Sensitive sequencing method for KRAS mutation detection by Pyrosequencing - PubMed (original) (raw)

Comparative Study

Sensitive sequencing method for KRAS mutation detection by Pyrosequencing

Shuji Ogino et al. J Mol Diagn. 2005 Aug.

Abstract

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.

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Figures

Figure 1

Figure 1

KRAS Pyrosequencing assay design. A: PCR amplifies a segment of KRAS exon 1 containing codons 12 and 13. The reverse PCR primer is biotinylated. B: KRAS-PF1 Pyrosequencing primer can detect common codon 12 mutations. C: KRAS-PF2 Pyrosequencing primer can detect the rest of the codon 12 mutations. D: KRAS-PF3 Pyrosequencing primer can detect codon 13 mutations. WT, wild type; Mut, mutant.

Figure 2

Figure 2

Pyrograms of wild-type and mutant KRAS. A: Wild-type codon 12 by the KRAS-PF1 primer. B: c.35G>T (codon 12 GTT) mutation by the KRAS-PF1 primer. C: c.35G>A (codon 12 GAT) mutation by the KRAS-PF1 primer. D: Wild-type codon 12 by the KRAS-PF2 primer. E: c.34G>T (codon 12 TGT) mutation by the KRAS-PF2 primer. F: c.34G>A (codon 12 AGT) mutation by the KRAS-PF2 primer. G: Wild-type codon 13 by the KRAS-PF3 primer. H: c.38G>A (codon 13 GAC) mutation by the KRAS-PF3 primer. Arrows indicate the presence of mutant alleles. Note that B, C, E, F, and H show the presence of wild-type sequence, which is derived from non-neoplastic cells in tumor (and a normal allele in tumor cells).

Figure 3

Figure 3

Cell line DNA mixing study.

Figure 4

Figure 4

Paraffin colon cancer DNA mixing study.

Figure 5

Figure 5

Allele quantifications by KRAS Pyrosequencing. The horizontal axis shows expected value of the fraction of mutant KRAS based on various cell line DNA mixtures. The vertical axis shows observed quantification results by Pyrosequencing. “Regular DNA” indicates results without WGA. The vertical bar from each dot indicates ±1 SD.

Figure 6

Figure 6

Comparison of Pyrosequencing and BigDye Terminator sequencing. A: Cell line DNA mixing study. A1: The c.35G>T (codon 12 GTT) mutation can be observed by both methods. A2: The c.35G>T (codon 12 GTT) mutation in the 2% mix can easily be observed by Pyrosequencing (left) but is subtle in BigDye sequencing (right). A3 and A4: The c.38G>A (codon 13 GAC) mutation can barely be observed by Pyrosequencing (left) but cannot be detected by BigDye sequencing (right). B: Paraffin colon tumor DNA mixing study. B1 and B2: The c.35G>T (codon 12 GTT) mutation can be barely observed by Pyrosequencing (left) but cannot be detected by BigDye sequencing (right). B3 and B4: The c.35G>A (codon 12 GAT) mutation can barely be observed by Pyrosequencing (left) but cannot be detected by BigDye sequencing (right). B5 and B6: The c.34G>A (codon 12 AGT) mutation can readily be observed by Pyrosequencing (left) but cannot be detected by BigDye sequencing (right).

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