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Clinical Chemistry 43: 936-942, 1997;
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(Clinical Chemistry. 1997;43:936-942.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Rapid and large-scale method to detect K-ras gene mutations in tumor samples

Evelyne Lopez-Crapez1,a, Camille Chypre2, Jacqueline Saavedra2, Joseph Marchand2 and Jean Grenier1

1 C.R.L.C. Val d'Aurelle, Laboratoire de Radioanalyse, 34094 Montpellier Cédex 5, France.

2 Cis Bio International, 91192 Gif-sur-Yvette Cédex, France.
a Author for correspondence. Fax 33 4 67 63 28 73.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have developed a rapid and large-scale method for the detection of K-ras gene mutations in tumors. First, DNA is amplified by an asymmetric PCR; second, the single-strand dinitrophenyl (DNP)-labeled amplified DNA is hybridized specifically to oligonucleotide probes affixed on a tube. Finally, perfectly matched duplexes are easily detected by a monoclonal anti-DNP antibody bearing125I. The usefulness of this technique is illustrated by analyzing K-ras codon 12 mutations in human colorectal samples. This reliable assay procedure can be applied to the rapid screening of virtually any genetic disease caused by previously described point mutations.


Key Words: indexing terms: immobilized oligonucleotide probes • point mutation • allele-specific oligonucleotide detection • polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The reference method for the identification of K-ras gene mutations is direct DNA sequencing (1). However, this approach is labor intensive, and therefore numerous alternative techniques have been developed. Some of these new methods are designed to scan DNA and reveal DNA sequence modifications without giving exactly their type. This group includes denaturing gradient gel electrophoresis (2), single-stand conformation polymorphism (3), and RNase mismatch cleavage (4). Other methods, allowing the detection of specific point mutations, include allele-specific oligonucleotide (ASO) hybridization (5), primer-mediated restriction fragment length polymorphism (RFLP) (6), allele-specific PCR (7), and mutant- enriched PCR (8)(9).1 These types of techniques are more attractive to use when specific point mutations have a clinical meaning, which seems to be the case for K-ras gene (10)(11).

Point mutations in ras genes have been found in many different human neoplasms and are frequent in adenocarcinomas of the pancreas, colon, and lung and certain types of hematological malignancies (12). As mutant ras alleles seem to be clinically useful tools for diagnostic and prognostic purposes, rapid and reliable methods are needed to analyze this potential molecular tumor marker.

Recently, we developed a simple method for the detection of p53 gene loss of heterozygosity by a tube-based assay (13). We describe here a mutation tube assay test (MUTA test), based on the same approach, to detect single point mutations in the 12th codon of the K-ras gene. After studying the correlation with direct DNA sequencing, we analyzed colorectal carcinoma DNAs extracted from microdissected slide sections and biopsies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
Common laboratory reagents were purchased from Sigma Chemical Co. (St. Louis, MO) and Boehringer Mannheim (Indianapolis, IN).

cell lines
Lung carcinoma cell line A549 [homozygous, GGT (gly) -> AGT (ser)], lymph node metastasis of colon adenocarcinoma cell line SW620 [homozygous, GGT (gly) -> GTT (val)], pancreatic carcinoma cell line MIA-PaCa-2 [homozygous, GGT (gly) -> TGT (asp)], colon adenocarcinoma cell lines SW1116 [heterozygous, GGT (gly) -> GCT (ala)] and LS174T [heterozygous, GGT (gly) -> GAT (asp)], displaying a homozygous or heterozygous mutation on the K-ras codon 12 sequence, and the prostate adenocarcinoma cell line PC3 (wild-type K-ras codon 12 sequence) were purchased from the American Type Culture Collection (Rockville, MD).

colorectal samples
Samples were obtained during surgery from 50 patients with colorectal cancer (C.R.L.C. Val d'Aurelle, Montpellier, France). Colorectal carcinoma and normal mucosa were resected from each patient. According to Dukes' classification, the tumors were staged from A to D (2 stage A, 22 stage B, 11 stage C, 15 stage D).

Each carcinoma was divided into two parts. One portion was directly frozen in liquid nitrogen; the other was embedded in OCT compound (Miles Diagnostics, Elkhart, IN; polyvinyl alcohol and polyethylene glycol in a nonreactive matrix) and six consecutive 15-µm thick sections were cut and then stored at -20 °C. One slide from each series was stained with hematoxylin and eosin and evaluated by light microscopy. With this stained slide as a guide, microdissections on the unstained cryostat sections were made to select neoplastic cells (14).

dna extraction
High-molecular-mass DNA from the blood of a volunteer donor, cell lines, frozen biopsies, and microdissection slides were prepared by proteinase K digestion and phenol–chloroform extraction as previously described (15).

oligonucleotides
The oligonucleotide primers and probes were synthesized with the use of an Applied Biosystems DNA synthesizer (Model 381A; Applied Biosystems, Foster City, CA) and purified by reversed-phase HPLC (Cis Bio International, Gif-sur-Yvette, France). Fig. 1 shows the oligonucleotide sequences and their location.



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  		Figure 1. DNA sequence and location of oligonucleotide primers and probes used for detection of K-ras mutations by the MUTA test and by direct sequencing.

Oligonucleotide labeling [biotin, dinitrophenol (DNP)] was done as previously described (16).

synthesis of dna sequences with base-specific k-ras mutations
Six synthetic DNAs corresponding to all K-ras codon 12 mutations were prepared according to the principle of site-specific mutagenesis and recombination via PCR (17).

dna sequencing
Sequence determination on DNA samples from patients was done on amplified (MD1/MD2) DNA by using [{alpha}-33P]dATP in the dideoxy chain termination method (18). The same sequencing method was applied to evaluate the synthesis of the six mutant DNAs (not shown).

dectection of k-ras mutations by muta test
The principle of the MUTA test that we have developed to analyze K-ras point mutations is described in Fig. 2 .



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  		Figure 2. MUTA test.

A portion of exon 1 of the K-ras gene encompassing the codon 12 sequence is amplified from DNA sample by using two primers [one is DNP-labeled ({square})]. Seven different oligonucleotide probes corresponding to the wild-type or mutated codon 12 sequence are immobilized by their biotin (•) extremity on avidin-coated tubes ( ). Each tube is then hybridized to a fraction of the asymmetric PCR product and washed under stringent conditions. DNA duplexes are detected by a 125I-labeled anti-DNP antibody ( ).

The DNA amplification reactions were carried out in a total volume of 50 µL containing 250 ng of extracted DNA or 10 µL of a 1:101 dilution of the selected synthetic DNA, 200 µmol/L of each dNTP, 10 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.1 g/L gelatin, 2 U of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT), and a 1:10 ratio of P3 ras 1 to 5' DNP-P3 ras 2.

The DNA was amplified through 32 cycles (94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min) with a 480 Perkin-Elmer DNA thermal cycler. Each extracted DNA was analyzed in triplicate, and the values in the tables represent the mean of these three independent MUTA tests. Experimental conditions to avoid PCR product carryover were applied (19).

To prepare the solid phase, avidin-coated tubes (Cis Bio International) were reacted for 1 h at 37 °C with 10 pmol of a capture biotinylated probe. After washing twice at room temperature, tubes were ready for subsequent hybridization.

Hybridization was performed by using 5 µL of the asymmetric PCR products without any additional post-PCR step. The reaction was carried out for 1 h at 45 °C in 100 µL of 10 mmol/L phosphate buffer, pH 7.4, containing 1 mol/L NaCl. After incubation, the tubes were washed three times for 20 min at 45 °C with 10 mmol/L phosphate buffer, pH 7.4, containing 150 mL/L formamide.

Fixed hybrids were then recognized by a 125I-labeled anti-DNP antibody (Cis Bio-International) (250 000 cpm/tube) for 1 h at 37 °C. After two washes of 5 min at room temperature with 300 µL of 10 mmol/L phosphate buffer, pH 7.4, containing 0.5 mL/L Tween 20, the bound radioactivity was directly measured.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
One genomic DNA extracted from the blood of a volunteer donor (wild-type codon 12 sequence) and the six synthetic DNAs (Fig. 1Up ), each carrying a specific K-ras codon 12 single mutation, were used to find optimal reaction conditions.

The asymmetric PCR allows the direct hybridization of amplified products to the oligonucleotide probes coated on the solid support without any denaturation step (20).

A reproducible signal was obtained when the ratio between the unmodified primer and the DNP-labeled primer was 1:10 and the amount of the less concentrated primer was at least 0.12 pmol. Furthermore, this PCR did not contain any spurious amplification products, as confirmed by 2% agarose gel electrophoresis (data not shown).

This assay is based on the use of oligonucleotide-coated tubes for hybridization of PCR-amplified K-ras gene. Our aim was to find the hybridization/washing procedure that allowed the detection of all seven possible sequences on codon 12 under the same operating conditions.

The influence of different products acting on the melting temperature (Tm), such as formamide, dimethyl sulfoxide, and tetramethylammonium chloride, which allows the same stability for CG and AT basepairs (21), was tested in the hybridization and washing buffers to obtain a specific hybridization.

The addition of formamide at a concentration of 150 mL/L to the washing buffer, and a 45 °C temperature during the hybridization and washing steps, allows a clear discrimination to be made between a fully matched hybrid (100% specific signal) and a 1-bp mismatched hybrid (a cpm value <12% of the specific signal) (Table 1 ).


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  		Table 1. Influence of 150 mL/L formamide addition in the hybridization and washing steps on the specificity of detection of the wild-type K-ras codon 12 sequence.1

Nevertheless, the interaction between probes forming CT mismatches in the first base of the codon was higher than probes forming other mismatches. Therefore, the effect of the length of the original M3 probe (20-mer) was studied (range from 20-mer to 12-mer) to increase the specificity of hybridization (Table 2 ).


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  		Table 2. M3 probe length vs hybridization.

The decrease from 20- to 19-mer allowed the reduction of the nonspecific hybridization. Moreover, not only the length of the probe seems to be important, but also the sequence. The 19-B M3 probe was more efficient than the 19-A M3 probe in discriminating between non-fully-matched hybrids. When the length of the probe was decreased, the specific hybridization, i.e., the hybridization of the M3 probe with the synthetic M3 DNA, also decreased (Table 2Up ). Neverthless, if we considered the 100% specific hybridization when the 20 M3 probe was used, 87% of the signal remained when the 19-B M3 probe was hybridized. The decrease in the length of the probe to 18-mer resulted in a 45% reduction of the signal.

muta characterization
To show the reliability of the procedure, different DNAs from tumor cell lines were analyzed.

To assess the specificity of the MUTA test for the detection of point mutations in genomic DNA, we studied DNA samples from tumor cell lines, each one carrying a different single mutation in the K-ras codon 12 gene.

The analysis of the MUTA test data is based on the calculation of a cutoff value. This value is determined as three times the mean of the three least important signals given by the hybridization of the seven probes. We considered the presence of a mutation when the cpm hybridization value for a selected probe was higher than the cutoff value. By using this method of calculation, K-ras codon 12 mutations (homozygous or heterozygous) for a DNA sample of interest can be determined. The results in Table 3 clearly indicate that all the types of mutations analyzed could be distinguished from the wild-type sequence.


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  		Table 3. Detection of K-ras gene mutations in tumoral cell lines by MUTA test.

Furthermore, several tumor cell lines have been reported to contain only the mutated K-ras gene. The MIA-PaCa-2 cell line (22) has been shown to have lost the wild-type sequence. The MUTA test confirmed the loss of the normal ras allele in this cell line. On the other hand, LS174T cell line (23) has been found to contain a normal allele in addition to the mutated K-ras gene. The presence of the normal allele in this cell line was also confirmed.

The sensitivity of the method was investigated by mixing DNAs from two different cell lines, PC3 containing the wild-type K-ras and MIA-PaCa-2 containing a (GGT -> TGT) mutation in codon 12 of K-ras. DNA from both cell lines were mixed in different proportions, amplified by asymmetric PCR, and then hybridized according to the developed method to an M3-containing mutation probe. The K-ras mutated sequence, present only in the cell line MIA PaCa-2, was still detected when this cell line represented only 5% of the starting material (Table 4 ). The presence of 10% mutant sequences was the limit of sensitivity by direct sequencing (data not shown).


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  		Table 4. Determination of sensitivity of MUTA test.

analysis of k-ras codon 12 mutations in colorectal tumors by muta test and direct sequencing
Fifty tumoral DNAs from 50 different patients were analyzed by both MUTA test and direct sequencing. The characteristics of seven representative patients are shown in Table 5 . Fig. 3 shows the data obtained from direct sequencing and MUTA test. A 100% correlation was obtained for the 50 DNA samples analyzed.


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  		Table 5. Characteristics of human tumoral samples.



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  		Figure 3. Detection of K-ras codon 12 mutations on human colorectal samples by both MUTA test and direct sequencing.

To increase the percentage of tumoral cells analyzed, microdissected cryostat sections were made from each colorectal biopsy. The results obtained with MUTA test on DNAs extracted from slides or biopsies are similar (Table 6 ).


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  		Table 6. Analysis of K-ras codon 12 mutations by MUTA test on DNA extracted from biopsies or microdissected cryostat sections.

K-ras gene mutations at codon 12 were found in 16 of the 50 carcinoma samples examined (32%). The mutations consisted of one M1 mutation (6.25%), no M2 mutation, one M3 mutation (6.25%), six M4 mutations (37.5%), one M5 mutation (6.25%), and seven M6 mutations (43.7%). No ras gene mutation was detected in any of the noncancerous tissues analyzed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Investigations to define new methods to detect small genetic alterations, especially single point mutations, have been thoroughly pursued. Here, an ASO method has been described to analyze K-ras gene mutations. This procedure is characterized by the hybridization of a PCR-amplified DNA to a panel of oligonucleotide probes specific to each K-ras codon 12 point mutation.

The ASO assay diagnoses known point mutations. The requirement for the use of oligonucleotide probes is to define their length and the conditions for the hybridization and posthybridization washes to be able to discriminate between wild-type and mutated sequences.

This method, currently based on the use of membrane as solid support (24)(25) and autoradiogram analysis, is tedious and seems difficult to be done by laboratory technicians unskilled in molecular biology. The MUTA test, combining a tube as solid support and numerical data as results, is similar to the immunoanalysis tests currently being carried out and could easily be introduced into clinical laboratories. Moreover, the MUTA test could be made semiautomated.

Other methods, such as the RFLP or the mutant-enriched PCR amplification, are often used but are not compatible with automation, and problems of partial digestion can be encountered. Moreover, the sensitivity of these procedures is limited by the ethidium bromide method of detection.

The MUTA test can easily detect point mutations in as little as 5% of the total DNA. The sensitivity of the method can be improved by an increase in the number of PCR reactions (26) and cycles. However, higher sensitivity also means a higher risk of false-positive signals (27) and misincorporation.

The asymmetric generated product is DNP-labeled and, at present, the detection system is based on a 125I-labeled anti-DNP antibody. Improvements in the procedure are in progress for the use of an anti-DNP antibody labeled with horseradish peroxidase, which would allow a colorimetric or luminescent signal.

To prevent false-negative signals due to the proportion of normal cells in tumor biopsies, we made microdissections on cryostat sections for all the tumors analyzed. This enrichment technique makes it possible to obtain a cell population composed of >80% tumoral cells. The results of analysis of K-ras codon 12 mutations in DNA samples coming from slides or biopsies show a perfect correlation. Nevertheless, the microdissection procedure is more attractive, as it requires less biological material and demonstrates the increase in sensitivity.

A 32% frequency of mutation was found, whatever the starting DNA material used. This value was essentially similar to that observed in various studies (28)(29).

Mutation detection on codon 13 and multiplex PCR amplification covering codons 12–13 and codon 61 are being developed to detect all K-ras gene mutations from only one PCR reaction.

In conclusion, we have demonstrated that the MUTA format is a sensitive, specific, and practical approach to detect and characterize codon 12 K-ras gene mutations. It is also more effective than the sequencing method in both the technical realization and the data analysis. This assay provides a means towards further understanding the implication of ras gene family mutations in the pathogenesis of cancer and in the diagnosis or early detection of different human cancers. Moreover, this format can be used to detect any described point mutation in a selected gene of interest.


   Acknowledgments
 
We thank Henri Pujol for his continued support. We thank Bernard Saint Aubert and Marc Ychou for providing colorectal tissues, and Joëlle Simony-Lafontaine for the anatomopathological analysis of biopsies. The technical work of Josette Noletti was appreciated.


   Footnotes
 
1 Nonstandard abbreviations: ASO, allele-specific oligonucleotide; RFLP, restriction fragment length polymorphism; MUTA, mutation tube assay; and DNP, dinitrophenol.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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