Degenerate Oligonucleotide-primed PCR Research Articles

Isolation and Functional Characterization of a Phenylalanine Ammonia-Lyase Gene (SsPAL1) from Coleus (Solenostemon scutellarioides (L.) Codd).

Phenylalanine ammonia-lyase (PAL) is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L.) Codd), a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1), suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins) biosynthesis in Coleus at the molecular level.

Interactive analysis and assessment of single-cell copy-number variations.

We present an open-source web platform, Ginkgo (http://qb.cshl.edu/ginkgo), for the analysis and assessment of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validate Ginkgo by reproducing the results of five major studies and examine the characteristics of three commonly used single-cell amplification techniques to conclude degenerate oligonucleotide-primed PCR to be the most consistent for CNV analysis.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

Use of laser microdissection for the construction of Humulusjaponicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis.

Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2) of Humulus japonicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of Humulus japonicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). Fast fluorescence in situ hybridization (FAST-FISH) using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, Humulus japonicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to Humulus lupulus and 88% to Cannabis sativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of Humulus japonicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in Humulus japonicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction.

A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

BackgroundSerine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.ResultsHere, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40°C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum’s SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.ConclusionsThis research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio .O ur results show that the composition of Characidium's W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.

Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions

Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the derivative chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effective labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identification of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis.

Identification of Diptera familias Families in Fauna of Thrace: First Record of Muscina Stabulans for Forensic Entomology

To establish a whole genome amplification testing system based on degenerate oligonucleotide primed-PCR (DOP-PCR) and to explore its reliability and sensitivity. DOP-PCR amplified production was detected by fluorescent labeled multiplex STR amplification and capillary electrophoresis detection system to determine reliability and sensitivity of DOP-PCR system. DOP-PCR system was successfully established and the detection sensitivity reached 5 cells (30 pg) by pretreatment of DOP-PCR and then detection of STR genotyping. The system established in this study is reliable and more testing sensitive for forensic trace evidence.

Does the S phase have an impact on the accuracy of comparative genomic hybridization profiles in single fibroblasts and human blastomeres?

To investigate if there is an association between single-cell replicative stage and the segmental chromosome imbalances detected by comparative genomic hybridization (CGH). First, 135 fibroblasts from cell-line GM03184 (Coriell) at three cell stages (G0/G1, S, and G2/M) were amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) or Sureplex and blindly analyzed by CGH. Second, 85 human blastomeres at the interphase and the metaphase stages, from 30 donated human cryopreserved embryos, were amplified by Sureplex and analyzed by CGH. Academic center for reproductive medicine. None. None. Incidence of aneuploidy and segmental imbalances detected at the different cell stages. In DOP-PCR amplifications of fibroblasts, an increased incidence of segmental abnormalities was detected in the S phase. In Sureplex amplifications of fibroblasts and blastomeres, no differences were detected between the different cell stages. A significantly increased incidence of structural abnormalities was seen in the aneuploid blastomeres. The segmental imbalances detected after Sureplex amplification in 73.3% of the cryopreserved embryos analyzed are mainly nontransitory. They correspond to segmental imbalances present in the cells due to chromosome instability, rather than to replicative DNA segments.

Chromosome painting of Z and W sex chromosomes in Characidium (Characiformes, Crenuchidae)

Some species of the genus Characidium have heteromorphic ZZ/ZW sex chromosomes with a totally heterochromatic W chromosome. Methods for chromosome microdissection associated with chromosome painting have become important tools for cytogenetic studies in Neotropical fish. In Characidium cf. fasciatum, the Z chromosome contains a pericentromeric heterochromatin block, whereas the W chromosome is completely heterochromatic. Therefore, a probe was produced from the W chromosome through microdissection and degenerate oligonucleotide-primed polymerase chain reaction amplification. FISH was performed using the W probe on the chromosomes of specimens of this species. This revealed expressive marks in the pericentromeric region of the Z chromosome as well as a completely painted W chromosome. When applying the same probe on chromosome preparations of C. cf. gomesi and Characidium sp., a pattern similar to C. cf. fasciatum was found, while C. cf. zebra, C. cf. lagosantense and Crenuchus spilurus species showed no hybridization signals. Structural changes in the chromosomes of an ancestral sexual system in the group that includes the species C. cf. gomesi, C. cf. fasciatum and Characidium sp., could have contributed to the process of speciation and could represent a causal mechanism of chromosomal diversification in this group. The heterochromatinization process possibly began in homomorphic and homologous chromosomes of an ancestral form, and this process could have given rise to the current patterns found in the species with sex chromosome heteromorphism.

Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata

Phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid biosynthesis, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds. In this study, we cloned a 2,326 bp full-length PAL2 gene from Lycoris radiata by using degenerate oligonucleotide primer PCR (DOP-PCR) and the rapid amplification of cDNA ends method. The cDNA contains a 2,124 bp coding region encoding 707 amino acids. The LrPAL2 shares about 77.0 % nucleic acid identity and 83 % amino acid identity with LrPAL1. Furthermore, genome sequence analysis demonstrated that LrPAL2 gene contains one intron and two exons. The 5' flanking sequence of LrPAL2 was also cloned by self-formed adaptor PCR (SEFA-PCR), and a group of putative cis-acting elements such as TATA box, CAAT box, G box, TC-rich repeats, CGTCA motif and TCA-element were identified. The LrPAL2 was detected in all tissues examined, with high abundance in bulbs at leaf sprouting stage and in petals at blooming stage. Besides, LrPAL2 drastically responded to MJ, SNP and UV, moderately responded to GA and SA, and a little increased under wounding. Comparison of LrPAL2 expression and LrPAL1 expression demonstrated that LrPAL2 can be more significantly induced than LrPAL1 under the above treatments, and LrPAL2 transcripts accumulated prominently at blooming stage, especially in petals, while LrPAL1 transcripts did not accumulated significantly at blooming stage. All these results suggested that LrPAL2 might play distinct roles in different branches of the phenylpropanoid pathway.

Whole genome amplification of degraded and nondegraded DNA for forensic purposes

Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to “recover” degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-012-0764-9) contains supplementary material, which is available to authorized users.

Genome-wide scan of the soybean genome using degenerate oligonucleotide primed PCR: an example for studying large complex genome structure

The availability of next generation sequencing instruments has made large-scale and genome-wide sequence analysis more feasible in a wild variety of species with large complex genomes, especially crops. This report suggested an approach for characterizing large complex genomes of less-studied/orphan crops. Degenerate oligonucleotide primed PCR (DOP-PCR) is a useful tool for the survey of genomes in less-studied/orphan crops, as prior sequence information is not necessary. Here, four different degenerate primers were redesigned from previously described DOP-PCR primers. The degeneracy of these primers was increased with the addition of two more ‘Ns’. The amplified DOP-PCR products from Sinpaldalkong 2, a soybean genotype, were applied to GS-FLX and the reads from Sinpaldalkong 2 were mapped against Williams 82 as a reference (http://www.phytozome.net/soybean.php), using the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/). These results suggest the identification of 4 single nucleotide polymorphisms between Sinpaldalkong 2 and Williams 82 and recent duplication of the soybean genome. The sequenced reads were subsequently assembled into contigs by Newbler under default conditions. A total of 29 Sinpaldalkong 2 contigs exhibited 95% similarity and < E-100 when mega-blasted with Williams 82 reference sequences. These contigs were mapped to the soybean chromosomes and positioned as clusters within each chromosome. Most of the contigs also showed similarity with the Arabidopsis RNase H domain-containing protein, suggesting a potential way to study retrotransposons in less-studied/orphan crops. Using these modified DOP-PCR primers and GS-FLX, it is possible to obtain insight into the large complex genomes of less-studied/orphan crops.

Comparison of Whole Genome Amplification Methods for Analysis of DNA Extracted from Microdissected Early Breast Lesions in Formalin-Fixed Paraffin-Embedded Tissue

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

Identification and characterization of a bacteria-like sequence in the genome of some Silene species

The aim of this work was to characterize a nucleotide sequence MK14 that originated from a plasmid library obtained via degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) amplification of laser microdissected Y-chromosomes of Silene latifolia. This sequence showed significant similarity to parts of two adjoining genes from bacterial representatives of the genus Ralstonia. MK14 sequence contains a part of a conserved domain, and phylogenetic analysis based on this region confirmed its relationship to Ralstonia-derived sequences. Genomic Southern blot analysis proved the presence of this fragment in the genome of S. latifolia. We hypothesize that this insertion is of bacterial origin, and was probably gained via horizontal gene transfer. Moreover, MK14 insertion is shared by some closely related Silene species, suggesting an ancient spontaneous transformation by an ancestor of bacteria from the genus Ralstonia.

DOP-PCR Amplification of Probe DNA for Whole-Mount FISH in Drosophila

It is sometimes useful to amplify DNA before labeling it. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) allows amplification of any complex DNA, regardless of sequence. DOP-PCR is performed using a single primer that has a fixed sequence at its 5' end and several degenerate bases near its 3' end that allow it to anneal at low stringency to many sites in complex DNA. Following several PCR cycles with an annealing temperature of only 30°C, the annealing temperature is raised so that only products with the primer sequence at each end are amplified in the reaction. This protocol describes the use of DOP-PCR to amplify probe DNA for whole-mount fluorescent in situ hybridization (FISH) in Drosophila. Primary DOP-PCR amplification may be used for microdissected material, small volumes of melted gel slice, or small quantities of other DNA template. A procedure for secondary DOP-PCR amplification is also provided.

Microdissection of Drosophila Polytene Chromosomes for DOP-PCR

This protocol describes a method for the microdissection of Drosophila polytene chromosomes. The microdissected DNA can be amplified by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and then used to prepare probes for fluorescent in situ hybridization (FISH).

The use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) technique to detect and isolate cytochrome P450 2E1 and 2A genes

Cytochrome P450 (CYP, P450) enzymes are widely studied for their involvement in the metabolism of drugs as well as endogenous substrates. Assessing some forms of P450 such as P450 2E1 and 2A phenotype may be of great importance to predict organism’s susceptibility to xenobiotic and environmental pollutants which are metabolically activated by these isoenzymes. In the present study, we designed universal degenerate oligonucleotide primers sets based on the highly conserved nucleotide sequences at the 5’ and 3’ cDNA ends of P450 2E1 and 2A gene family from Camelus dromedaries (Arabian camel), Bos Taurus (cow), Capra hircus (domestic goat), Ovis aries (domestic sheep), Oryctolagus cuniculus (European rabbit), Xenopus tropicalis (Western clawed frog), Rattus norvegicus (Norway rat), Mus musculus (house mouse) and Mesocricetus auratus (golden hamster). We successfully demonstrated that these primers sets were able to detect the expression of P450 2E1 and 2A genes from these eukaryotic organisms. Moreover, we amplified 1070 and 1200 nucleotides of both P450 2E1 and 2A from these organisms and this represent two third of the actual size of these P450 isoforms. The nucleotide sequences of these genes showed percent identities ranging from 80 to 85%. The degenerate primers designed in this study allowed the detection and amplification of known and unknown P450s belonging to the gene families 2E1 and 2A from different organisms. Key words : Cytochrome P450, xenobiotics, degenerate primers, mixed function oxidases.

Quantification of trace-level DNA by real-time whole genome amplification.

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Differential Display System with Vertebrate-Common Degenerate Oligonucleotide Primers: Uncovering Genes Responsive to Dioxin in Avian Embryonic Liver

To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.