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1.  COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions 
PLoS ONE  2014;9(4):e94103.
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.
doi:10.1371/journal.pone.0094103
PMCID: PMC3984089  PMID: 24728321
2.  TEMPERATURE-TOLERANT COLD-PCR ELIMINATES TEMPERATURE STRINGENCY AND ENABLES ROBUST MUTATION ENRICHMENT 
Clinical chemistry  2012;58(7):1130-1138.
BACKGROUND
Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3°C. This precludes simultaneous mutation enrichment in sequences of substantially different melting-temperature (Tm) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT-COLD-PCR) approach that reduces this obstacle.
METHODS
Thermo-cycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR are described. This approach enables enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation is provided for KRAS and TP53 exons 6–9 using dilutions of mutated DNA, clinical cancer samples and plasma-circulating DNA.
RESULTS
A single thermocycling program with a denaturation-temperature window of 2.5–3.0°C enriches mutations in all DNA amplicons simultaneously, despite their different Tms. Mutation enrichments of 6–9-fold were obtained using TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other two forms of COLD-PCR, fast-COLD-PCR and ice-COLD-PCR.
CONCLUSIONS
Low-level mutations in diverse amplicons with different Tm can be mutation-enriched via TT-COLD-PCR provided that their Tms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons, and increases significantly the versatility of COLD-PCR.
doi:10.1373/clinchem.2012.183095
PMCID: PMC3418919  PMID: 22587896
COLD-PCR; mutation detection; mutation enrichment; low-abundance mutations; cancer
3.  ENABLING CLINICAL CANCER GENOMICS FOR RARE MUTATIONS: COLD-PCR MAGNIFIES MUTATIONS PRIOR TO TARGETED AMPLICON RE-SEQUENCING 
Clinical chemistry  2011;58(3):580-589.
Despite widespread interest in the application of next-generation-sequencing (NGS) to the mutation profiling of individual cancer specimens, the onset of personalized clinical genomics is currently stalled due in part to technical hurdles. As tumors are genetically-heterogeneous and often mixed with normal/stromal cells, the resulting low-abundance DNA somatic mutations often produce ambiguous results or fall below the current NGS detection limit, thus hindering mutation calling that abides to clinical sensitivity/specificity standards. Here we examine the feasibility of applying COLD-PCR, a form of PCR that magnifies selectively the mutations, to boost the detection of unknown rare somatic mutations prior to applying NGS-based amplicon re-sequencing to clinical samples. We amplified DNA from serially-diluted mutation-containing human cell-lines into wild-type (WT) DNA, as well as lung adenocarcinoma and colorectal cancer specimens using COLD-PCR or conventional PCR for comparison. Following individual amplification of TP53, KRAS, IDH1, and EGFR regions, PCR products were barcoded, pooled for library preparation and sequenced on the Illumina-HiSeq2000 platform. Regardless of sequencing depth, sequencing errors dictated a mutation-detection limit of ~1–2% mutation abundance in conventional PCR amplicons analyzed by NGS. In contrast, COLD-PCR amplicons enabled genuine mutations to exceed the sequence noise levels, thus allowing reliable identification of mutation abundances of ~0.04%. Sequencing depth was not a significant factor in the identification of COLD-PCR-magnified mutations. The analyzed clinical specimens revealed several TP53 and KRAS missense mutations that could not be called following NGS of conventional amplicons, yet were clearly detectable in COLD-PCR amplicons. Extensive tumor heterogeneity in the TP53 gene was revealed in some samples. As cancer care shifts toward personalized intervention, based on the unique genetic abnormalities in each patient’s tumor genome, we anticipate that COLD-PCR-NGS will elucidate the role of rare mutations in tumors, enable NGS-based analysis of diverse clinical specimens and the broad inter-phasing of NGS with clinical practice.
doi:10.1373/clinchem.2011.176198
PMCID: PMC3418918  PMID: 22194627
COLD-PCR; mutation enrichment; low-abundance mutations; next generation sequencing; cancer
4.  Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion 
PLoS ONE  2012;7(12):e51362.
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 (Tc) 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.
doi:10.1371/journal.pone.0051362
PMCID: PMC3516544  PMID: 23236486
5.  COLD-PCR: improving the sensitivity of molecular diagnostics assays 
The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.
doi:10.1586/erm.10.115
PMCID: PMC3111913  PMID: 21405967
cancer; coamplification at lower denaturation temperature; COLD-PCR; denaturation temperature; low-abundance mutations; variant and mutation enrichment
6.  Two-round COLD-PCR-based Sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma 
Human mutation  2009;30(11):1583-1590.
Reliable identification of cancer-related mutations in TP53 is often problematic as these mutations can be randomly distributed throughout numerous codons and their relative abundance in clinical samples can fall below the sensitivity limits of conventional sequencing. To ensure the highest sensitivity in mutation detection, we adapted the recently described COLD-PCR method to employ two consecutive rounds of COLD-PCR followed by Sanger sequencing. Using this highly sensitive approach we screened 48 non-microdissected lung-adenocarcinoma samples for TP53 mutations. Twenty-four missense/frameshift TP53 mutations throughout exons 5–8 were identified in 23 of 48 (48%) lung-adenocarcinoma samples examined, including 8 low-level mutations at an abundance of ~1–17%, most of which would have been missed using conventional methodologies. The identified alterations include two rare lung-adenocarcinoma mutations, one of which is a ‘disruptive’ mutation currently undocumented in the lung cancer mutation-databases. A sample harboring a low-level mutation (~2% abundance) concurrently with a clonal mutation (80%-abundance) revealed intra-tumoral TP53 mutation heterogeneity. The ability to identify and sequence low-level mutations in the absence of elaborate micro-dissection, via COLD-PCR-based Sanger-sequencing, provides a platform for accurate mutation profiling in clinical specimens and the use of TP53 as a prognostic/predictive biomarker, evaluation of cancer risk, recurrence, and further understanding of cancer biology.
doi:10.1002/humu.21112
PMCID: PMC2784016  PMID: 19760750
lung cancer; TP53; low-level mutation; intra-tumor heterogeneity; COLD-PCR
7.  Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations 
Nucleic Acids Research  2010;39(1):e2.
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.
doi:10.1093/nar/gkq899
PMCID: PMC3017621  PMID: 20937629
8.  Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes 
BMC Genomics  2010;11:485.
Background
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.
Results
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 C. virginica, C. gigas, and C. hongkongensis. 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.
Conclusions
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.
doi:10.1186/1471-2164-11-485
PMCID: PMC2996981  PMID: 20813041
9.  COLD-PCR–Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations 
Clinical chemistry  2009;55(12):2130-2143.
BACKGROUND:
Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature–PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples.
METHODS:
Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6–8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced.
RESULTS:
Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons.
CONCLUSIONS:
COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.
doi:10.1373/clinchem.2009.131029
PMCID: PMC2828872  PMID: 19815609
10.  PCR-Based Methods for the Enrichment of Minority Alleles and Mutations 
Clinical chemistry  2009;55(4):632-640.
BACKGROUND:
The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze.
CONTENT:
Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change.
SUMMARY:
This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.
doi:10.1373/clinchem.2008.113035
PMCID: PMC2811432  PMID: 19201784

Results 1-10 (10)