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s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis.

Li J, Berbeco R, Distel RJ, Jänne PA, Wang L, Makrigiorgos GM - Nucleic Acids Res. (2007)

Bottom Line: Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor) and oligonucleotide tails are enzymatically attached at the position of mutations.A novel application of PCR enables selective amplification of mutation-containing DNA fragments.Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA.

ABSTRACT
The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.

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Related in: MedlinePlus

Detection of p53 exon 8 mutations using s-RT-MELT. (A) dHPLC chromatograms of the products obtained using the standard Surveyor™-dHPLC approach (28), versus the new technology on a sample containing a p53 exon 8 14522G > A mutation or a wild-type sample. Curves 1 and 2: Standard Surveyor™-dHPLC on wild- type and mutant samples, respectively. Curves 3–7: s-RT-MELT products when real-time PCR is performed at different denaturation temperatures. (B) Real-time differential melting curves for a PCR denaturation temperature of 88°C. (C) Sequencing of the s-RT-MELT-generated PCR fragment for a PCR denaturation temperature of 88°C. Direct sequencing of the same PCR product from genomic DNA is also depicted. D. dHPLC chromatograms of s-RT-MELT products obtained using serial dilution of DNA from SW480 cells in wild-type DNA. Real-time PCR was performed at 88°C denaturation temperature. (E) Melting curve analysis of the s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA at 88°C denaturation temperature. (F) s-RT-MELT-sequencing of s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA. (G) Predicted-versus-observed minimum denaturation temperatures for generation of s-RT-MELT products following real-time PCR. The influence of the GC-clamp length (no GC-clamp; 26-nt GC-clamp; or 117-nt GC-clamp), and the position of the mutation along the sequence are depicted. (H) s-RT-MELT screening of unknown p53 exon 8 mutations of 48 colon and lung tumor DNA: representative results from mutation-positive samples and wild-type sample are depicted. (I) Sequencing of low-level p53 exon 8 mutation (colon tumor sample CT20) by direct sequencing and by s-RT-MELT sequencing.
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Figure 2: Detection of p53 exon 8 mutations using s-RT-MELT. (A) dHPLC chromatograms of the products obtained using the standard Surveyor™-dHPLC approach (28), versus the new technology on a sample containing a p53 exon 8 14522G > A mutation or a wild-type sample. Curves 1 and 2: Standard Surveyor™-dHPLC on wild- type and mutant samples, respectively. Curves 3–7: s-RT-MELT products when real-time PCR is performed at different denaturation temperatures. (B) Real-time differential melting curves for a PCR denaturation temperature of 88°C. (C) Sequencing of the s-RT-MELT-generated PCR fragment for a PCR denaturation temperature of 88°C. Direct sequencing of the same PCR product from genomic DNA is also depicted. D. dHPLC chromatograms of s-RT-MELT products obtained using serial dilution of DNA from SW480 cells in wild-type DNA. Real-time PCR was performed at 88°C denaturation temperature. (E) Melting curve analysis of the s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA at 88°C denaturation temperature. (F) s-RT-MELT-sequencing of s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA. (G) Predicted-versus-observed minimum denaturation temperatures for generation of s-RT-MELT products following real-time PCR. The influence of the GC-clamp length (no GC-clamp; 26-nt GC-clamp; or 117-nt GC-clamp), and the position of the mutation along the sequence are depicted. (H) s-RT-MELT screening of unknown p53 exon 8 mutations of 48 colon and lung tumor DNA: representative results from mutation-positive samples and wild-type sample are depicted. (I) Sequencing of low-level p53 exon 8 mutation (colon tumor sample CT20) by direct sequencing and by s-RT-MELT sequencing.

Mentions: To provide initial proof of principle for unknown mutation scanning using s-RT-MELT we utilized cell lines and tumor samples containing sequencing-identified mutations at several positions of p53 exon 8. Figure 2A depicts dHPLC chromatograms of the products obtained using a sample containing a p53 exon 8 G > A mutation or a wild-type sample. The standard Surveyor™-dHPLC approach (28) was first employed to identify the mutation following PCR amplification of exon 8 from genomic DNA. The resulting dHPLC traces contain a single product for the wild-type and two products for the mutation-containing sequences (Figure 2A, curves 1 and 2, respectively). Next, s-RT-MELT was used to screen the same p53 exon 8 sequence. Following PCR amplification with GC/M13-modified primers we cross-hybridized PCR products and exposed them to Surveyor™ and TdT tailing. The subsequent real-time PCR was run at different denaturation temperatures and the products were examined either via dHPLC or via real-time melting-curve analysis. At the standard denaturation temperature of 94°C the mutation-containing sample contains two peaks, corresponding to the anticipated amplification of both Surveyor™-digested and un-digested fragments (Figure 2A, curve 3). However, when the PCR denaturation temperature is lowered (e.g. 86–88°C) a single PCR product is generated for the mutant sample, while the wild-type sample demonstrates no product (Figure 2A, curves 4–7). In Figure 2B, real-time differential melting curves for the PCR reaction run at 88°C are depicted. A peak corresponding to the PCR product from the mutant sample is again clearly evident, which is absent in the wild-type sample. Finally, Figure 2C depicts sequencing of the s-RT-MELT-generated PCR fragment, as well as the direct sequencing from genomic DNA. The G > A mutation is evident in both samples. In the s-RT-MELT product the anticipated addition of the poly-A tail at the 3′-position next to the mutation is illustrated.Figure 2.


s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis.

Li J, Berbeco R, Distel RJ, Jänne PA, Wang L, Makrigiorgos GM - Nucleic Acids Res. (2007)

Detection of p53 exon 8 mutations using s-RT-MELT. (A) dHPLC chromatograms of the products obtained using the standard Surveyor™-dHPLC approach (28), versus the new technology on a sample containing a p53 exon 8 14522G > A mutation or a wild-type sample. Curves 1 and 2: Standard Surveyor™-dHPLC on wild- type and mutant samples, respectively. Curves 3–7: s-RT-MELT products when real-time PCR is performed at different denaturation temperatures. (B) Real-time differential melting curves for a PCR denaturation temperature of 88°C. (C) Sequencing of the s-RT-MELT-generated PCR fragment for a PCR denaturation temperature of 88°C. Direct sequencing of the same PCR product from genomic DNA is also depicted. D. dHPLC chromatograms of s-RT-MELT products obtained using serial dilution of DNA from SW480 cells in wild-type DNA. Real-time PCR was performed at 88°C denaturation temperature. (E) Melting curve analysis of the s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA at 88°C denaturation temperature. (F) s-RT-MELT-sequencing of s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA. (G) Predicted-versus-observed minimum denaturation temperatures for generation of s-RT-MELT products following real-time PCR. The influence of the GC-clamp length (no GC-clamp; 26-nt GC-clamp; or 117-nt GC-clamp), and the position of the mutation along the sequence are depicted. (H) s-RT-MELT screening of unknown p53 exon 8 mutations of 48 colon and lung tumor DNA: representative results from mutation-positive samples and wild-type sample are depicted. (I) Sequencing of low-level p53 exon 8 mutation (colon tumor sample CT20) by direct sequencing and by s-RT-MELT sequencing.
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Figure 2: Detection of p53 exon 8 mutations using s-RT-MELT. (A) dHPLC chromatograms of the products obtained using the standard Surveyor™-dHPLC approach (28), versus the new technology on a sample containing a p53 exon 8 14522G > A mutation or a wild-type sample. Curves 1 and 2: Standard Surveyor™-dHPLC on wild- type and mutant samples, respectively. Curves 3–7: s-RT-MELT products when real-time PCR is performed at different denaturation temperatures. (B) Real-time differential melting curves for a PCR denaturation temperature of 88°C. (C) Sequencing of the s-RT-MELT-generated PCR fragment for a PCR denaturation temperature of 88°C. Direct sequencing of the same PCR product from genomic DNA is also depicted. D. dHPLC chromatograms of s-RT-MELT products obtained using serial dilution of DNA from SW480 cells in wild-type DNA. Real-time PCR was performed at 88°C denaturation temperature. (E) Melting curve analysis of the s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA at 88°C denaturation temperature. (F) s-RT-MELT-sequencing of s-RT-MELT products obtained using serial dilution of SW480 in wild-type DNA. (G) Predicted-versus-observed minimum denaturation temperatures for generation of s-RT-MELT products following real-time PCR. The influence of the GC-clamp length (no GC-clamp; 26-nt GC-clamp; or 117-nt GC-clamp), and the position of the mutation along the sequence are depicted. (H) s-RT-MELT screening of unknown p53 exon 8 mutations of 48 colon and lung tumor DNA: representative results from mutation-positive samples and wild-type sample are depicted. (I) Sequencing of low-level p53 exon 8 mutation (colon tumor sample CT20) by direct sequencing and by s-RT-MELT sequencing.
Mentions: To provide initial proof of principle for unknown mutation scanning using s-RT-MELT we utilized cell lines and tumor samples containing sequencing-identified mutations at several positions of p53 exon 8. Figure 2A depicts dHPLC chromatograms of the products obtained using a sample containing a p53 exon 8 G > A mutation or a wild-type sample. The standard Surveyor™-dHPLC approach (28) was first employed to identify the mutation following PCR amplification of exon 8 from genomic DNA. The resulting dHPLC traces contain a single product for the wild-type and two products for the mutation-containing sequences (Figure 2A, curves 1 and 2, respectively). Next, s-RT-MELT was used to screen the same p53 exon 8 sequence. Following PCR amplification with GC/M13-modified primers we cross-hybridized PCR products and exposed them to Surveyor™ and TdT tailing. The subsequent real-time PCR was run at different denaturation temperatures and the products were examined either via dHPLC or via real-time melting-curve analysis. At the standard denaturation temperature of 94°C the mutation-containing sample contains two peaks, corresponding to the anticipated amplification of both Surveyor™-digested and un-digested fragments (Figure 2A, curve 3). However, when the PCR denaturation temperature is lowered (e.g. 86–88°C) a single PCR product is generated for the mutant sample, while the wild-type sample demonstrates no product (Figure 2A, curves 4–7). In Figure 2B, real-time differential melting curves for the PCR reaction run at 88°C are depicted. A peak corresponding to the PCR product from the mutant sample is again clearly evident, which is absent in the wild-type sample. Finally, Figure 2C depicts sequencing of the s-RT-MELT-generated PCR fragment, as well as the direct sequencing from genomic DNA. The G > A mutation is evident in both samples. In the s-RT-MELT product the anticipated addition of the poly-A tail at the 3′-position next to the mutation is illustrated.Figure 2.

Bottom Line: Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor) and oligonucleotide tails are enzymatically attached at the position of mutations.A novel application of PCR enables selective amplification of mutation-containing DNA fragments.Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA.

ABSTRACT
The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.

Show MeSH
Related in: MedlinePlus