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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

s-RT-MELT for rapid mutation scanning using enzymatic selection and real-time DNA-melting. (A) General outline of the approach. The dotted line contains the new steps involved in s-RT-MELT relative to previous approaches, i.e. the addition of a 3′-polynucleotide tail followed by real-time PCR that enables selective amplification of the Surveyor™-cut sequences and real time melting curve analysis. (B) Detailed outline of the procedure used to selectively amplify the mutation-containing fragments in s-RT-MELT.
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Figure 1: s-RT-MELT for rapid mutation scanning using enzymatic selection and real-time DNA-melting. (A) General outline of the approach. The dotted line contains the new steps involved in s-RT-MELT relative to previous approaches, i.e. the addition of a 3′-polynucleotide tail followed by real-time PCR that enables selective amplification of the Surveyor™-cut sequences and real time melting curve analysis. (B) Detailed outline of the procedure used to selectively amplify the mutation-containing fragments in s-RT-MELT.

Mentions: The s-RT-MELT assay converts PCR fragments generated at positions of mutations by the Surveyor™ enzyme to fully amplifiable sequences that enable selective PCR amplification in a subsequent quantitative PCR detection method. Following denaturation and re-annealing of PCR products that leads to formation of cross-hybridized sequences at the positions of mutations (Figure 1A) the sample is exposed to Surveyor™ endonuclease that recognizes base pair mismatches or small loops with high specificity (28) and generates a break on both DNA strands 3′ to the mismatch. The resulting DNA fragments participate in a terminal transferase (TdT) reaction that leads to polynucleotide ‘tailing’ (sequential addition of adenine, poly-A-tail) at the 3′-ends. A real-time PCR reaction is subsequently performed using adjusted conditions that enable selective amplification of the mutant-only fragments, followed by real-time melting curve analysis for identification of mutations in the presence of SYBR-GREEN™ or LC-GREEN™ DNA dye.Figure 1.


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)

s-RT-MELT for rapid mutation scanning using enzymatic selection and real-time DNA-melting. (A) General outline of the approach. The dotted line contains the new steps involved in s-RT-MELT relative to previous approaches, i.e. the addition of a 3′-polynucleotide tail followed by real-time PCR that enables selective amplification of the Surveyor™-cut sequences and real time melting curve analysis. (B) Detailed outline of the procedure used to selectively amplify the mutation-containing fragments in s-RT-MELT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: s-RT-MELT for rapid mutation scanning using enzymatic selection and real-time DNA-melting. (A) General outline of the approach. The dotted line contains the new steps involved in s-RT-MELT relative to previous approaches, i.e. the addition of a 3′-polynucleotide tail followed by real-time PCR that enables selective amplification of the Surveyor™-cut sequences and real time melting curve analysis. (B) Detailed outline of the procedure used to selectively amplify the mutation-containing fragments in s-RT-MELT.
Mentions: The s-RT-MELT assay converts PCR fragments generated at positions of mutations by the Surveyor™ enzyme to fully amplifiable sequences that enable selective PCR amplification in a subsequent quantitative PCR detection method. Following denaturation and re-annealing of PCR products that leads to formation of cross-hybridized sequences at the positions of mutations (Figure 1A) the sample is exposed to Surveyor™ endonuclease that recognizes base pair mismatches or small loops with high specificity (28) and generates a break on both DNA strands 3′ to the mismatch. The resulting DNA fragments participate in a terminal transferase (TdT) reaction that leads to polynucleotide ‘tailing’ (sequential addition of adenine, poly-A-tail) at the 3′-ends. A real-time PCR reaction is subsequently performed using adjusted conditions that enable selective amplification of the mutant-only fragments, followed by real-time melting curve analysis for identification of mutations in the presence of SYBR-GREEN™ or LC-GREEN™ DNA dye.Figure 1.

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