Experimental analysis of variance for DNA hybridization: II. Precision

Using DNAs from the Virginia opossum (Didelphis virginiana), we estimated the variance components for two classes of replicate hybrids: different drivers matched to the same tracer and different homoduplexes made from tracers matched to identical drivers. A nested analysis of variance (ANOVA) was used to partition total variance among four levels: Individuals, extracts, preparations, and different aliquots from the same preparation. The variance contributed by these levels depended on the kind of hybrid replicate (driver or tracer) and on the index of thermal stability (T_mode, T_m, T₅₀H, or Normalized Percentage Hybridization). For replicate drivers, significant variance contributions were made by (1) individuals to T_m, (2) extracts to T_mode and NPH but not T_m, and (3) different preparations to NPH. The composite T₅₀H measure calculated from both T_m and NPH revealed effects from both constituent indices. For replicate tracers, preparation error was the single most consistent effect across all indices, followed by extract effects for those indices that incorporated a measure of percent hybridization (T₅₀H NPH). Total variance of the four indices was qualitatively similar for both drivers and tracers: T_mode ranked lowest, followed in order by T_m, T₅₀H, and NPH, with the variance of NPH being as much as 100 times greater than for T_mode. These results provide guidelines for the design of experiments to generate DNA hybridization-based phylogenies and to assess their robustness with bootstrapping. Replicate drivers for a distance matrix based on T_m should use different individuals, whereas one based on T_mode could minimally use different extracts from the same individual. Thus, T_mode may be the index of choice for DNA hybridization experiments when material, time, and money are limited.Correspondence to: R. Bleiweiss     

A comparison of solution and membrane-bound DNA × DNA hybridization,as used to infer phylogeny

A new method of membrane-bound DNA × DNA hybridization was devised to accommodate the study of small quantities of DNA obtained from museum specimens for phylogeny reconstruction. Membranebound, single-stranded target genomic DNAs were competitively hybridized with a total genomic DNA probe to form hybrid duplexes required for the DNA dissociation experiments. We compared the thermal elution profiles derived from dissociating duplexes made with probes of whole genomic, single-copy, and repetitive DNA, as well as solution DNA × DNA hybridization using sc tracer. Quantitatively, pairwise indices of genetic distance derived from duplexes made with genomic probes depended entirely on hybridization of repetitive sequences, but a parallel set of experiments using repetitive and sc probes produced qualitatively similar results. The indices of genetic distance generated by the membrane-bound hybrids form an internally consistent, resolved tree which is in agreement with the solution DNA × DNA hybridization trials and traditional views of the phylogeny of the taxa under study.Correspondence to: P. Houde     

Phylogenetic relationships of the endemic malagasy carnivoreCryptoprocta ferox (Aeluroidea): DNA/DNA hybridization experiments

A molecular and morphological study of several living aeluroid Carnivora was completed to evaluate the evolutionary relationships of the endemicCryptoprocta ferox, a carnivore living on the island of Madagascar. The molecular analysis, based on DNA/DNA hybridization experiments, suggests thatCryptoprocta is more closely related to the Herpestidae (as represented byMungos andIchneumia) than it is to the Viverrinae (Genetta), Paradoxurinae (Paguma, Paradoxurus), Felidae (Felis, Panthera), or Hyaenidae (Crocuta). Based on bootstrapping procedures applied to the individual DNA/DNA results, three branching patterns were observed which differ only by the relative position of the Felidae within the Aeluroidea. The amounts of genetic divergence measured between pairs of compared taxa have been transformed into millions years datings by the molecular clock concept, and this was done by establishing a molecular time scale based on the fossil record of the aeluroid Carnivora.     

Experimental analysis of variance for DNA hybridization: I. Accuracy

We used tissues of the Virginia opossum (Didelphis virginiana) to examine the experimental accuracy of DNA hybridization statistics of thermal stability (T_mode, T_m, T₅₀H, and NPH) with respect to systematic biases in counting radioactivity in elution fractions, and column position and loading order of hybrids in the thermal elution device. We failed to detect any change in the mean melting temperatures among five replicate ¹²⁵I-labeled hybrids counted over 72 h. Furthermore, column position in the automated thermal elution device (TED) did not bias the statistics of aliquots loaded over a few minutes from a single large mother hybrid. On the other hand, the normalized percentage hybridization (NPH) increased as much as 3–5% for aliquots loaded during 1 h from a similar mother hybrid. A parallel but less consistent increase was noted for T₅₀H, which incorporates a measure of NPH. This NPH effect disappeared when hybrids were prepared individually and diluted and loaded in turn—the usual procedure in our laboratory. Replicate distances measured as NPH appear to be sensitive to departures from the normal-distribution assumption of least-squares regression. We recommend that replicate cell values of NPH be transformed to improve their fit to a normal distribution prior to analysis by least-squares phylogenetic algorithms such as those available in Felsenstein's PHYLIP package. Thus, potential sources of inaccuracy in DNA hybridization data can be avoided with simple precautions.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

Molecular homology and DNA hybridization

Summary We reviewed the concept of homology, which can broadly be defined as a correspondence between characteristics that is caused by continuity of information (Van Valen 1982). The concept applies widely in molecular biology when correspondence is taken to mean a genetic relationship resulting from a unique heritable modification of a feature at some previous point in time. Such correspodence can be established for features within a single organism as well as between organisms, making paralogy a valid form of molecular homology under this definition. Molecular homology can be recognized at a variety of organizational levels, which are intedependent. For example, the recognition of homology at the site level involves a statement of homology at the sequence level, and vice versa. This hierarchy, the potential for nonhomologous identity at the site level, and such processes as sequence transposition combine to yield a molecular equivalent to complex structural homology at the anatomical level. As a result, statements of homology between heritable units can involve a valid sense of percent homology.We analyzed DNA hybridization with respect to the problems of recognizing homology and using it in phylogenetic inference. Under a model requiring continuous divergence among compared sequences, DNA hybridization distances embed evolutionary hierarchy, and groups inferred using pairwise methods of tree reconstruction are based on underlying patterns of apomorphic homology. Thus, symplesiomorphic homology will not confound DNA hybridization phylogenies. However, nonhomologous identities that act like apomorphic homologies can lead to inaccurate reconstructions. The main difference between methods of phylogenetic analysis of DNA sequences is that parsimony methods permit hypotheses of nonhomology, whereas distance methods do not.This article was presented at the C.S.E.O.L. Conference on DNA-DNA Hybridization and Evolution, Lake Arrowhead, California, May 11–14, 1989     

The phylogeny of the hominoid primates: a statistical analysis of the DNA-DNA hybridization data

Sibley and Ahlquist compared the single-copy nuclear DNA sequences of the hominoid primates using DNA-DNA hybridization. From this data set they estimated a phylogeny that clusters man and chimpanzees using a distance Wagner procedure. However, no assessment of statistical confidence in this estimated phylogeny was made, despite the fact that their data set contains internal inconsistencies concerning the correct branching order. This paper presents a modification of Pielou's Q- statistic that allows one to make nonparametric tests of phylogenetic relationship from distance data. The results of this analysis indicate that the estimated phylogeny of Sibley and Ahlquist is without statistical significance owing to the internal inconsistencies of the data set. A survey and additional analyses of other types of molecular data indicate that the phylogeny that clusters chimpanzees and gorillas and has the human lineage splitting off earlier is statistically consistent with all the molecular data (including the DNA-DNA hybridization data), whereas the phylogeny estimated by Sibley and Ahlquist can be rejected at the 5% level using the data on restriction- endonuclease sites in the mitochondrial genome.      

DNA hybridization,cladistics, and the phylogeny of phalangerid marsupials

Summary Single-copy DNA/DNA hybridization experiments and numerical cladistic analyses of anatomical characters were used to investigate relationships among nine phalangerid (Marsupialia) species from four different genera. Both rate-dependent and rate-independent analyses of molecular data indicate that species ofTrichosurus form one clade and thatStrigocuscus, Phalanger, andSpilocuscus form a second. Within the latter group,Spilocuscus is excluded from aStrigocuscus-Phalanger calde, which, in turn, is not fully resolved on a jackknife strict consensus tree. Minimum-length Dollo, Wagner, and Camin-Sokal parsimony trees based on 35 anatomical characters, in contrast, suggest placement ofStrigocuscus withTrichosurus rather than withSpilocuscus andPhalanger. However, there are two derived characters that support the alternative arrange ofStrigocuscus withSpilocuscus andPhalanger and one character that further unitesStrigocuscus andPhalanger. Thus, DNA hybridization results are not inconsistent with the distribution of derived character states among anatomical characters, only with minimum-length trees based on character data.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system

We find that the catalytic activity of gold nanoparticles (GNPs) on luminol-H₂O₂ chemiluminescence (CL) system is greatly enhanced after it is aggregated by 0.5 M NaCl. We use this observation to design a CL detection of DNA hybridization. It is based on that the single- and double-stranded oligonucleotides have different propensities to adsorb on GNPs in colloidal solution, and the hybridization occurred between the probe DNA and target DNA can result in aggregation of the GNPs, producing strong CL emission. In the assay, no covalent functionalization of the GNPs, the probe, or the target DNA is required. The assay, including hybridization and detection, occurs in homogenous solution. The detection limit of target DNA (3σ) was estimated to be as low as 1.1 fM. The sensitivity was increased more than 6 orders of magnitude over that of GNPs-based colorimetric method. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.     

Estimation of hominoid phylogeny from a DNA hybridization data set

Summary Analysis of the expanded data set of Sibley and Ahlquist (1987) on primate phylogeny using a maximum likelihood mixed model analysis of variance method shows that there is significant evidence for resolving theHomo-Pan-Gorilla trifurcation in favor of aHomo-Pan clade. The resulting tree is close to that estimated by Sibley and Ahlquist (1984). The mixed model can be used to test a number of hypotheses about the existence of components of variance and the linearity of the relationship between branch length and expected distance. No evidence is found that there is a variance component for extract, or for the individual from which the extract was taken. A variance component for experiment does seem to exist, presumably arising as a result of error of measurement of the common standard from which all values in the same experiment were substracted. There is significant evidence that the relationship between total branch length between species and their expected distances is nonlinear, or else that the measurement error on larger distances is greater than on smaller ones. Allowing for the nonlinearity might cause one to infer the time of distant common ancestors as less remote than the measured hybridization values would imply if used directly.     

Phylogenetic relationships based on two mitochondrial genes and hybridization patterns in Anatidae

We produced DNA sequence data from two mitochondrial genes (cytochrome b and the NADH dehydrogenase subunit 2) to reconstruct the phylogenetic relationships among 121 species of the Anseriformes (waterfowls including ducks, geese, swans, the magpie goose and screamers). Phylogenetic analyses converged into a congruent topology and defined several well-supported clades. We calibrated a molecular clock and reconstructed ancestral biogeographical areas using Bayesian inference supporting an austral continental (Gondwanaland) origin of the waterfowls. Ducks, swans and geese might have diversified during the Miocene (23–5 Myr ago) reaching northern distributions in Holarctic and Afrotropical regions. The evolution of hybridization patterns in Anseriformes has been investigated using a cladistic analysis (morphology), which may underestimate or overestimate the phylogenetic divergence among species, or restricted only to ducks. Using a phylogenetic framework, genetic-based distances and a Bayesian time calibration, our data support the hypothesis based on immunological distances of slow rate of appearance of reproductive incompatibilities in waterfowls compared with other vertebrates and the view that these birds may be like frogs in having lost their interspecific hybridization potential more slowly than mammals.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.     

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5′-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.     

The use of <Emphasis Type="Italic">gyrB</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1.     

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation‐sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.     

Reticulate hybridization of <Emphasis Type="Italic">Alpinia</Emphasis> (Zingiberaceae) in Taiwan

Reticulate hybridization is a complicated and creative mechanism in plant evolution that can cause interference in phylogenetic studies. Based on observations of intermediate morphology, low pollen fertility, and overlapping distributions of putative parent species, Yang and Wang (Proceedings of the cross-strait symposium on floristic diversity and conservation. National Museum of Natural Science, Taichung, Taiwan, pp 183–197, 1998) first proposed reticulate hybridization of Alpinia in Taiwan. In the present study, molecular tools were used to explore relationships between four parental species and their homoploidy hybrids, and the impact of hybridization on phylogeny reconstruction. Based on DNA markers, maternal heritance of the chloroplast genome, and additivity of nuclear ribosomal internal transcribed spacer, the present results provide strong support for the hybridization hypothesis. Co-existence of parental ribotypes within hybrids revealed that these hybridization events were current, while reciprocal and introgressive hybridization were inferred from chloroplast DNA data. Furthermore, iterative hybridizations involving more than two parental species may occur in notorious hybrid zones. Ecological, phenological, and physiological evidence provides insight into why such frequent hybridization occurs in Taiwanese Alpinia. In the phylogenetic tree of the Zerumbet clade reconstructed in this study, the chloroplast sequences from one hybrid species were not grouped into a subclade, implying instability caused by hybridization. Failure to find morphological apomorphies and biogeographical patterns in this clade was likely partially due to reticulate hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

MOLECULAR EVOLUTIONARY DIVERGENCE AMONG NORTH AMERICAN CAVE CRICKETS. II. DNA-DNA HYBRIDIZATION

Single-copy DNA divergence among 23 populations of cave crickets belonging to two genera (Euhadenoecus and Hadenoecus) has been determined by DNA-DNA hybridization employing the TEACL method. These same populations have been studied for allozyme variation (Caccone and Sbordoni, 1987). In addition, a European relative (Dolichopoda laetitiae) has been included as an outgroup for rooting the phylogeny. One of the most remarkable findings is the large degree of DNA divergence among these species and populations. A ΔT_m of up to 5°C has been found between populations of the same species; even further divergence is indicated by a lowered normalized percentage of reassociation. A phylogeny was constructed and tested for synchrony of rates, i.e., a molecular clock. Statistically, we could not reject the clock hypothesis. Attempts to calibrate the clock led to the conclusion that these insects are among the fastest evolving (with respect to single-copy DNA) groups yet studied—at least as fast as Drosophila and sea urchins—where a ΔT_m of 1°C indicates 0.5 to 1.5 MY since the last common ancestor. In general, the phylogeny derived from the DNA data agrees with that derived from isozymes. Nei's D and ΔT_m are correlated; in this group a D of 0.1 corresponds to a ΔT_m of about 1.5°C. This indicates that, relative to total single-copy DNA, the protein-coding regions of the genome are slowly evolving.