Stacking Interactions in RNA and DNA: Roll-Slide Energy Hyperspace for Ten Unique Dinucleotide Steps

Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by B97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. (c) 2014 Wiley Periodicals, Inc. Biopolymers 103: 134-147, 2015.

Genetic diversity and proviral DNA load in different neural compartments of HIV-1 subtype C infection

In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.

Snaps and mends: DNA breaks and chromosomal translocations

Integrity in entirety is the preferred state of any organism. The temporal and spatial integrity of the genome ensures continued survival of a cell. DNA breakage is the first step towards creation of chromosomal translocations. In this review, we highlight the factors contributing towards the breakage of chromosomal DNA. It has been well-established that the structure and sequence of DNA play a critical role in selective fragility of the genome. Several non-B-DNA structures such as Z-DNA, cruciform DNA, G-quadruplexes, R loops and triplexes have been implicated in generation of genomic fragility leading to translocations. Similarly, specific sequences targeted by proteins such as Recombination Activating Genes and Activation Induced Cytidine Deaminase are involved in translocations. Processes that ensure the integrity of the genome through repair may lead to persistence of breakage and eventually translocations if their actions are anomalous. An insufficient supply of nucleotides and chromatin architecture may also play a critical role. This review focuses on a range of events with the potential to threaten the genomic integrity of a cell, leading to cancer.

DNA Elasticity from Short DNA to Nucleosomal DNA

Active biological processes like transcription, replication, recombination, DNA repair, and DNA packaging encounter bent DNA. Machineries associated with these processes interact with the DNA at short length (<100 base pair) scale. Thus, the study of elasticity of DNA at such length scale is very important. We use fully atomistic molecular dynamics (MD) simulations along with various theoretical methods to determine elastic properties of dsDNA of different lengths and base sequences. We also study DNA elasticity in nucleosome core particle (NCP) both in the presence and the absence of salt. We determine stretch modulus and persistence length of short dsDNA and nucleosomal DNA from contour length distribution and bend angle distribution, respectively. For short dsDNA, we find that stretch modulus increases with ionic strength while persistence length decreases. Calculated values of stretch modulus and persistence length for DNA are in quantitative agreement with available experimental data. The trend is opposite for NCP DNA. We find that the presence of histone core makes the DNA stiffer and thus making the persistence length 3-4 times higher than the bare DNA. Similarly, we also find an increase in the stretch modulus for the NCP DNA. Our study for the first time reports the elastic properties of DNA when it is wrapped around the histone core in NCP. We further show that the WLC model is inadequate to describe DNA elasticity at short length scale. Our results provide a deeper understanding of DNA mechanics and the methods are applicable to most protein-DNA complexes.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.

The Foraging Ecology of the Mountain Long-Eared Bat Plecotus macrobullaris Revealed with DNA Mini-Barcodes

10 p.

Ancient DNA from Hunter-Gatherer and Farmer Groups from Northern Spain Supports a Random Dispersion Model for the Neolithic Expansion into Europe

10 p.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

[ENG]Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs

Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

Recognition of double helical DNA by purine oligonucleotides via triple helix formation

Oligonucleotide-directed triple helix formation is one of the most versatile methods for the sequence specific recognition of double helical DNA. Chapter 2 describes affinity cleaving experiments carried out to assess the recognition potential for purine-rich oligonucleotides via the formation of triple helices. Purine-rich oligodeoxyribonucleotides were shown to bind specifically to purine tracts of double helical DNA in the major groove antiparallel to the purine strand of the duplex. Specificity was derived from the formation of reverse Hoogsteen G•GC, A•AT and T•AT triplets and binding was limited to mostly purine tracts. This triple helical structure was stabilized by multivalent cations, destabilized by high concentrations of monovalent cations and was insensitive to pH. A single mismatched base triplet was shown to destabilize a 15 mer triple helix by 1.0 kcal/mole at 25°C. In addition, stability appeared to be correlated to the number of G•GC triplets formed in the triple helix. This structure provides an additional framework as a basis for the design of new sequence specific DNA binding molecules.

In work described in Chapter 3, the triplet specificities and required strand orientations of two classes of DNA triple helices were combined to target double helical sequences containing all four base pairs by alternate strand triple helix formation. This allowed for the use of oligonucleotides containing only natural 3'-5' phosphodiester linkages to simultaneously bind both strands of double helical DNA in the major groove. The stabilities and structures of these alternate strand triple helices depended on whether the binding site sequence was 5'-(purine)_m (pyrimidine)_n-3' or 5'- (pyrimidine)_m (purine)_n-3'.

In Chapter 4, the ability of oligonucleotide-cerium(III) chelates to direct the transesterfication of RNA was investigated. Procedures were developed for the modification of DNA and RNA oligonucleotides with a hexadentate Schiff-base macrocyclic cerium(III) complex. In addition, oligoribonucleotides modified by covalent attachment of the metal complex through two different linker structures were prepared. The ability of these structures to direct transesterification to specific RNA phosphodiesters was assessed by gel electrophoresis. No reproducible cleavage of the RNA strand consistent with transesterification could be detected in any of these experiments.

Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes

Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean nuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form independent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the Holliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

I. Studies on bacteriophage DNA's and minicircular DNA's in M. lysodeikticus and E. coli 15. II. Flow dichroism of DNA solutions

Part I

Chapter 1.....A physicochemical study of the DNA molecules from the three bacteriophages, N1, N5, and N6, which infect the bacterium, M. lysodeikticus, has been made. The molecular weights, as measured by both electron microscopy and sedimentation velocity, are 23 x 10⁶ for N5 DNA and 31 x 10⁶ for N1 and N6 DNA's. All three DNA's are capable of thermally reversible cyclization. N1 and N6 DNA's have identical or very similar base sequences as judged by membrane filter hybridization and by electron microscope heteroduplex studies. They have identical or similar cohesive ends. These results are in accord with the close biological relation between N1 and N6 phages. N5 DNA is not closely related to N1 or N6 DNA. The denaturation T_m of all three DNA's is the same and corresponds to a (GC) content of 70%. However, the buoyant densities in CsCl of Nl and N6 DNA's are lower than expected, corresponding to predicted GC contents of 64 and 67%. The buoyant densities in Cs₂SO₄ are also somewhat anomalous. The buoyant density anomalies are probably due to the presence of odd bases. However, direct base composition analysis of N1 DNA by anion exchange chromatography confirms a GC content of 70%, and, in the elution system used, no peaks due to odd bases are present.

Chapter 2.....A covalently closed circular DNA form has been observed as an intracellular form during both productive and abortive infection processes in M. lysodeikticus. This species has been isolated by the method of CsC1-ethidium bromide centrifugation and examined with an electron microscope.

Chapter 3.....A minute circular DNA has been discovered as a homogeneous population in M. lysodeikticus. Its length and molecular weight as determined by electron microscopy are 0.445 μ and 0.88 x 10⁶ daltons respectively. There is about one minicircle per bacterium.

Chapter 4.....Several strains of E. coli 15 harbor a prophage. Viral growth can be induced by exposing the host to mitomycin C or to uv irradiation. The coliphage 15 particles from E. coli 15 and E, coli 15 T^- appear as normal phage with head and tail structure; the particles from E. coli 15 TAU are tailless. The complete particles exert a colicinogenic activity on E.coli 15 and 15 T^-, the tailless particles do not. No host for a productive viral infection has been found and the phage may be defective. The properties of the DNA of the virus have been studied, mainly by electron microscopy. After induction but before lysis, a closed circular DNA with a contour length of about 11.9 μ is found in the bacterium; the mature phage DNA is a linear duplex and 7.5% longer than the intracellular circular form. This suggests the hypothesis that the mature phage DNA is terminally repetitious and circularly permuted. The hypothesis was confirmed by observing that denaturation and renaturation of the mature phage DNA produce circular duplexes with two single-stranded branches corresponding to the terminal repetition. The contour length of the mature phage DNA was measured relative to φX RFII DNA and λ DNA; the calculated molecular weight is 27 x 10⁶. The length of the single-stranded terminal repetition was compared to the length of φX 174 DNA under conditions where single-stranded DNA is seen in an extended form in electron micrographs. The length of the terminal repetition is found to be 7.4% of the length of the nonrepetitious part of the coliphage 15 DNA. The number of base pairs in the terminal repetition is variable in different molecules, with a fractional standard deviation of 0.18 of the average number in the terminal repetition. A new phenomenon termed "branch migration" has been discovered in renatured circular molecules; it results in forked branches, with two emerging single strands, at the position of the terminal repetition. The distribution of branch separations between the two terminal repetitions in the population of renatured circular molecules was studied. The observed distribution suggests that there is an excluded volume effect in the renaturation of a population of circularly permuted molecules such that strands with close beginning points preferentially renature with each other. This selective renaturation and the phenomenon of branch migration both affect the distribution of branch separations; the observed distribution does not contradict the hypothesis of a random distribution of beginning points around the chromosome.

Chapter 5....Some physicochemical studies on the minicircular DNA species in E. coli 15 (0.670 μ, 1.47 x 10⁶ daltons) have been made. Electron microscopic observations showed multimeric forms of the minicircle which amount to 5% of total DNA species and also showed presumably replicating forms of the minicircle. A renaturation kinetic study showed that the minicircle is a unique DNA species in its size and base sequence. A study on the minicircle replication has been made under condition in which host DNA synthesis is synchronized. Despite experimental uncertainties involved, it seems that the minicircle replication is random and the number of the minicircles increases continuously throughout a generation of the host, regardless of host DNA synchronization.

Part II

The flow dichroism of dilute DNA solutions (A₂₆₀≈0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5-160 sec.^-1 were studied. The DNA samples were whole, "half," and "quarter" molecules of T4 bacteriophage DNA, and linear and circular λb₂b₅c DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear A DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.

Differenzierung von Pangasius (Pangasius hypophthalmus) und Asiatischem Rotflossenwels (Hemibagrus wyckioides) durch Protein- und DNA-Analyse

In the last years farmed Pangasius (Tra-Pangasius, Pangasius hypophthalmus) from Vietnam has reached a considerable market share, whereas aquaculture of Asian Redtail Catfish (Hemibagrus wyckioides) is in its infancy. Recently it has been detected by food control authorities in Hamburg, that Pangasius fillets have been mislabelled and sold as fillets produced from Asian Redtail catfish. The necessity to improve the analytical methods for differentiation of Pangasius and Redtail Catfish prompted us to evaluate the suitability of isoelectric focusing (IEF) and DNA-analysis for identification of the two species. IEF of water soluble proteins was found to be a fast, reliable and economical method for differentiation of raw fillets of Pangasius and Redtail Catfish, as long as reference material is available. PCR-based DNA analysis was performed as follows: (i) amplification of a 464 bp segment of the cytochrome b gene; (ii) sequencing of the PCR product; (iii) comparison of the sequence with entries in GenBank using BLAST. The sequences of both species differed considerably, allowing the unequivocal differentiation between P. hypophthalmus and H. wyckioides. Kurzfassung Pangasius (Schlankwels, Tra-Pangasius, Pangasius hypophthalmus) hat sich innerhalb weniger Jahre zu einem bedeutenden Zuchtfisch entwickelt, während die Aquakultur des Asiatischen Rotflossenwelses (Hemibagrus wyckioides) in Vietnam noch in einem relativ kleinen Maßstab stattfindet. Kürzlich wurde von der Lebensmittelüberwachung in Hamburg nachgewiesen, dass im Handel erhältliche Filets mit der Deklaration „Rotflossenwels“ aus Pangasius hergestellt worden waren. Vor diesem Hintergrund wurden zwei Methoden auf ihre Eignung zur Differenzierung von Pangasius und Rotflossenwels geprüft. Es zeigte sich, dass sowohl die isoelektrische Fokussierung (IEF) wasserlöslicher Proteine als auch die PCR-basierte DNA-Analyse zur Unterscheidung beider Arten gut geeignet ist. Die IEF stellt eine schnelle und kostengünstige Untersuchungsmethode dar, die allerdings Referenzmaterial benötigt. Mit Hilfe der PCR (Polymerase-Kettenreaktion) wurde ein Abschnitt des Cytochrom b-Gens vervielfältigt und sequenziert. Die Sequenzen von P. hypophthalmus und H. wyckioides wiesen beträchtliche Unterschiede auf. Es wird diskutiert, wie sich durch Vergleich dieser Sequenzen mit Einträgen in Gendatenbanken unbekannte Proben beider Arten sicher zuordnen lassen.