Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

<p>Abstract</p> <p>Background</p> <p>Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters.</p> <p>Results</p> <p>In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters <it>C. virginica, C. gigas</it>, and <it>C. hongkongensis</it>. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes.</p> <p>Conclusions</p> <p>Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.</p

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

Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules

Detection of un-methylated DNA within different abundances of methylated DNA background by conventional or <i>fast</i>-COLD-MS-PCR.

<p><b>Panel A.</b> Post-PCR melting profile of the 255 bp bisulfite-converted-specific amplicon after conventional PCR or COLD-PCR. Serial dilutions of un-methylated (U) to methylated (M) genomic DNA are depicted (top half). Higher abundances of un-methylated DNA can be discriminated from methylated DNA by the melt peak, whereas lower abundances are only detectable if <i>fast</i>-COLD-MS-PCR replaces conventional PCR. <b>Panel B.</b> Sanger sequencing results of the 0.05% un-methylated (U): methylated (M) DNA sample as amplified by conventional and <i>fast</i>-COLD-MS-PCR are shown (bottom half). Chromatograms are aligned and compared using SeqDoc, and the CpG methylation positions are revealed in the middle panel.</p

Melting profiles of bisulfite-converted DNA from clinical samples following conventional PCR.

<p>Post-PCR melting profiles of the 255 bp bisulfite-converted <i>MGMT</i> gene amplicon after conventional PCR. Examples of fully un-methylated DNA samples isolated from infant blood (<b>Panel A</b>) and glioma samples (<b>Panel B</b>) are depicted. 100% methylated (M) and 100% un-methylated (U) DNA controls are used as reference standards, demonstrating a ∼5°C melting temperature difference among the two. <b>Panel C.</b> A glioma sample with mixed methylation/unmethylation pattern is shown.</p

Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p