Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis - PubMed (original) (raw)

Comparative Study

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

Matilde E Lleonart et al. Nucleic Acids Res. 2004.

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.

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Figures

Figure 1

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 with the enzyme BpmI. p indicates a 5′-phosphate group. Bold letters indicate the mutation introduced in the primer to amplify the DNA.

Figure 2

Figure 2

(a) Negative electrospray mass spectrum of the wild-type-s oligonucleotide shown in Figure 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 the solvent conditions described herein, the [M-2H]2– ion peak at m/z 1279.6 is the most intense ion, and sodium and other metal-adduct ions (see inset) are kept to a minimum. (b) CID mass spectrum of the [M-2H]2– oligonucleotide ion (m/z 1279.6) shown in Figure 1. Fragment ion peaks in the mass spectrum give oligonucleotide sequence information. (c) The [an–Bn]– series of fragment ions gives sequence information about the 5′ end of the molecule; the wx– series of fragment ions gives sequence information about the 3′ end.

Figure 2

Figure 2

(a) Negative electrospray mass spectrum of the wild-type-s oligonucleotide shown in Figure 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 the solvent conditions described herein, the [M-2H]2– ion peak at m/z 1279.6 is the most intense ion, and sodium and other metal-adduct ions (see inset) are kept to a minimum. (b) CID mass spectrum of the [M-2H]2– oligonucleotide ion (m/z 1279.6) shown in Figure 1. Fragment ion peaks in the mass spectrum give oligonucleotide sequence information. (c) The [an–Bn]– series of fragment ions gives sequence information about the 5′ end of the molecule; the wx– series of fragment ions gives sequence information about the 3′ end.

Figure 2

Figure 2

(a) Negative electrospray mass spectrum of the wild-type-s oligonucleotide shown in Figure 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 the solvent conditions described herein, the [M-2H]2– ion peak at m/z 1279.6 is the most intense ion, and sodium and other metal-adduct ions (see inset) are kept to a minimum. (b) CID mass spectrum of the [M-2H]2– oligonucleotide ion (m/z 1279.6) shown in Figure 1. Fragment ion peaks in the mass spectrum give oligonucleotide sequence information. (c) The [an–Bn]– series of fragment ions gives sequence information about the 5′ end of the molecule; the wx– series of fragment ions gives sequence information about the 3′ end.

Figure 3

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 ions were monitored for each DNA sample.

Figure 4

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

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) and antisense (1236.3→659.4) mutation. When the wild-type DNA in this sample is cut with BstN1 during PCR amplification (b), these two oligonucleotides increase in intensity relative to wild-type oligonucleotides. For DNA sample 946459N (c), a sample treated under the same conditions but containing no mutated DNA, only wild-type- specific oligonucleotides are observed even after BstN1 treatment (d).

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