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1.  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
2.  Discovering the RNA Transcription Landscape using Directional Approaches 
High-throughput complementary DNA sequencing (RNA-Seq) is a powerful technique that allows for sensitive digital quantification of transcript levels. Moreover, RNA-Seq enables the detection of non-canonical transcription start sites and termination sites, alternative splice isoforms and transcript mutation and edition. Standard “next-generation” RNA-sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. In this approach, the synthesis of randomly primed double-stranded cDNA followed by addition of adaptors for sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Our objective was to address this need by developing a novel streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information while maintaining even coverage of transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differential tagging of the first strand cDNA ends. As a result, we have enabled strand specific mRNA sequencing, as well as whole transcriptome sequencing (Total RNA-Seq) from ribosomal-depleted samples. Total RNA-Seq provides a much broader picture of expression dynamics including discovery of antisense transcripts. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.
PMCID: PMC3635307
3.  A Fast Solution to NGS Library Prep with Low Nanogram DNA Input 
Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.
PMCID: PMC3635320
4.  Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations 
Nucleic Acids Research  2012;41(3):e50.
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.
doi:10.1093/nar/gks1250
PMCID: PMC3561944  PMID: 23258702
6.  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
7.  Removal of Oxidative DNA Damage via FEN1-Dependent Long-Patch Base Excision Repair in Human Cell Mitochondria ▿ †  
Molecular and Cellular Biology  2008;28(16):4975-4987.
Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase γ and interferes with SP-BER by covalently trapping polymerase γ during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.
doi:10.1128/MCB.00457-08
PMCID: PMC2519700  PMID: 18541666
8.  ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis▿ †  
Molecular and Cellular Biology  2007;27(24):8834-8847.
Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T½ fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T½) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.
doi:10.1128/MCB.00974-07
PMCID: PMC2169401  PMID: 17938202
9.  5-Halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2) 
Nucleic Acids Research  2005;33(9):3057-3064.
Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals.
doi:10.1093/nar/gki612
PMCID: PMC1140371  PMID: 15917437
10.  DNA ligases ensure fidelity by interrogating minor groove contacts 
Nucleic Acids Research  2004;32(15):4503-4511.
DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3′-hydroxyl and 5′-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3′ side of the ligase junction, but tolerant of mispairs on the 5′ side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3′ side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson–Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson–Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson–Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3′ side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.
doi:10.1093/nar/gkh781
PMCID: PMC516055  PMID: 15328364

Results 1-10 (10)