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The discriminatory power of the noncoding control region (CR) of domestic dog mitochondrial DNA alone is relatively low. The extent to which the discriminatory power could be increased by analyzing additional highly variable coding regions of the mitochondrial genome (mtGenome) was therefore investigated. Genetic variability across the mtGenome was evaluated by phylogenetic analysis, and the three most variable ~1 kb coding regions identified. We then sampled 100 Swedish dogs to represent breeds in accordance with their frequency in the Swedish population. A previously published dataset of 59 dog mtGenomes collected in the United States was also analyzed. Inclusion of the three coding regions increased the exclusion capacity considerably for the Swedish sample, from 0.920 for the CR alone to 0.964 for all four regions. The number of mtDNA types among all 159 dogs increased from 41 to 72, the four most frequent CR haplotypes being resolved into 22 different haplotypes.

The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. Here we show the presence of multiple genome wide copy number polymorphisms (CNPs) covering 82 genes, the most extensive spanning a cumulative size of 110kilobases. CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation, cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread. Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.

Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA.Preservation of expression ratios after amplification was examined comparing log(2) ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach.Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

Background The PTCH tumour suppressor gene is involved in the development of nearly all basal cell carcinomas (BCCs) of the skin and a fraction of squamous cell carcinomas (SCCs). A nonconservative Pro/Leu nucleotide polymorphism within PTCH exon 23 at codon 1315 was recently reported to be potentially important for the development of breast epithelial cell cancers. Objectives Accordingly, the status of PTCH codon 1315 was analysed for a possible association with the development of nonmelanoma skin cancers (NMSCs) in a pilot study. Because skin cancer risk is affected by specific population-dependent phenotypes such as skin and hair colour, codon 1315 was also analysed for normal allele frequency variation in human populations having differing extents of eumelanin vs. phaeomelanin. Methods The single nucleotide polymorphism in codon 1315 of the human PTCH gene was analysed in genomic DNA from six different populations comprising 472 blood samples and from 170 patients in four different categories with NMSC. Polymerase chain reaction and pyrosequencing were used to determine the allele frequencies. Allelic loss was furthermore determined in tumours following microdissection. Results The Pro/Pro genotype frequency ranged from 30% to 65% between populations, with a significant trend for a reduced frequency of the Pro/Pro genotype in populations having lighter pigmentation (P = 0.020). Pro/Pro frequency showed an increasing trend with increasing tumour case severity (P = 0.027). In 260 samples from 180 Swedish patients with NMSC and a control group of 96 healthy ethnically matched volunteers, no statistically significant pairwise differences between groups were detected in the PTCH codon 1315 allelic distribution, neither was a difference seen for multiple or early onset cases of BCC in the Swedish population. In Swedish patients with single tumours, allelic loss (loss of heterozygosity) was observed in 20 of 30 (67%) patients with BCC and four of 22 (18%) patients with SCC, with no preference in the allele lost. In contrast, the Pro/Pro genotype was frequent in seven U.S. patients having multiple independent BCCs. One of these patients was heterozygous, enabling allelic loss studies. Of 20 independent tumours, 11 had lost an allele; 10 of the 11 had lost Leu, suggesting nonrandom loss that favoured retention of Pro (P = 0.0059). Conclusions Our results indicate an association between the eumelanin-to-phaeomelanin shift and a shift from the Pro/Pro genotype to Leu-containing genotypes. Failure to lose Pro during the shift to phaeomelanin may be associated with an increased population risk for BCC and increased individual risk for multiple BCC. During development of a tumour, the effect of Pro may be magnified by loss of the Leu allele.

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.

Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.

Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3′ ends and then PCR amplification of the 3′-end signature tag population. This method minimizes biased amplification that may occur during parallel amplification of long and short templates. The amplified tags can be either cloned and sequenced or labeled and hybridized to DNA arrays to identify the expressed transcripts. To verify that the relative levels between transcripts in different mRNA/cDNA populations are maintained during the amplification protocol, we have used the Affymetrix oligonucleotide platform and real-time PCR.

BACKGROUND:Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. RESULTS:The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. DISCUSSION:In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.

The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and parapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with severalBordetellaspecies by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay. Five different sequence types of the amplified 239- or 249-bp region were found among the 33Bordetella pertussis,B. parapertussis, andB. bronchisepticaAmerican Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis withTaqI,BglI, andHaeII, which gave four different patterns, can be performed to reliably identifyB. pertussis,B. parapertussis, andB. bronchiseptica.

Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one or both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Furthermore, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.

A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin-biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9-36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 microm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5-8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50-100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (>70°C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hotstart PCR with improved amplification performance.

Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region. QC 20130405. Updated from submitted to published.

We isolated and characterized a Vibrio cholerae O139 Bengal-specific DNA region by arbitrary PCR. The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide or lipopolysaccharide biosynthesis. In order to evaluate the possibility that this region could be used for the specific detection of V. cholerae O139 Bengal, a PCR system was established. The specificity and sensitivity of the PCR were investigated by analyzing 240 strains within the family Vibrionaceae and 178 stains of other gram-negative bacteria. All V. cholerae O139 Bengal strains tested were positive, and none of the 384 control strains were amplified. The sensitivity of the assay was 10(2) CFU/ml.

In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.

Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.

The aim of this study was to evaluate whether HIV-1 or cytomegalovirus (CMV) may contribute to oral lesions frequently found in patients with the acquired immunodeficiency syndrome (AIDS). Saliva samples from 63 HIV-1 positive patients and 21 healthy controls were tested for the presence of HIV-1 and CMV using the polymerase chain reaction (PCR) and virus isolation. CMV IgG titres in serum were also compared in the different groups. HIV-1 RNA, but not DNA, was detected in saliva from 15% (9 out of 59) of the HIV-infected patients. There was no correlation between the presence of HIV-1 RNA and oral symptoms in the patients. CMV DNA was detected significantly more frequently in samples from HIV-1 seropositive than from seronegative patients. CMV was detected in saliva from AIDS patients more often than in saliva from patients with mild or no symptoms. CMV IgG titres were also significantly higher in symptomatic than in asymptomatic patients. There was a significant correlation between the presence of CMV DNA and necrotizing gingivitis, and oral Kaposi's sarcoma in the patients, and also between these lesions and the stage of disease. This does not prove that CMV causes these oral lesions, but a direct or indirect role for CMV cannot be excluded.

BACKGROUND: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. PRINCIPAL FINDINGS: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. CONCLUSIONS: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

BACKGROUND: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. CONCLUSIONS/SIGNIFICANCE: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

An essential question in human biology is how cells and tissues differ in gene and protein expression and how these differences delineate specific biological function. Here, we have performed a global analysis of both mRNA and protein levels based on sequence-based transcriptome analysis (RNA-seq), SILAC-based mass spectrometry analysis and antibody-based confocal microscopy. The study was performed in three functionally different human cell lines and based on the global analysis, we estimated the fractions of mRNA and protein that are cell specific or expressed at similar/different levels in the cell lines. A highly ubiquitous RNA expression was found with >60% of the gene products detected in all cells. The changes of mRNA and protein levels in the cell lines using SILAC and RNA ratios show high correlations, even though the genome-wide dynamic range is substantially higher for the proteins as compared with the transcripts. Large general differences in abundance for proteins from various functional classes are observed and, in general, the cell-type specific proteins are low abundant and highly enriched for cell-surface proteins. Thus, this study shows a path to characterize the transcriptome and proteome in human cells from different origins.

A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor β-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene ( PfDHFR/TS ). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser 108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum .

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.

We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin-coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE-based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand-held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off-the-shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point-of-care tests.

We describe a rapid solid phase assay for detection and sequencing of DNA sequences based on selective introduction of biotin and isotope into the specific DNA fragment amplified by the polymerase chain reaction (PCR). A two-step PCR procedure is used to lower the background signal. The in vitro amplified material is immobilized on magnetic beads with covalently coupled streptavidin and the amount of bound label is measured. Samples identified as positive can be analysed by direct solid phase DNA sequencing. A strategy is also described to use general primers for detection, capturing and sequencing, which are not homologous to the specific sequence to be detected. The concept has been optimized using oligonucleotides specific for Staphylococci and Streptococci, respectively. Here, we show that the assay can be used for detection of Plasmodium falciparum in clinical samples.

We have previously demonstrated the use of pyrosequencing to investigate NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog] mutations in melanoma biopsies. Here, we expanded the analysis to include BRAF (V-raf murine sarcoma viral oncogene homolog B1), another member of the Ras-Raf-mitogen-activated protein kinase (MAPK) signalling pathway, and analysed a total of 294 melanoma tumours from 219 patients. Mutations in BRAF exons 11 and 15 were identified in 156 (53%) tumours and NRAS exon 2 mutations in 86 (29%) tumours. Overall, mutations in NRAS or BRAF were found in 242 of 294 tumours (82%) and were found to be mutually exclusive in all but two cases (0.7%). Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis. Association with preexisting nevi was significantly higher in BRAF mutated tumours (P=0.014). In addition, tumours with BRAF mutations showed a significantly more frequent moderate to pronounced infiltration of lymphocytes (P=0.013). NRAS mutations were associated with a significantly higher Clark level of invasion (P=0.022) than BRAF mutations. Age at diagnosis was significantly higher in tumours with NRAS mutations than in those with BRAF mutations (P=0.019). NRAS and BRAF mutations, however, did not influence the overall survival from time of diagnosis (P=0.7). In conclusion, the separate genotypes were associated with differences in several key clinical and pathological parameters, indicating differences in the biology of melanoma tumours with different proto-oncogene mutations.

Background There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers. Results The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated. Conclusion The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

Background Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromosome sequence and the search for polymorphisms therein. The genome has been only partially sequenced for one male dog, disallowing mapping of the sequence into specific chromosomes. However, by comparing the male genome sequence to the complete female dog genome sequence, candidate Y-chromosome sequence may be identified by exclusion. Results The male dog genome sequence was analysed by Blast search against the human genome to identify sequences with a best match to the human Y chromosome and to the female dog genome to identify those absent in the female genome. Candidate sequences were then tested for male specificity by PCR of five male and five female dogs. 32 sequences from the male genome, with a total length of 24 kbp, were identified as male specific, based on a match to the human Y chromosome, absence in the female dog genome and male specific PCR results. 14437 bp were then sequenced for 10 male dogs originating from Europe, Southwest Asia, Siberia, East Asia, Africa and America. Nine haplotypes were found, which were defined by 14 substitutions. The genetic distance between the haplotypes indicates that they originate from at least five wolf haplotypes. There was no obvious trend in the geographic distribution of the haplotypes. Conclusion We have identified 24159 bp of dog Y-chromosome sequence to be used for population genetic studies. We sequenced 14437 bp in a worldwide collection of dogs, identifying 14 SNPs for future SNP analyses, and giving a first description of the dog Y-chromosome phylogeny.

The arginine variant of the p53 codon 72 polymorphism as well as anogenital and epidermodysplasia verruciformis (EV) types of human papilloma virus (HPV) are suggested to confer increased risk for developing cutaneous squamous cell carcinoma (SCC). In this pilot study, we analysed the p53 codon 72 genotype distribution in 106 microdissected samples from normal and tumour tissues of 53 cases of cutaneous SCC and 96 controls from Sweden. Both normal and tumour samples from cases of SCC were screened for anogenital and EV HPV. The p53 Arg allele was not associated with the development of cutaneous SCC. Anogenital HPV (44%) was more prevalent than EV HPV (12%). Data also indicate that anogenital HPV is more common in tumour samples, but HPV infection was not identified as a significant risk factor for developing SCC. The presence of anogenital HPV, but not EV HPV might be a risk factor for development of cutaneous SCC.

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.

Sun exposure is accepted as the major risk factor for developing skin cancer, the most common cancer in the western world. Ultraviolet-B (UV-B) radiation is considered the causative agent, but recently several findings suggest a role also for ultraviolet-A (UV-A) radiation. Repeated suberythemal doses of ultraviolet-A1 (UV-A1) on healthy human skin induce an increase of p53 immunoreactive cells in epidermis, which may indicate cell cycle arrest and/or occurrence of p53 mutations.We have investigated the possible mutagenic effect of UV-A1 on skin by sequencing exons 4-11 and adjacent intron sequence of the p53 gene in immunoreactive single cells from three healthy individuals. Previously unexposed buttock skin was irradiated three times a week for 2 weeks with physiological fluences (40 J/cm2) of UV-A1. Punch biopsies were taken before and at different time-points after the exposure, and from these single p53 immunoreactive cells were isolated by using laser-assisted microdissection.Three mutations--all being indicative of oxidative damage and most likely related to UV-A exposure--were found among the 37 single cells from exposed skin, whereas no mutations were found in the 22 single cells taken before exposure.The findings indicate a mutagenic effect of low-dose UV-A1 on healthy human skin, which further demonstrates the importance of considering UV-A when taking protective measures against skin cancer.

Background: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras.Methods: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively.Results: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified.Conclusion: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have shown previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrays using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.

Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the field of tumor pathology.

Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid copurification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations.

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR—the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.