysis of the PCR products can distinguish the specific mutant PCR product from wild-type and other mutants.
Thus, mutation-specific probes seem to be a way to avoid the problem with polymerase-introduced errors. However, the hybridization probe cannot distinguish a real mutation in the original template from one introduced by the polymerase during PCR. In view of our results we emphasize the possibility for false-positives due to polymerase-induced errors matching the probe.
In their discussion of possible limitations, they mention the possibility that errors introduced by Taq polymerase can lead to false-positive results when the amount of mutant template is less than 0.
The strategy with mutation-specific hybridization probes also suffers from the drawback that multiple hybridization probes are needed to cover all possible mutations in K- ras codon 12 and codon There are 12 possible missense mutations in codons 12 and 13, of which several are common.
This would require up to 12 different mutation-specific probes to cover the mutational spectrum. In contrast, our single PNA clamp PCR assay detected different mutations in codons 12 and 13 with similar efficiency results not shown. Using amplification curves to detect mutations introduces another challenge. The amplification curves of a sample must be compared with the amplification curve of wild-type template to judge whether it has a mutation or not.
In a context of minimal residual disease detection, this could be applied to obtain a relative measure for the number of tumor cells present in the clinical sample. However, DNA polymerase errors need consideration also for other types of assays based on selective amplification of genetic variants, such as allele-specific amplification- and restriction endonuclease-mediated selective amplification-PCR. It is likely that both of these methods will benefit from high-fidelity DNA polymerases.
Thus, continued effort in the development of DNA polymerase systems with higher fidelities is in the interest of several fields of molecular diagnostics. National Center for Biotechnology Information , U. Journal List J Mol Diagn v.
J Mol Diagn. Author information Article notes Copyright and License information Disclaimer. Box , Stavanger, Norway on. Accepted Feb Published by Elsevier Inc. All rights reserved. This article has been cited by other articles in PMC. Abstract Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level.
Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. References 1. Distinguishing pancreatic carcinoma from other periampullary carcinomas by analysis of mutations in the Kirsten-ras oncogene. Ann Surg. Molecular prognostic markers in pancreatic cancer. J Hepatobiliary Pancreat Surg. Bos JL. Cancer Res. Detection of isolated tumor cells by polymerase chain reaction-restriction fragment length polymorphism for K-ras mutations in tissue samples of colorectal cancer patients.
Clin Cancer Res. Genetic alterations during colorectal-tumor development. N Engl J Med. Ray A, Norden B. Peptide nucleic acid PNA : its medical and biotechnical applications and promise for the future. Thenmalarchelvi R, Yathindra N. New insights into DNA triplexes: residual twist and radial difference as measures of base triplet non-isomorphism and their implication to sequence-dependent non-uniform DNA triplex.
Nucleic Acids Res. Edited by Nielsen PE. Wymondham, Horizon Bioscience Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes. Int J Cancer.
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