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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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Multiplex s-RT-MELT or OpenArray™-based s-RT-MELT. (A) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 2) or exon 9 mutation (curve 3) or wild-type (curve 1). (B) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 3) or 10-fold diluted into wild-type exon 8 mutation (curve 2) and wild-type (curve 1). (C) OpenArray™ based s-RT-MELT PCR growth curves for p53 exon 8 using DNA from lung and colon surgical specimens and cell lines. (D). Melting curves obtained following OpenArray™ based s-RT-MELT of p53 exon 8 using DNA from lung and colon surgical specimens and cell lines.
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Figure 4: Multiplex s-RT-MELT or OpenArray™-based s-RT-MELT. (A) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 2) or exon 9 mutation (curve 3) or wild-type (curve 1). (B) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 3) or 10-fold diluted into wild-type exon 8 mutation (curve 2) and wild-type (curve 1). (C) OpenArray™ based s-RT-MELT PCR growth curves for p53 exon 8 using DNA from lung and colon surgical specimens and cell lines. (D). Melting curves obtained following OpenArray™ based s-RT-MELT of p53 exon 8 using DNA from lung and colon surgical specimens and cell lines.

Mentions: A significant potential advantage of enzymatic mutation scanning is the ability to screen several sequences simultaneously for mutations. To demonstrate that s-RT-MELT can be used for parallel scanning of mutations in several PCR products, we mixed equimolar amounts of PCR products from p53 exons 5–9 containing mutations either in exon 8 or in exon 9. We then formed ‘cross-hybridized sequences’ and screened the mixture for mutations in p53 exons 5–9 in a single tube using s-RT-MELT, as depicted in Figure 1A. Following real-time PCR and melting curve analysis, the exon 8 or exon 9 mutants were clearly distinguished from the wild-type sample (Figure 4A, curves 1–3). Next, the mutant exon 8 DNA sample was first diluted 10-fold into wild-type exon 8 and the equimolar mixture of p53 exons 5–9 was prepared and screened again in a single tube via s-RT-MELT. The exon 8 mutation was again distinguished from the wild-type mixture of exons (Figure 4B, curves 1–3). Since >80% of p53 mutations in human tumors are encountered in exons 5–9 (45), the multiplex single-tube s-RT-MELT reaction could be used to identify most p53 mutations encountered in clinical tumor samples. Combined with multiplex PCR directly from genomic DNA, this approach could result to a convenient, high-throughput method for mutation scanning.Figure 4.


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)

Multiplex s-RT-MELT or OpenArray™-based s-RT-MELT. (A) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 2) or exon 9 mutation (curve 3) or wild-type (curve 1). (B) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 3) or 10-fold diluted into wild-type exon 8 mutation (curve 2) and wild-type (curve 1). (C) OpenArray™ based s-RT-MELT PCR growth curves for p53 exon 8 using DNA from lung and colon surgical specimens and cell lines. (D). Melting curves obtained following OpenArray™ based s-RT-MELT of p53 exon 8 using DNA from lung and colon surgical specimens and cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1919510&req=5

Figure 4: Multiplex s-RT-MELT or OpenArray™-based s-RT-MELT. (A) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 2) or exon 9 mutation (curve 3) or wild-type (curve 1). (B) Melting curves obtained following multiplex s-RT-MELT for mixture of p53 exons 5–9 (exon 8 mutation, curve 3) or 10-fold diluted into wild-type exon 8 mutation (curve 2) and wild-type (curve 1). (C) OpenArray™ based s-RT-MELT PCR growth curves for p53 exon 8 using DNA from lung and colon surgical specimens and cell lines. (D). Melting curves obtained following OpenArray™ based s-RT-MELT of p53 exon 8 using DNA from lung and colon surgical specimens and cell lines.
Mentions: A significant potential advantage of enzymatic mutation scanning is the ability to screen several sequences simultaneously for mutations. To demonstrate that s-RT-MELT can be used for parallel scanning of mutations in several PCR products, we mixed equimolar amounts of PCR products from p53 exons 5–9 containing mutations either in exon 8 or in exon 9. We then formed ‘cross-hybridized sequences’ and screened the mixture for mutations in p53 exons 5–9 in a single tube using s-RT-MELT, as depicted in Figure 1A. Following real-time PCR and melting curve analysis, the exon 8 or exon 9 mutants were clearly distinguished from the wild-type sample (Figure 4A, curves 1–3). Next, the mutant exon 8 DNA sample was first diluted 10-fold into wild-type exon 8 and the equimolar mixture of p53 exons 5–9 was prepared and screened again in a single tube via s-RT-MELT. The exon 8 mutation was again distinguished from the wild-type mixture of exons (Figure 4B, curves 1–3). Since >80% of p53 mutations in human tumors are encountered in exons 5–9 (45), the multiplex single-tube s-RT-MELT reaction could be used to identify most p53 mutations encountered in clinical tumor samples. Combined with multiplex PCR directly from genomic DNA, this approach could result to a convenient, high-throughput method for mutation scanning.Figure 4.

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