PCR amplification following restriction to detect site-specific DNA methylation

A procedure to test for DNA methylation at sites recognized by methylation-sensitive restriction endonucleases is described. The procedure is based on the assumption that the polymerase chain reaction (PCR) will amplify sequences between two primers only if the target DNA is intact after digestion. A carrot (Daucus carota) cell line that is heterozygous for two sequenced alleles ofDc8, a gene which is expressed during the later stages of embryogenesis provided an ideal source of DNA for developing and testing protocols. The promoters of the two alleles differs significantly in length between two sites used for primers, and only one promoter has a GATC (Sau 3A1 orMbo I) site. This allowed development of a protocol where only the sequence lacking the GATC site was amplified to detectable levels following digestion of DNA withMbo I which is insensitive to symmetric methylation with^m4C or^m5C.     

Gene amplification (PCR) to detect and differentiate mycoplasmas in porcine mycoplasmal pneumonia

The causative agent of porcine mycoplasmal pneumonia, Mycoplasma hyopneumoniae, is difficult and time-consuming to isolate. Serological identification using antibodies induced by the disease is confused by cross-reaction with a closely related organism, Myc. flocculare . From pig lungs obtained at slaughter for meat and deemed free of acute disease, it was possible to detect by culture both Myc. hyopneumoniae and Myc. flocculare . This study has improved on an earlier PCR detection of DNA from the former species by using a nested PCR capable of detecting the purified DNA equivalent to one mycoplasmal genome. With this PCR assay both mycoplasma species were detected and differentiated directly from lung tissue.     

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Metagenomics approaches have been developing rapidly in marine sciences. However, the application of these approaches to marine eukaryotes, and in particular to the smallest ones, is challenging because marine microbial communities are dominated by prokaryotes. One way to circumvent this problem is to separate eukaryotic cells using techniques such as single-cell pipetting or flow cytometry sorting. However, the number of cells that can be recovered by such techniques remains low and genetic material needs to be amplified before metagenomic sequencing can be undertaken. In this methodological study, we tested the application of whole-genome amplification (WGA) to photosynthetic eukaryotes. We performed various optimization steps both on a mixture of known microalgal strains and on natural photosynthetic eukaryote populations sorted by flow cytometry. rRNA genes were used as markers for assessing the efficiency of different protocols. Our data indicate that WGA is suitable for the amplification of photosynthetic eukaryote genomes, but that biases are induced, reducing the diversity of the initial population. Nonetheless, this approach appears to be suitable for obtaining metagenomics data on microbial eukaryotic communities.     

A rapid procedure to detect in situ DNA/RNA hybrids

We developed a new method for detecting DNA/RNA hybrids formed $\text{in}\text{situ}$ using $\text{anti-}\text{DNA}\text{RNA}$ antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesised $\text{in}\text{vitro}$ from cloned $\text{Drosophila}$ histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.     

Use of bromovirus RNA3 hybrids to study template specificity in viral RNA amplification.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

Use of DNA barcoding to detect invertebrate invasive species from diapausing eggs

The global transhipment of ballast water and associated flora and fauna by cargo vessels has increased dramatically in recent decades. Invertebrate species are frequently carried in ballast water and sediment, although identification of diapausing eggs can be extremely problematic. Here we test the application of DNA barcoding using mitochondrial cytochrome c oxidase subunit I and 16S rDNA to identify species from diapausing eggs collected in ballast sediment of ships. The accuracy of DNA barcoding identification was tested by comparing results from the molecular markers against each other, and by comparing barcoding results to traditional morphological identification of individuals hatched from diapausing eggs. Further, we explored two public genetic databases to determine the broader applicability of DNA barcodes. Of 289 diapausing eggs surveyed, sufficient DNA for barcoding was obtained from 96 individuals (33%). Unsuccessful DNA extractions from 67% of eggs in our study were most likely due to degraded condition of eggs. Of 96 eggs with successful DNA extraction, 61 (64%) were identified to species level, while 36% were identified to possible family/order level. Species level identifications were always consistent between methodologies. DNA barcoding was suitable for a wide range of taxa, including Branchiopoda, Copepoda, Rotifera, Bryozoa and Ascidia. Branchiopoda and Copepoda were respectively the best and worst represented groups in genetic databases. Though genetic databases remain incomplete, DNA barcoding resolved nearly double the number of species identified by traditional taxonomy (19 vs. 10). Notorious invaders are well represented in existing databases, rendering these NIS detectable using molecular methods. DNA barcoding provides a rapid and accurate approach to identification of invertebrate diapausing eggs that otherwise would be very difficult to identify.     

Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA)

Background  

MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample.     

Use of ribosomal DNA sequence data to characterize and detect a neogregarine pathogen of Solenopsis invicta (Hymenoptera: Formicidae)

A neogregarine parasite of the red imported fire ant, Solenopsis invicta, was discovered recently in Florida and tentatively placed in the Mattesia genus based on morphological characterization. S. invicta infected with this Mattesia species exhibited a characteristic yellowing of the cuticle which was designated Mattesia "yellow-head disease" (YHD). The 18S rRNA gene sequence from Mattesia YHD was elucidated and compared with the neogregarine pathogens, Mattesia geminata and Ophriocystis elektroscirrha. The sequence data support the previous conclusion that Mattesia YHD is a new species that infects S. invicta. Furthermore, high sequence identity between Mattesia YHD, M. geminata (95.7%), and O. elektroscirrha (86.2%) correctly place the YHD organism in the Mattesia genus and Neogregarinorida order. Oligonucleotide primer pairs were designed to unique areas of the 18S rRNA genes of Mattesia YHD and S. invicta. Multiplex PCR resulted in sensitive and specific detection of Mattesia YHD infection of S. invicta.     

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.     

Use of random amplified polymorphic DNA analysis to detect genetic variation inPyrus species

Pyrus communis L. is the most important pear species for European production. Very few cultivars satisfy standards for fruit quality and clonal fidelity; thus, accurate verification of cultivar identity for checking propagation material and patent protection is important. We evaluated the randomly amplified polymorphic DNA (RAPD) technique for its ability to identify genetic differences among standard pear (Pyrus communis L.) cultivars, William, Passa Crassana, and Conference, and three gamma-ray induced variants. To identify genotype-specific markers, we used thirty 10-mer and two 11-mer sequences, annealing temperatures from 36–45°C, 2Taq polymerases (AmpliTaq and Stoffel fragment, both from former Perkin Elmer Cetus), and 2–4 replicate amplifications. Of the 32 primers (30 from Operon Technologies, Alameda, CA, USA), very few distinguished William from Passa Crassana, and only 1 could clearly differentiate all 3 cultivars. Two primers that did not reveal polymorphisms when used singly, generated polymorphic patterns that distinguished standard from gamma-ray-treated material when used in combination. We show that RAPD analyses can discriminate pear genotypes and suggest this technique as a reliable and inexpensive method for marker-facilitated screening of propagation material and for patent protection.     

Radical mechanism of cyanophage phycoerythrobilin synthase (PebS)

PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.     

Use of stains to detect fingermarks

Abstract

Detection of fingermarks at a crime scene or on related items is of prime interest for forensic investigators, mainly for identification purposes. Most of the fingermarks are invisible to the naked eye, however. The application of detection techniques is required to establish visual contrast between the secretion residue and the underlying substrate. We give here a review of the field related to the concept of using stains to detect fingermarks. A distinction has been made between the physically driven classical detection techniques, the chemically driven ones, and those based on nanostructured materials, an emerging field in forensic science.     

Inhibition of DNA amplification in marine fish larvae preserved in formalin

Recent advances in molecular biology open the possibility touse formalin-preserved specimens stored in ichthyoplankton collectionsfor population genetics studies. Nine DNA extraction techniqueswere tested on Engraulis mordax larvae preserved in bufferedformalin. However, none of the DNA extracts resulted in thepositive amplification of mitochondrial DNA (mtDNA) (NADH1,16srRNA, 12srRNA and control region fractions). An experimentwith different length-time exposure to formalin done with Cynoscionparvipinnis larvae allowed us to confirm the difficulty of amplifyingmtDNA from larvae preserved in formalin for long time periodsand the possibility of DNA extraction and amplification fromshort-term (less than 48 h) formalin-fixed marine fish larvaepreserved in ethanol (70%). We discuss the possible influenceof physical–chemical complexes associated with the durationof preservation to inhibition of amplification reactions.