Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion

Background: Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens. Methods: Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature $(T_c)$ corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion. Results: Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined. Conclusions: TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube

Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods

COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes

Caracterización molecular de aislados de "Mycobacterium avium" subespecie "paratuberculosis"

Tesis de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 09-07-2010Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

El IVA en la venta de energía eléctrica

<div><p>Non-small cell lung cancer (NSCLC) patients treated with small molecule <i>EGFR</i> inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on <i>EGFR</i> exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, <u>Di</u>fferential <u>S</u>trand <u>Se</u>paration at <u>C</u>ritical <u>T</u>emperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M <i>EGFR</i> mutation. DISSECT pre-enriches low-abundance T790M <i>EGFR</i> mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.</p></div

Cell lines used in the present study.

<p>Cell lines used in the present study.</p

T790M<i>EGFR</i> mutant enrichment analysis by HRM and Sanger Sequencing.

<p>(A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M <i>EGFR</i> mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C>T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C>T.</p

Schematic of procedure.

<p>(A) Flowchart diagram of <i>EGFR</i> exon 20 mutant enrichment by DISSECT followed by PNA-LNA PCR. (B) Schematic design of <i>EGFR</i> exon 20 primers, LNA probe, and PNA clamp during PNA-LNA PCR procedure.</p

Temperature-tolerant-fast-COLD-PCR in emulsion: Overview of the steps involved.

<p>Multiplex pre-amplification from genomic DNA; emulsification with gene-specific primers; mixing into a single tube; and temperature-tolerant emulsion-based fast-COLD-PCR.</p

Comparison of PNA-LNA sensitivity of detection for the T790M<i>EGFR</i> mutation.

<p>(A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.</p