Jan 17, 1994 - Radiolabeled Polymerase Chain Reaction Assay for Detection of ras ..... The PCRfragment with wiki-type Kl-ras codon 12 is digested. 114 bp.
CLIN. CHEM.4015,705-709 (1994)
#{149} Molecular
Pathology
Radiolabeled Polymerase Chain Reaction Assay for Detection of ras Oncogene Point Mutations in Tumors Yoichi Aoki,’
J. C. Lee,1 Shiv PiIlai,’
Kurt J. Isselbacher,’
The human ras gene plays a fundamental role in the
transduction of extracellular signals to the nucleus, thereby regulating cell growth and differentiation. Point mutations in the ras gene convert it into a transforming oncogene that has been found in many solid and hematologic malignancies. We describe a rapid and sensitive
assaybasedon a radiolabeledpolymerasechainreaction followed by restriction enzyme digestion that we have adaptedfor differentiating between the wild-type and mutant ras genes. This assay shouldprove useful in the analysisof ras gene point mutations in clinical tumor specimensin which ras oncogene activation is an early
event in carcinogenesis. IndexingTerms: hybridization assays/restriction enzymes/cancer/ single-strand conformation polymorphism
The human ras gene family includes the Ha-, Ki-, and N-ras genes, all of which encode homologous GTP binding proteins that play a role in signal transduction. Point mutations in the ras gene, particularly in codons 12, 13, or 61, convert the gene into a transforming oncogene, and have been described in a variety of tumors (1, 2). Numerous techniques have been used for the detection of ras point mutations in tumor specimens. The biological assay based on DNA transfection into NIH/3T3 cells and subsequent identification of cells that either form morphologically transformed foci or induce tumors in nude mice is too laborious to be applicable for large-scale clinical screening. Bos et al. (3,4) developed a dot-blot procedure for the identification of ras gene point mutations by hybridization to specific oligonucleotide probes. Jiang et al. (5, 6) devised a nonradioactive method, using polymerase chain reaction (PCR)-amplifled DNA with mismatched Ki-ras primers followed by restriction enzyme digestion to distinguish between mutant and wild-type alleles. Kumar and Barbacid (7) described an assay involving PCR-amplifled Ki-ras DNA fragments and a liquid hybridization technique with labeled oligonucleotide probes. Others have used a PCRlSouthern-based assay (8). ‘Massachusetts General Hospital Cancer Center, Harvard Medical School, 149 13th St., Charlestown, MA 02129. 2(j Unit, Jackson 7, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114. 3Mdress correspondence to this author at: Gastrointestinal Unit, Jackson 7, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114. Received November 23, 1993; accepted January 17, 1994.
and Anil K. Rustgi”2’3
By using radioactive dNTPs and mismatched Ki-ras primers, we have adapted a PCR-based assay to distinguish between wild-type and mutant DNA templates after restriction enzyme digestion. We have compared this assay with other techniques and show that it is a rapid and sensitive method for detecting ras gene point mutations in a heterogeneous cell population.
Materials and Methods Colon carcinoma cell lines and genomic DNA preparation. Fourteen colon carcinoma cell lines (COLO32OHSR, DiFi, Caco-2, HCT116, C0L0205, TM, LoVo, DLD-1, COLO2O1, SK-CO1, LS-174T, SW1417, SW480, and HT29) were obtained from American Type Culture Collection(Rockville, MD). High-molecular-mass DNA was prepared from these cell lines (9). PCR amplification. One microgram of genomic DNA was amplified in a 100-zL reaction volume containing 10 mmo]/L Tris-HC1 (pH 9.0), 50 mmol/KC1, 1.5 mmol/L MgC12, 0.1 g/L gelatin, 1.25 mmol/L dNTPs, 1 jmol/L PCR primers, and 2.5 U of Taq polymerase (Promega, Madison, WI). We used 5’-ACTGAATATAAACCTTGTGGTAGTTGGACCT-3’ (KRAS5’) as the 5’ pruner and 5’-TCAAAGAATGGTCCTGGACC-3’ (KRAS3’) as the 3’ primer (6). A PCR cycle consisted of 1 mm of denaturation at 94#{176}C, 2 mm of annealing at 55#{176}C, and 3 mm of elongation at 72#{176}C. Samples were subjected to 35 cycles of amplification. Restriction enzyme analysis. Aliquots (10 L) of PCRamplified samples were digested with the restriction enzyme BstNI in a total volume of 20 pL under conditions recommended by the supplier (New England Biolabs; Beverly, MA). Digestion mixtures were incubated at 60#{176}C for 2-3 h. DNA was electrophoresed through an 8% native acrylamide gel for 4-5 h. Gels were stained with ethidium bromide and photographed on an ultraviolet light transilluminator. Liquid hybridization and probe shift assay. This assay was based primarily on the previously described method (7). Five microliters of the PCR sample was digested with 2 L (16 U) of BstNI at 60#{176}C for 12-16 h to detect the Ki-ras codon-12 mutation. For detection of the Kiras codon-13 mutation, 10 of PCR product was digested with 2 L (8 U) of HphI (New England Biolabs) at 37#{176}C for 14-16 h. The digest was mixed with 5 x iO dpm of the Ki-ras first exon probe (5’-CGAATATGATCCAACAATAG-3’) (KRASY) (Fig. 1) in a 30-pL reaction volume containing 0.75 mo]IL NaCl. The probe was endCLINICALCHEMISTRY, Vol. 40, No. 5,
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labeled with [y-32P]dATP (6000 Cimol/L) (New England h. The digests were run on an 8% native acrylainide gel Nuclear, Boston, MA) and T4 polynucleotide kinase for 4-5 h. The gel was wrapped in plastic wrap and (Pharmacia LKB Biotechnology, Piscataway, NJ). Reexposed to film at room temperature for 2-6 h. Single-strand conformation polymorphism (SSCP) actions, overlaid with mineral oil, were denatured at 97#{176}C for 10 miii and rapidly cooled to the annealing analysis. Ki-ras codon-12 mutation was detected with a temperature of 55#{176}C. Hybridization was carried out at modified version of the SSCP method (10, 11). Following radioactive PCR as described above, 2 j.&Lof the PCR 55#{176}C for 2 h and terminated by cooling on ice. Separation was carried out on an 8% native acrylaimde gel by product was mixed with 8 L of loading buffer (950 electrophoresis. After electrophoresis, the gel was mLJL formamide, 20 mmol/L EDTA, 0.5 g/L xylene cyanol, 0.5 g/L bromphenol blue) and incubated at 95#{176}C for placed in a plastic wrap and exposed to Amersham (Arlington Heights, IL) Hyperfilm’TM at room temperature 5 miii. We loaded 2 L of each sample on a 6% acrylfor 4-6 h. amide gel containing 100 mLfL glycerol. The gel was Southern hybridization with specific oligonucleotide run at 40W constant power for 3-4 h at 4#{176}C with lx probes. Southern hybridization for detecting the Ki-ras TBE (89 mmol/L Tris-borate, 2 mmol/L EDTA, pH 8.3) value mutation at codon 12 was performed according to as running buffer. The gel was dried and exposed to film the previously described method (8, 10). Briefly, 50 iL at -70#{176}C for 14 h. of each PCR sample was run on a 2% agarose gel, and, Results after incubation in 16 mmol/L HC1 for 30 min, the gel was transferred to Immobion N (Millipore, Bedford, Detection of Ki-ras codon-12 and -13 mutations in MA) in 0.4 mol/L NaOH. The oligonucleotide used for colon carcinoma cell lines. Genomic DNA prepared from hybridization was 5’-GGAGCTGTTGGCGTAGGCAA. 14 colon carcinoma cell lines was amplified with the primers KRAS5’ and KRAS3’, generating a DNA frag3’ for the valine codon-12-mut.ant Ki-ras. The oligonument of 157 nucleotides. The KRAS5’ primer is 30 nucleotide was labeled with 32P and T4 polynucleotide kinase to a specific activity of _108 dpm/ig. Hybridizacleotides long and incorporates a cytosine residue at the tion was performed at 52#{176}C for 6 h. After hybridization, first position of codon 11 and terminates adjacent to the membranes were rinsed in 3x SSC (450 mmol/L codon 12 (Fig. 1). A single nucleotide substitution is also sodium chloride, 18 mmoWL sodium citrate, and 1 incorporated into the KRAS3’ primer as a positive conmmol/L Tris-HC1, pH 7.2), 1 g/L sodium dodecyl sulfate trol for BstNI cleavage. The Ki-ras codon-12 mutation at room temperature for 5 mm, and then washed at 56#{176}C was detected with BstNI digestion and liquid hybridizain 3x SSC, 1 gIL sodium dodecyl sulfate for 30 miii. The tion in the SW480, SK-CO1, and LS-174T cell lines (Fig. filters were then exposed to film at -70#{176}C for 2-6 h. 2). Upon incubation with BstNI, fragments encoding Radioactive PCR. Radioactive PCR was carried out in wild-type codon-12 sequences were cleaved twice, rea 100-.tL volume with the following modifications: The sulting in the largest band of 114 nucleotides. Fragconcentration of cold dCTP was reduced to 0.25 mmolJL, ments containing mutations at either the first or second positions of codon 12 were cleaved only once, resulting and 0.5 .tL of [a-32P]dCTP (3000 Cimol/L) (New Enin a longer band of 143 nucleotides (Fig. 1). In the gland Nuclear) was added to the reactions. After amplification, 5 iL of each PCR sample was digested with 2 SW480 cell line, only the mutant band was observed and this is consistent with the previously reported results tL (16 U) of BstNI in a 20-pL volume at 60#{176}C for 14-16
K-ras codon 12
K-ras codon 13
Undigested DNA KRAS5
-
KR4SYprobe
KRAS3
BatNi
wild-type
BstNl
digested DNA
ll4bp
l5lbp
I14PP
KRASY probe
KRASY probe
Mutant digested DNA I
BstNl
114PP
lhl
I
43bp1
114 bp KRASY probe
KRASY probe
codon 10
11
12
13
GGA GCT GGT GGC KRAS5’ primer generatedsequence GGA T (GT GGC BstNl Codon 13 Asp mutant sequence GGA GCT G(T (aC HphI wild-type sequence
706
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CHEMISTRY, Vol. 40, No. 5, 1994
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Fig. 1. Fragmentsizes created by restrictionenzyme digestionin Ki-ras codons 12 and 13. The PCRfragment with wiki-type Kl-ras codon 12 is digested twiceby BstNI, once atthe BstNI site that overlaps codon 12 and once at an,ntemal control site at the 3’ end, creating three bands of 114,29, and 14 bp. A mutation in eitherof the fIrsttwo bases of l10% molecule, it is possible to detect the mutation analysis (11, 12). SSCP is limited in detection mutation when wild-type ras molecules >90% of the clinical sample.
band with 1:10 diluof mutant by SSCP of the ras constitute
Discussion Many molecular biological approaches have been used to analyze point mutations in the ras gene and applied to
0
022990000.I 0
0 0
C)
Fig. 5. Detectionof Ki-ras codon-12 mutation with the Southernblot
assay. SW480DNA
was mixed with HT29 DNA in 10-fold serial dilutions. Mixtures were amplified andafter gel electrophoresis, samples were hybridized with the sequence-specific oligonucleotide for the mutant valine in KI-ras codon 12. Ratios of SW480to H129 DNA are given above the sample lanes.
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CLINICALCHEMISTRY, Vol. 40, No. 5,
1994
Fig. 7. Detectionof Ki-ras codon-12 mutationwith SSCP analysis. SW480 DNA and HT29 DNA were mixed in 10-fold serial dilutions. Mixtures were labeled by incorporating 32P during PCR with KRAS5’ and KRAS3’ primers. The PCR product was subjected to SSCP analysis.
Table 1. Compa rison of radioactive methods. Uquld hybrIdization
Southern hybridization
Radlolabeled
Probe required
1
7
-
Hybridizationrequired lime required
1
7
-
24 h
3 days Clear (high stringency) 1:102_103
Band signal
Sensitivity
Background
signal
i:i0-i0
the early diagnosis of tumors or to differentiating benign from malignant conditions (13). Using radioactive dNTPs and mismatched Ki-ras oligonucleotides, we have adapted a PCR-based technique to differentiate between wild-type and mutant Ki-rns DNA templates. The PClllSouthern assay involves amplification of Kii-as codons 12 or 13 and detection of mutant molecules by hybridization with labeled oligonucleotideprobes. The specificity of the method depends on conditions that ensure that the mutant oligonucleotide provides a detectable signal only by hybridizing to mutant molecules. Stringent hybridization and washing conditions are required for the appropriate signal-to-noise ratio. Radiolabeled PCR followed by restriction enzyme digestion proved to be faster and more sensitive than the PCRI Southern assay. A liquid hybridization approach involving PCR amplification with mismatched oligonucleotides, diagnostic restriction enzyme digestion, and detection with a radiolabeledprobe was extensivelyexamined. The radiolabeled PCR assay we describe is an adaptation of this technique. Although the liquid hybridization assay is as quick and sensitive as the radiolabeled PCR method, its applicability is limited by higher background noise, making interpretation difficult. Furthermore, hybridization and washing steps are obviated in the radiolabeled PCR method. An SSCP-based approach has low sensitivitywhen compared with all other techniques tested, and thus its application to clinical material is limited. We conclude that the radiolabeled PCR method followed by diagnostic restriction enzyme digestion is a rapid and sensitive means of detecting ras gene point mutations (Table 1). This method has the advantages of no hybridization steps and a high signal-to-noise ratio. Conditions for all methods were meticulously optimized as reported in the literature. The radiolabeled PCR
PCR
SSCP -
24 h Clear i:i0-10
24 h
Clear 1:10
method is especially useful for analyzing clinical samples in which mutant ras alleles represent a small percentage of available i-as molecules.Such clinical situations are important in the early detection of cancer and in the differentiation of benign from malignant disease. Applications in pancreatic cancer and colon cancer are under investigation. This work was supported in part by the American Cancer Society (IRG) and the Blair Foundation.
References 1. Vogelstein ations during
B, Fearon ER, colorectal
Hamilton
SR, et al. Genetic alterN Engl J Med 1988;
tumor development.
319:525-32. 2. Boa JL. ras oncogene in human cancer; a review. Cancer Boa 1989;49:4682-9. 3. Vries MV, Bogaard ME, van den Elst H, van Boom JH, van der Eb AJ, Boa JL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligonucleotides. Gene 1986;50:313-20. 4. Boa JL, Fearon ER, Hamilton SR. et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987;327:293-7. 5. Jiang W, Kahn SM, Guillem JG, Lu S, Weinstein lB. Rapid detection of ras oncogenes in human tumors: applications to colon, esophageal, and gastric cancer. Oncogene 1989;4:923-8. 6. Kahn S, Jiang W, Weinstein lB. PCR-based assays for the detection of ras mutations. Amplifications 1990;4:22-6. 7. Kumar R, Barbacid M. Oncogene detection at the single cell leveL Oncogene 1988;3:647-51. 8. Sidransky D, Tokino T, Hamilton SR, et at. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 1992;256:102-5. 9. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning a laboratory manual, Vol. 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989:9.14-9.23. 10. Sidransky D, Eschenbach AV, Tsai YC, et at. Identification of p53 gene mutations in bladder cancers and urine samples. Science 1991;252:706-9. 11. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989;5:874-9.
12. Orita M, Iwahara H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 1989;86:2766-70. 13. Capon DJ, Seeburg PH, McGrath ,JP, et at. Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations. Nature 1983;304:507-13.
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