uracil-DNA Glycosylase Treatment Research Articles

Uracil base PCR implemented for reliable DNA walking

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

Biochemical reconstitution of heat-induced mutational processes.

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase δ. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase ζ on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase δ or ε.

Qualification of tumour mutational burden by targeted next-generation sequencing as a biomarker in hepatocellular carcinoma.

Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). Eligible patients (n=29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P<.0001), estimated deamination counts (median 1335.50 vs 0, P<.0001) and C>T transitions at CpG sites (median 60.3% vs 9.1%, P=.002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P=.041) and deamination counts (median 6393.5 vs 328.5, P=.041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.

Abstract 1996: Tumor mutational burden is affected by next-generation sequencing enrichment method in highly deaminated samples from solid tumors

Abstract Background: Tumor mutational burden (TMB) is currently under investigation as a biomarker for predicting response to anti-cancer immunotherapy. TMB can be defined as the number of somatic nonsynonymous mutations per Mb in a cancer genome, therefore DNA artifacts could lead to inaccurate measurements. Cytosine deamination (C:G&amp;gt;T:A) is a well-known phenomenon in formalin-fixed, paraffin-embedded (FFPE) samples, producing errors in next-generation sequencing (NGS). Herein, we measured TMB in FFPE samples using two comprehensive NGS panels for routine diagnostics implementation. Methodology: TMB assessment was performed on FFPE samples by amplicon-based target enrichment on Thermo Fisher's Oncomine™ Tumor Mutation Load (OTML; n=9) and/or hybrid capture-based on Illumina's TruSight™ Oncology 500 Assay (TSO500; n=8), according to manufacturer's instructions. TMB values were compared for uracil-DNA glycosylase (UDG) treatment prior to PCR amplification and enrichment method. Samples were also compared according to their previous results in a clinically-certified NGS and an in-house microsatellite instability (MSI) assay. TMB &amp;gt;=10 mutations/Mb was considered high, and MSI &amp;gt;=20% was considered unstable. Results and Discussion: The concordance between TMB values for OTML and TSO500 was 75% (n=4) and 62.5% (n=8) with and without UDG treatment, respectively. UDG reduced artifactual C:G&amp;gt;T:A for both enrichment approaches, reinforcing its presence in FFPE DNA; however, there was no alterations on TMB results for both methods (TSO500 n=5; OTML n=7). C:G&amp;gt;T:A variants could be successfully filtered by low allelic frequency (&amp;lt;5%) or low quality (&amp;lt;q20) in non-treated samples. For OTML, a high baseline noise was observed in samples with high deamination scores (&amp;gt;60), leading to TMB overestimation (&amp;gt;350), which could not be corrected even after UDG treatment and manufacture's bioinformatic adjustment. Interestingly, the same samples presented low TMB values for TSO500. Samples with low deamination rates were not influenced by UDG treatment or bioinformatics adjustment regardless of enrichment method. TMB values from a certified NGS was completely concordant for TSO500 (n=4) but only 25% for OTML (n=4). MSI was fully concordant between certified NGS and TSO500 (n=4) and between in-house method and TSO500 (n=4) (value not analyzed by OTML assay). No QC parameter predicted differences between high and low deaminated samples. Conclusions: NGS enrichment methods strongly influenced TMB assessment. Hybrid-based capture showed better performance than amplicon-based. UDG treatment and higher allelic frequency cutoff could minimize false positives at variant calling level, although no significant effect could be observed for general TMB values. Future experiments should cover other alternatives to distinct FFPE DNA damages. Citation Format: Michele A. Pereira, Feliciana L. Marinho, Joice P. Silva, Renato Puga, Maíra C. Freire, Mariana R. Monteiro, Rafael L. Guedes, Luiz H. Araujo. Tumor mutational burden is affected by next-generation sequencing enrichment method in highly deaminated samples from solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1996.

A time-dependent fluorescent biosensor for uracil-DNA glycosylase detection based on the uracil inhibition effect towards archaebacterial DNA polymerases

Uracil-DNA glycosylase (UDG) plays important role in DNA lesion repair. It can remove the uracil lesion in DNA and is involved in multiple biological processes. UDG activity detection methods based on DNA probe are developed fast recently due to its high sensitivity and simplicity. They typically use two strategies: the conformational change or the local environmental change of the DNA probe after UDG cleavage. Here we proposed a new strategy for UDG activity detection, which utilized uracil-containing oligo to inhibit archaebacterial DNA polymerases, and after UDG treatment the archaebacterial DNA polymerases could be activated to generate linear or exponential amplification fluorescent signal. The exponential methods showed a time-dependent signal output and achieved limit of detection of 0.0025 U/mL. More importantly, the feature of time-dependence allowed us to easily modulate the resolution or the dynamic range of our method to meet different demands in various occasions. The strategy was also demonstrated to be highly selective toward DNase and exonucleases and had been applied to detect the UDG activity in cell lysate.

RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs.

Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part. After the reverse transcription, uracil-DNA glycosylase (UDG) treatment was used to remove the dU base and destroy the stem-loop structure of RT product. Enzymatic assay confirmed that the recombinant UDG could efficiently eliminate the dU base in the oligonucleotide. Transcriptions of two small RNAs (TFF and ryeA) in Escherichia coli were detected by RT-qPCR with different primers. Results showed that the use of the chimeric dU stem-loop primer and UDG treatment could enhance the detection specificity and sensitivity about 1.1- to 3.4-fold, compared to those with traditional stem-loop primer and linear primer. Total RNA of 1-10pg was enough for efficient detection with the chimeric stem-loop primers. In a word, this strategy could promote the RT-qPCR detection efficiency on the transcription of bacterial small RNAs even in trace samples and can facilitate the detection of exiguous change in cellular metabolism.

Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine

Deamination of nucleotides causes C:G>T:A changes in formalin-fixed, paraffin-embedded (FFPE) tissue samples and produces false positives during next-generation sequencing (NGS). Uracil DNA glycosylase (UDG) helps eliminate this issue, but the effect of UDG in different tissue preparation conditions has not been rigorously studied. To investigate whether UDG can reduce false-positive single-nucleotide variant (SNV) calls, we used tumor and normal tissues from gastric adenocarcinoma patients prepared using different fixation times and pH conditions. FFPE tumor blocks >10 years were also evaluated for the comparison. We performed semiconductor-based NGS to evaluate nucleotide changes and used UDG to test deamination-related effects. Sequencing quality parameters mildly worsened with prolonged fixation time, acidic pH, and delayed fixation. SNV calls and C:G>T:A changes increased after >48 hours of fixation. In both recently prepared and old FFPE tissue blocks, UDG treatment reduced deamination-induced nucleotide changes. In the recently prepared samples, both high-quality SNVs and mean target coverage were remarkably increased on treatment with UDG. However, the quality of NGS results from old-age samples varied irrespective of UDG treatment. In conclusion, based on our findings, we believe that when performing NGS on recently embedded blocks, it is important to consider that certain poorly fixed samples may be at the risk of being deaminated, which can be corrected with UDG treatment.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination.

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination

In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2U Bst DNA polymerase and 4mM MgSO4 for 1h at 60–62°C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.

Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts. We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs. Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the background sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil. UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.

Abstract 54: Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates.

Abstract Formalin-fixed and paraffin-embedded (FFPE) tissues are commonly used for the detection of mutational biomarkers in cancer patients. One of the challenges associated with FFPE DNA in genetic testing is its high number of sequence artefacts, thus increasing the risk of false positive results particularly for low level mutations. We have previously shown that uracil lesions in FFPE DNA due to deamination of cytosines are the major cause of C:G&amp;gt;T:A sequence artefacts and pretreatment of damaged FFPE DNA with uracil-DNA glycosylase (UDG) prior to PCR amplification markedly reduces the C:G&amp;gt;T:A sequence artefacts using methodologies suitable for the testing of limited numbers of genomic loci. As the number of cancer associated mutations increases, massively parallel sequencing (MPS) approaches are increasingly necessary. In particular, the use of targeted amplicon based strategies is being implemented in many laboratories. We set out to test whether sequence artifacts were a serious problem and whether the UDG pretreatment strategy is also applicable to a amplicon based targeted MPS approach (Truseq Cancer Panel). Samples not treated with UDG showed multiple sequence artefacts with a significant portion of changes greater than background sequencing error, with C:G&amp;gt;T:A changes forming the vast majority of artefacts. Treatment of the samples with UDG prior to sequencing dramatically reduced the majority of C:G&amp;gt;T:A changes without affecting the detection of known SNPs and somatic mutations. Elimination of artefactual changes by UDG treatment also improved the accurate detection of low-level sequence variants. Interestingly, most C:G&amp;gt;T:A changes that occurred in the context of CpG dinucleotides were resistant to the UDG treatment as a likely consequence of 5-methyl cytosine being deaminated to thymine. This approach will greatly facilitate accurate detection of mutations in FFPE samples using amplicon-based approaches which is imperative for personalised cancer medicine, especially when working with samples of low tumour purity. This work also has implications for choice of tissue and protocols for the assessment of heterogeneity in tumours. Citation Format: Hongdo Do, Stephen Q. Wong, Jason Li, Alexander Dobrovic. Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2013-54 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

A Highly Sensitive Electrochemical Platform for the Assay of Uracil-DNA Glycosylase Activity Combined with Enzymatic Amplification

Uracil-DNA glycosylase (UDG) plays a crucial role in DNA lesion repair because it is one of the most important base excision repair (BER) enzymes. Quantitative analysis of UDG activity is of fundamental importance in bioanalysis. Here, an electrochemical sensing platform combined with enzymatic amplification was developed for simple and sensitive assay of UDG activity and its inhibition. This strategy relies on the release of a biotinylated signal probe from the electrode surface, due to the lowered melting temperature of the duplex UDG substrate after UDG treatment. A biotin modification was used as a tracer in the signal probe and streptavidin-alkaline phosphatase (SA-ALP) was taken as a reporter molecule. Upon reacting with UDG, the loss of biotin label led to a decrease in the amount of bound SA-ALP on the electrode surface, resulting in a weaker electrochemical signal. This strategy allowed for a simple, cost-effective, sensitive and selective assay for UDG with a wide linear response range from 0.01 to 10 U/mL and a low detection limit of 0.0079 U/mL. In addition, the effects of drugs on UDG activity have also been investigated. The proposed strategy not only provides a universal platform for the assay of BER enzymes, but also shows potential application for drug screening.

Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

Non-reproducible sequence artefacts are frequently detected in DNA from formalinfixed and paraffin-embedded (FFPE) tissues. However, no rational strategy has been developed for reduction of sequence artefacts from FFPE DNA as the underlying causes of the artefacts are poorly understood. As cytosine deamination to uracil is a common form of DNA damage in ancient DNA, we set out to examine whether treatment of FFPE DNA with uracil-DNA glycosylase (UDG) would lead to the reduction of C>T (and G>A) sequence artefacts. Heteroduplex formation in high resolution melting (HRM)-based assays was used for the detection of sequence variants in FFPE DNA samples. A set of samples that gave false positive HRM results for screening for the E17K mutation in exon 4 of the AKT1 gene were chosen for analysis. Sequencing of these samples showed multiple non-reproducible C:G>T:A artefacts. Treatment of the FFPE DNA with UDG prior to PCR amplification led to a very marked reduction of the sequence artefacts as indicated by both HRM and sequencing analysis, indicating that uracil lesions are the major cause of sequence artefacts. Similar results were shown for the BRAF V600 region in the same sample set and EGFR exon 19 in another sample set. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA as it did not affect the detection of true KRAS codon 12 and true EGFR exon 19 and 20 mutations. We conclude that uracil in FFPE DNA leads to a significant proportion of sequence artefacts. These can be minimised by a simple UDG pretreatment which can be readily carried out, in the same tube, as the PCR immediately prior to commencing thermal cycling. HRM is a convenient way of monitoring both the degree of damage and the effectiveness of the UDG treatment. These findings have immediate and important implications for cancer diagnostics where FFPE DNA is used as the primary genetic material for mutational studies guiding personalised medicine strategies and where simple effective strategies to detect mutations are required.

Abstract 2098: Reducing sequence artefacts from FFPE DNA for cancer diagnostics

Abstract Non-reproducible sequence artefacts have been frequently detected in DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Studies of ancient DNA reveal that deamination of cytosine to uracil is a major cause of DNA damage. We hypothesised that similar lesions may occur in FFPE DNA and examined the role of uracil lesions on generation of transitional G:C to A:T artefacts in FFPE DNA by treatment with uracil DNA glycosylase (UDG). High resolution melting (HRM)-based assays were used for rapid and cost-effective screening of artefacts. The artefact rate was assessed by limited copy number-HRM (LCN-HRM) in FFPE DNA samples in which multiple G:C to A:T artefacts were found. The effect of UDG treatment on detection of true sequence changes was examined by testing mutational hotspots at the EGFR, KRAS and AKT1 genes. A high HRM positive rate as judged by the formation of heteroduplexes was found in FFPE DNA by LCN-HRM. This was markedly reduced after treatment with UDG. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA and did not affect the detection of true KRAS codon 12 and EGFR exon 19 and 20 mutations. In conclusion, this study reports for the first time that uracil lesions due to cytosine deamination is one of the major causes of artefacts in FFPE DNA and the use of uracil-DNA-glycosylase that removes uracil lesions on DNA can lead to a significant reduction of artefacts. This finding will thus greatly improve biomarker studies using DNA samples derived from FFPE tissues, which has immediate implications especially to diagnostic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2098. doi:1538-7445.AM2012-2098

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material.

High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers

MicroRNAs are small noncoding RNAs that serve as important regulators of eukaryotic gene expression and are emerging as novel diagnostic and therapeutic targets for human diseases. Robust and reliable detection of miRNAs is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Existing methods for miRNA quantification rely on fluorescent probes for optimal specificity. In this study, we developed a high-performance real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay that allows specific and rapid detection of mature miRNAs using a fast thermocycling profile (10 sec per cycle). This assay exhibited a wide dynamic range (>7 logs) and was capable of detecting miRNAs from as little as 1 pg of the total RNA or as few as 10 cells. The use of modified reverse-transcription oligonucleotides with a secondary structure and hemi-nested reverse PCR primers allowed excellent discrimination of mature miRNAs from their precursors and highly homologous family members using SYBR Green I. Using a novel approach involving uracil-DNA glycosylase treatment, we showed that carryover of the reverse transcription oligonucleotide to the PCR can be successfully eliminated and discrimination between miRNA homologs could be further enhanced. These assays were further extended for multiplexed detection of miRNAs directly from cell lysates without laborious total RNA isolation. With the robust performance of these assays, we identified several miRNAs that were regulated by glial cell-line-derived neurotrophic factor in human glioblastoma cells. In summary, this method could provide a useful tool for rapid, robust, and cost-effective quantification of existing and novel miRNAs.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B

Hepatitis B virus (HBV) integration into the host genome is frequently detected in HBV positive hepatocellular carcinoma (HCC) in China. The aim of this study is to carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI (national center for biotechnology information) BLAST and MapViewer search were used for identification of HBV location on human genomes. In 40 HBsAg positive HCC samples, 34 (85%) were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 (96%) cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death. Three genes, myeloid/lymphoid or mixed-lineage leukemia 4, G protein alpha transducing activity polypeptide 1 and fibronectin, were found to be recurrently targeted by HBV. HBV-Alu-PCR is a powerful tool for the study of HBV integration sites. Truncated X is a major form existed in the HBV integrants. HBV integration is not distributed evenly throughout the host genome and viral insertional mutagenesis may play an important role in the development of HCC.

Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA

E. coli nucleoside diphosphate kinase ( EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK ( MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung − strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.

Photochemical production of uracil quantified in bromodeoxyuridine-substituted SV40 DNA by uracil DNA glycosylase and a lysyl-tyrosyl-lysine tripeptide.

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechst dye 33258 results in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB) yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis. Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is approximately 5-fold more efficient than SSB formation. Formation of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.