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Nucleic Acids Res. 2004; 32(5): e53.
Published online Mar 22, 2004. doi:  10.1093/nar/gnh051
PMCID: PMC390351

Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis

Abstract

Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10–12% of wild-type DNA.

INTRODUCTION

A single base change in DNA can lead to altered cellular behavior, inducing oncogenesis, if crucial genes are modified. Mutations in the c-K-ras gene contribute to the development of colon cancer, and such mutations seem to appear early in the colon tumorigenesis pathway (1,2). Detection of such mutations at low levels would be beneficial for early diagnosis, prognosis and the evaluation of therapeutic outcome in cancer treatments (3). The possibility of low-frequency mutation detection is of particular interest for early detection of malignant diseases, for evaluation of surgical margins and for monitoring relapse. The c-K-ras gene is commonly mutated in human cancers, especially in lung and colon tumors (4,5). c-K-ras point mutations have been described at codons 12, 13, 59 and 61 (6,7) and, depending on the location and the kind of mutation, histopathological differences have been described in animal models (8) and humans in different kinds of cancer (6,9). Most K-ras mutations are localized at codon 12 and they have been associated with tumor progression and shortened patient survival in colon (10) and non-small cell lung cancer (NSCLC) (11), although, in some cases, such association has not been detected.

At the moment, a number of methods for detecting single base mutations in DNA sequences have been described. Those methods include the amplification refractory mutation system (12), single-strand conformation polymorphism (SSCP) and allele-specific amplification (13), mismatch amplification mutation assay (14), oligonucleotide ligation assay (15), ligation chain reaction (16), enriched PCR (17), PCR primer-introduced restriction analysis with enrichment of mutant alleles (18), PCR-based assays (19), point mutation detection using exonuclease amplification-coupled capture technique (20), restriction fragment length polymorphism (RFLP) (7,21) and denaturing gradient gel electrophoresis (DGGE) (22). These techniques have several disadvantages for automating DNA diagnosis, which in some cases include the requirement for various hybridization conditions, the use of two PCR amplifications and, finally, the need for electrophoresis. No ideal method for diagnosis, in which minimal steps are required to give maximal sensitivity, is in universal use for cases of known mutations in a single codon.

Previous studies have shown that mass spectrometric analysis of PCR products is feasible for detecting point mutations in DNA (23). Several mass spectrometric approaches for analyzing short oligonucleotides have been used: electrospray ionization mass spectrometry (ESI-MS) (24) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) (2527) by using purified or intact PCR products (28).

Short oligonucleotide mass analysis, previously described as SOMA (23), an approach which uses mass spectrometric analysis of PCR-amplified oligonucleotides, has previously been successfully used to selectively and sensitively detect variant sequences involving a single base change at a codon, for example codon 1307 of the APC gene (26), codon 249 of the p53 gene (2931) and certain polymorphisms with a prognostic value (27). This technique exploits the utility of mismatched bases within primer sequences described for the PCR approach. PCR amplification and subsequent restriction digestion generate small (7- to 15mer) DNA sequences of wild-type and/or mutated genomic DNA in sufficient quantities for sensitive and specific sequence analysis by ESI-MS/MS.

The validity of the mutant DNA presence given by ESI-MS/MS was confirmed by a previously described PCR strategy using BstN1 (3234). This strategy, known as restriction endonuclease-mediated selective PCR, exploits the thermostable properties of BstN1 and Taq polymerase, allowing enhanced amplification of mutant K-ras, when compared with a PCR where no mutant enhanced amplification has been performed.

We used SOMA to selectively detect two c-K-ras codon 12 mutant sequences in DNA from 121 colon adenocarcinomas. In this study, we provide detailed information on how the SOMA method can also be used to detect specific mutations even when multiple mutations are possible in a codon, and we compared these results with sequencing. However, looking solely at the two most common mutations in a single codon, as we did, does not rule out other mutations at the same codon or other mutations at other codons, such as 13, 59 or 61 in the case of K-ras (68). We raise the importance of the SOMA method as a technique that can be used for routine diagnosis in clinical practice.

MATERIALS AND METHODS

Samples

Samples of tumor biopsy DNA from patients diagnosed with colon adenocarcinoma had been collected at the Department of Pathology at Clinica Puerta de Hierro (Madrid) in relation to a previous study involving other genetic alterations (35).

SOMA PCR

PCR primers which amplify a 93 bp DNA sequence around codon 12 are shown in Figure Figure1.1. Both the 43mer forward (F1) and 42mer reverse (R1) primers introduce the six-base recognition sequence CTGGAG for the type II restriction enzyme BpmI (New England Biolabs, Beverly, MA) 17–22 and 15–20 bases, respectively, from the 3′ end of each primer. PCR was performed with Taq-Platinum (Invitrogen, Cergy Pointoise, France) in a 25 µl total volume using 150 ng of genomic DNA. Thermal cycling conditions for all primer sets were 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s. (Perkin Elmer Thermal Cycler).

Figure 1
Forward (F1 and F2) and reverse (R1) SOMA PCR primers that were used. Primers amplify a 93 bp DNA fragment in the K-ras gene. The inset shows the two wild-type 8mer SOMA oligonucleotides which are produced by restriction digestion of the amplified DNA ...

Restriction digestion of this amplified DNA with BpmI produces an 8mer DNA sequence (Fig. (Fig.1,1, inset) with the codon 12 sequence located at the 5′ end of the sense oligonucleotide. Amplification and restriction digestion are independent of the eight-base sequence so a mixture of wild-type and mutated DNA sequences is produced.

The restriction enzyme BstN1 is commonly used to enhance K-ras codon 12 mutation (3234). Forward primer (F2), a modification of primer F1 involving replacement of a G with a C three bases from its 3′ end (Fig. (Fig.1),1), produces amplified DNA containing a CĈWGG recognition sequence for the restriction enzyme BstNI (New England Biolabs). As it is heat stable, BstN1 can be included in the PCR mix (2 U per reaction) to destroy wild-type DNA during each amplification cycle, enhancing amplification of mutated over wild-type DNA. Thermal cycling conditions using BstN1 were the same as those described above except that the elongation step was done at 60°C.

Preparation and purification of SOMA oligonucleotides

Following PCR amplification, DNA was cleaned up by phenol/chloroform extraction and ethanol precipitation by adding one-third volume of 7.5 M ammonium acetate (to reduce the level of sodium adduct ions) and 6 vol of ethanol, and holding the solution at –80°C for 1 h. A 2 µl aliquot of See-DNA (Amersham, Orsay, France) was added to the PCR products to enhance precipitation. DNA was precipitated after centrifugation and the resulting pellet was digested with 8 U of BpmI for 2 h at 37°C in a final reaction volume of 50 µl. This procedure produces an 8mer DNA sequence (Fig. (Fig.1).1). The 8mer DNA sequence was then purified using 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1 by vol) (Sigma, St Louis, MO) and precipitated again in the presence of 2 µl of See-DNA, one-third volume of 7.5 M ammonium acetate and 6 vol of ethanol. After washing the colored pellet with 70% ethanol, samples were allowed to air dry. Before HPLC-MS analysis, samples were resuspended in 6 µl of a solution of aqueous 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma) and methanol (85:15, v/v).

HPLC conditions

Before introduction into the mass spectrometer, oligonucleotides were further purified by HPLC, carried out at ~20 µl/min on a 15 cm × 800 µm i.d. Vydac C-18 reversed phase column (5 µm, 300 Å pore size) (LC Packings, Amsterdam, The Netherlands). To obtain this flow rate, an HP1100 pump (Agilent, Palo Alto, CA) operating at 0.32 ml/min was connected to an LC Packings Accurate microflow splitter. HPLC solvents were prepared from a stock solution of aqueous 0.8 M HFIP, adjusted to pH 7.0 with triethylamine, then diluted to 0.4 M (with water for solvent A and methanol for solvent B) as described previously (24). An initial mobile phase concentration of 35% B was held for 2 min, then programmed to 65% B in 9 min. The entire 5 µl sample was injected onto the HPLC under these initial conditions, as the oligonucleotides are concentrated at the head of the column and low molecular weight salts and other impurities are washed away.

Mass spectrometric analysis of SOMA oligonucleotides

Mass chromatograms were obtained on a LC Quattro mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ionization source operated in the negative ionization mode. The electrospray capillary was held at –5 kV and the cone potential was typically 77 V. Ion source temperature was 100°C and the electrospray desolvation temperature was 215°C. Argon pressure in the collision cell was ~5 × 10–3 mBar.

As the 8mer DNA elutes into the mass spectrometer, it dissociate into a pair of sense and antisense 8mer oligonucleotides for each wild-type (12GGT) or mutated sequence selected. Electrospray ionization of such an 8mer oligonucleotide produces a series of multiply charged ions. For example, the negative electrospray mass spectrum for the wild-type sense (WT-s) K-ras oligonucleotide (Fig. (Fig.2a)2a) shows a major peak for the [M-2H]2– ion at m/z 1279.6. A smaller signal is present for the triply charged [M-3H]3– ion at m/z 852.8; the singly charged [M-H] molecular ion at m/z 2560.6 is absent. To maximize the sensitivity of the method, HFIP was added to the HPLC mobile phase to increase the relative intensity of doubly charged ions and to minimize sodium adduction (29).

Figure 2Figure 2Figure 2
(a) Negative electrospray mass spectrum of the wild-type-s oligonucleotide shown in Figure Figure1.1. The mass of this peak depends on the number of G, C, A and T bases present in the oligonucleotide. It gives no information about sequence. Under ...

To obtain sequence information, the first sector of the tandem mass spectrometer is set to selectively pass ions with a mass-to-charge ratio corresponding to the [M-2H]2– ion of a chosen oligonucletide. The beam of ions is then collided, at an energy of 37 V, with a curtain of argon gas to produce a spectrum of sequence-specific CID fragment ions (Fig. (Fig.2b)2b) which are analyzed by the second sector of the tandem mass spectrometer. An ax–Bx series of fragment ions is formed from the 5′ end of the oligonucleotide and a wx series of ions from the 3′ end (Fig. (Fig.22c).

Fragment ions specific for wild-type and mutated sense and antisense oligonucleotides are monitored as the compounds elute into the mass spectrometer. For specific detection of aspartate and serine mutation (Fig. (Fig.3),3), the mass spectrometer was programmed to acquire data in the multiple reaction monitoring mode monitoring five sequence-specific [M-2H]2– ion fragments for the sense (s) and antisense (as) SOMA oligonucleotides: 12GGT(WT)-s, 1279.6→1139.2 or 650.4 (both fragment ions are sequence specific); 12AGT-s/12GAT-s, 1271.8→1123.7 or 650.4 (either sequence can produce this fragment ion); 12AGT-as, 1236.3→963.6; 12GAT-as, 1236.3→659.4; and 12GGT(WT)-as, 1228.8→659.4.

Figure 3
SOMA mass chromatograms illustrating the specific detection of 12AGT and 12GAT variants in the DNA from eight tumor samples shown by sequencing to contain (a) wild-type DNA, (b) wild-type and 12AGT DNA and (c) wild-type and 12GAT DNA. Five CID fragment ...

Sequencing

Primers used for PCR amplification of a region of exon 1 of the K-ras gene which includes codon 12 produced a 240 bp DNA fragment (sense, 5′-CCTTATGTGTGACATGTTC-3′; and antisense, 5′-CTCATGAAAATGGTCAGAG-3′). The reaction was carried out in a final volume of 20 µl with a primer concentration of 0.25 µM under the following PCR conditions: 96°C for 5 min, followed by 35 cycles of 96°C for 30 s, 55°C for 20 s and 60°C for 4 min. Samples from the PCR were purified with QIAquick gel extraction columns (Qiagen, Courtabeuf, France). The sequencing reaction was based on the rhodamine terminator using fluorescent ddNTPs. Samples were run on a 4.8% polyacrylamide gel (250 µl of APS 10% + 35 µl of TEMED) during 3.5 h at 3000 V and 51°C and read using a Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA).

Comparison of SOMA with sequencing

Sequencing and SOMA require approximately the same time to perform. Both methods involve: DNA extraction, PCR amplification, purification and injection into the mass spectrometer (10 min per sample) or sequencer (30–60 min per sample). SOMA requires an additional restriction digestion after the PCR amplification, which increases the analysis time by 1–2 h. The cost of consumable items for SOMA and sequencing is comparable; the cost of the tandem mass spectrometer used in this study is about twice that of a capillary electrophoresis sequencer.

RESULTS AND DISCUSSION

Comparison of SOMA with sequencing

The major objective of this work was to show that the SOMA technique can detect specific point mutations of well-known genes, such as codon 12 of the K-ras gene, even when present at levels below 10–12% of wild-type DNA (Fig. (Fig.3).3). Detection of a specific mutation by SOMA does not interfere with the detection of other mutations when the latter occur in the same codon. Tumor biopsy DNA samples from 121 patients diagnosed with colon adenocarcinoma were analyzed by SOMA at codon 12 of the c-K-ras gene, and controlled by sequencing in separate PCRs.

We focused on differentiating the aspartate from the serine mutation versus other variants occurring at codon 12 of the K-ras gene (Fig. (Fig.3).3). In other variant groups, we considered wild-type and/or other mutants less frequent than aspartic or serine at codon 12. We performed the study in such a manner because we focused on the predominant mutational pattern as described in the literature for K-ras (36,37). Under these conditions, by SOMA we detected four samples with the 12AGT mutation, 26 samples with the 12GAT mutation, versus 91 samples that were considered as other variants (wild-type and/or other mutations).

After sequencing, we detected four samples containing the 12AGT mutation and 23 samples containing the 12GAT mutation instead of the 26 previously detected by SOMA. Moreover, sequencing revealed 16 samples containing other mutations at codon 12, and finally eight samples contained a G→A mutation at codon 13 (13GAC) versus 70 wild-type samples.

Detection of low levels of mutated DNA by SOMA

An excess amount of wild-type DNA may hamper mutant DNA detection. Different approaches involving PCR, as mentioned above, have been considered to avoid wild-type DNA interference for an accurate detection.

For three tumor DNA samples out of 121, 12GAT mutant DNA was detected by SOMA but was not confirmed by sequencing (Fig. (Fig.4).4). Experiments were done in triplicate. In the SOMA method, F1 and R1 primers were used (Fig. (Fig.1).1). We hypothesized that when the 12GAT mutation present in the DNA extracted from tumor tissue is <10–12% of wild type, it may not be detected by sequencing. To confirm this hypothesis, we again used the SOMA method but with a different approach. Given the advantage seen by other authors using BstN1 for c-K-ras point mutation analysis, a forward SOMA primer (F2) was designed which inserts a CĈWGG recognition sequence for the restriction enzyme BstN1 into the amplified 93mer DNA fragment (Fig. (Fig.1).1). In this approach, F2 and R1 primers were used. As BstN1 is active at 60°C, it can be added to the PCR mix to efficiently and selectively cut wild-type codon 12 sequence DNA during PCR amplification (3234). Figure Figure55 shows the SOMA traces for DNA from one of the three samples suspected to contain 12GAT mutant DNA (note that SOMA fragment ions different from those used in Fig. Fig.3,3, but specific, were used to monitor sense wild-type and mutated DNA). When BstN1 is added to the PCR mix (Fig. (Fig.5b),5b), peaks for the wild-type sense and antisense DNA disappear and 12GAT-specific peaks increase in intensity when compared in the absence of BstN1 (Fig. (Fig.5a5a and b). On the other hand, for a sample analyzed under the same conditions and containing only wild-type DNA, the addition of BstN1 makes no difference to the intensity of 12GAT-specific peaks (Fig. (Fig.5c5c versus d). In this way, the presence of 12GAT mutant DNA was confirmed in the three samples which were negative by sequencing. Surprisingly, SOMA in the three samples gave an indication of mutant DNA before the selective PCR containing BstN1 was added to the PCR mix. Although detection limits for these codon 12 K-ras mutations were not precisely determined, one can estimate a level of 10–12% mutated DNA in this DNA sample (Fig. (Fig.5a)5a) by comparing relative peak areas for wild-type and 12GAT SOMA peaks (Fig. (Fig.3c).3c). Elimination of wild-type DNA with BstN1 increases 12GAT-specific peak areas by about a factor of 10. Thus, we can reliably measure mutated DNA at a level of 1–2% relative to wild-type. Our data support a previous report (30) showing that SOMA is more sensitive than other molecular biology methods for detecting point mutations in tumor DNA.

Figure 4
Sequencing chromatogram for DNA sample 953068T shown by SOMA to contain 12% 12GAT-mutated DNA. Primers used for sequencing are shown in Materials and Methods.
Figure 5
SOMA mass chromatograms showing the effect of elimination of wild-type DNA by restriction digestion with BstN1 during PCR amplification. DNA sample 953068T (a) shows small peaks for SOMA oligonucleotides specific for the 12GAT sense (1271.8→650.4) ...

The use of BstN1 increases the sensitivity of detecting mutant alleles up to more than 10 times when comparing the increase in the area of the peaks of the mutant alleles before and after adding BstN1 (Fig. (Fig.5).5). Moreover, the clear disappearance of the wild-type alleles suggests the reliability of using this method as a routine diagnostic test.

We consider that codon 12 K-ras gene mutation analysis is more challenging than p53 249 mutation due to the presence of several mutations in a single codon, as occurs with codon 12. Up to now, the high sensitivity of the SOMA technique was strictly based upon the fortuitous presence of a restriction endonuclease site that was either created or destroyed by the mutation of interest (31). In this study, we show that SOMA analysis allows the introduction of more than one target for restriction enzymes in a primer. This fact does not limit mutation detection by the SOMA method in a well-known hot-spot containing natural targets for restriction enzymes.

SOMA analysis of G to T mutations in K-ras codon 12

In addition to 12GAT or 12AGT mutant DNA, sequencing revealed the presence of 12GTT and 12TGT mutant sequences among the 121 tumor DNA samples. For specific detection of these G to T mutations, the mass spectrometer was programmed to acquire data in the multiple reaction monitoring mode, monitoring five sequence-specific [M-2H]2– ion transitions: 12GGT(wild-type)-s, 1279.6→650.4; 12GTT-s/12TGT-s, 1267.3→650.4; 12TGT-as, 1240.8→972.6; 12GTT-as, 1240.8→948.6; and 12GGT(wild-type)-as, 1228.8→659.4, and there was no interference from wild type. Several randomly chosen samples were tested with the masses corresponding to the above-mentioned mutations that may occur at codon 12. All the samples analyzed correlated with sequencing (data not shown).

When comparing our results with patient survival, no correlation was found between the presence of any specific c-K-ras mutation and survival. The failure to find any correlation could be due to the limited number of samples studied in this report.

In summary, we described a SOMA technique for analyzing p53 or K-ras mutations previously (23,29); however, we did not detect any samples in disagreement for any specific mutation when comparing SOMA with sequencing. We report here that in three samples, when tumor DNA is present at <10–12% that of wild type, the validity of SOMA is precise and accurate regardless of whether selective PCR is performed. In this study, we have shown that SOMA can specifically detect multiple codon 12 K-ras mutations in DNA, in the presence of a 10-fold excess of wild-type DNA.

By monitoring SOMA 8mer DNA for both the sense and antisense strands for each case, possible interference from unexpected variants in nearby codons is avoided. As the molecular weights of wild-type and mutated DNA sequences differ, a high background of wild-type DNA does not interfere with detection of mutant sequences by SOMA. When, in addition, wild-type DNA is eliminated by BstN1 restriction digestion during PCR amplification, mutations can be detected in the presence of a 100-fold excess of wild-type DNA.

An exhaustive c-K-ras hot-point mutation analysis should include codons 13, 59 and 61. Multiple PCR analyzed by mass spectrometry has been performed for analyzing different hot-spots at the DNA (38). Therefore, in the case of K-ras, we consider it could be feasible to amplify the four hot-spot codons in a multiplex PCR. The analysis of such a sample by SOMA would simplify the time-consuming effort considerably. This study did not quantitatively analyze wild-type versus mutant DNA. We are currently developing a modified SOMA technique using an internal standard DNA plasmid which will quantitatively measure mutated DNA. The SOMA method is widely applicable for the detection of mutant K-ras DNA admixed with large numbers of normal DNA, and could be used for routine K-ras analysis for diagnosis in stool or circulating DNA in the blood as a marker for early detection of cancer (3941).

ACKNOWLEDGEMENTS

The authors wish to thank Dr Zhao-Qi Wang, Chief of the Gene–Environment Interactions Unit, IARC, for his scientific support, Brigitte Chapot and Liliane Garren for technical assistance, all members of the GEI unit (IARC), and Jose Antonio Leal and Laura Casado for help with information technology. Financial support for this work was provided in part by DHHS grant P01 ES06052.

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