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

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

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

Understanding How Disease and Environment Combine to Structure Resistance in Estuarine Bivalve Populations

Delaware Bay oyster (Crassostrea virginica) populations are influenced by two lethal parasites that cause Dermo and MSX diseases. As part of the US National Science Foundation Ecology of Infectious Diseases initiative, a program developed for Delaware Bay focuses on understanding how oyster population genetics and population dynamics interact with the environment and these parasites to structure he host populations, and how these interactions might modified by climate change. Laboratory and field studies undertaken during this program include identifying genes related to MSX and Dermo disease resistance, potential regions for refugia and the mechanisms that allow them to exist, phenotypic and genotypic differences in oysters from putative refugia and high-disease areas, and spatial and temporal variability in the effective size of the spawning populations. Resulting data provide inputs to oyster genetics, population dynamics, and larval growth models that interface with a three-dimensional circulation model developed for Delaware Bay. Reconstruction of Lagrangian particle tracks is used to infer transport pathways of oyster larvae and MSX and Dermo disease pathogens. Results emerging from laboratory, field, and modeling studies are providing an understanding of long-term changes in Delaware Bay oyster populations that occur as the oyster population responds to climate, environmental, and biological variability

PCR thermocycling conditions utilized in the present work.

1<p>Conditions from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury4" target="_blank">[22]</a>.</p

Temperature-tolerant COLD-PCR in emulsion, TT-fast-eCOLD-PCR: Enrichment of mutations in multiple DNA sequences in a single tube.

<p>A 5% mutation abundance was evaluated for <i>TP53</i> gene exons 6–9 by conventional PCR (left panels) and TT-fast-eCOLD-PCR (right panels). Duplicate experiments are depicted in each case. The enrichment of the mutations in all four exons is estimated from the chromatograms.</p

Primer sequences used in this study.

1<p>Oligonucleotides (F) forward or (R) reverse.</p>2<p>Oligonucleotide sequences described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Fredriksson1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury4" target="_blank">[22]</a>.</p>3<p>Oligonucleotides from Castellanos et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-CastellanosRizaldos1" target="_blank">[16]</a>.</p>4<p>Primer sequences described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Li1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051362#pone.0051362-Milbury3" target="_blank">[17]</a>.</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