Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products.

A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles.     

Influence of the PCR artifacts on the genotyping efficiency by the microsatellite loci using native polyacrylamide gel electrophoresis

Formation of different types of artifacts during amplification has been studied using different classes of molecular-genetic markers (Indel and SSR). It has been shown that DNA heteroduplexes are formed during amplification of heterozygous samples as fragments of both target genes and microsatellite loci. Electrophoresis of the amplification products of homozygous samples by microsatellite loci in native polyacrylamide gel has revealed specific additional fragments that do not belong to the class of heteroduplex DNA. It has been supposed that the additional fragments belong to a special type of homoduplex DNA—nonlinear homoduplexes. The analysis has revealed that the formation of nonlinear homoduplex DNA takes place on the 20–25 cycle of the PCR at the amplification of both experimental samples and individual DNA fragments cut from the gel.     

Synthesis and in vitro characterization of a dendrimer-MORF conjugate for amplification pretargeting

Amplification pretargeting can play an important role in molecular imaging by significantly increasing the accumulation of signal in target tissues. Multiple-step amplification pretargeting offers the potential to greatly improve target localization of effector molecules through the intermediate use of polymers conjugated with multiple copies of complementary oligomers. In this study, PAMAM dendrimer generation 3 (G3) was conjugated with multiple copies of a phosphorodiamidate morpholino (MORF) oligomer. Characterization of the conjugate by native-PAGE and SE-HPLC demonstrated that the conjugation was successful. The average numbers of MORF groups in the G3-MORF conjugate, both attached and accessible to the (99m)Tc labeled complementary MORF (cMORF), were determined. The antitumor antibody CC49 was conjugated with both MORF and cMORF (collectively (c)MORF) at an average of about one group per molecule. Nine of the 32 carboxyl groups of the dendrimer were modified with MORF, of which 90% were accessible in solution to (99m)Tc-cMORF. After purification, the G3-MORF was radiolabeled with tracer (99m)Tc-labeled cMORF (i.e., G3-MORF/(99m)Tc-cMORF) and added to the antibody CC49 previously conjugated with cMORF (i.e., CC49-cMORF/G3-MORF/(99m)Tc-cMORF), the complex demonstrated a single peak on SE-HPLC as evidence of complete hybridization between G3-MORF/(99m)Tc-cMORF and CC49-cMORF. The CC49-(c)MORF were bound to both Protein G and Protein L coated plates, and G3-MORF was added to hybridize with CC49-cMORF before the (99m)Tc-cMORF was added to test amplification pretargeting. In comparison to conventional pretargeting without the G3-MORF, the signal was amplified about 6 and 14 times, respectively, showing that the G3-MORF participated in amplifying the signal. Further amplification studies using the CC49-(c)MORF for LS174T tumor cells in tissue culture also demonstrated clear evidence of signal amplification.     

DNA homoduplexes containing no pyrimidine nucleotide

We show using polyacrylamide gel electrophoresis that guanine+adenine repeat strands of DNA associate into homoduplexes at neutral pH and moderate ionic strength. The homoduplexes melt in a cooperative way like the Watson-Crick duplex, although they contain no Watson-Crick base pair. Guanine is absolutely needed for the homoduplex formation and the homoduplex stability increases with the guanine content of the repeat. The present results have implications for the nature of the first replicators, as well as regarding forces stabilizing the duplexes of DNA.     

99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

PurposeRadiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.Methods and resultsIdentical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were ^99mTc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of ^99mTc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8 pmol, respectively, per 10¹⁰ cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90 min for both ^99mTc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of ^99mTc-study MORF was significantly higher (p = 0.009) than that of the control in the thigh with sterile inflammation.ConclusionA ^99mTc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.     

Thermodynamics of sequence-specific binding of PNA to DNA

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6.5+/-0.3 kJ mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M(-1) bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T(m)) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M(-1) per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.     

Genetic Relatedness of Type 1 and Type 2 Herpes Simplex Viruses

The extent of homology between herpes simplex virus(1) and(2) (HSV-1 and HSV-2) deoxyribonucleic acid (DNA) was measured in two ways: (i) by determination of the relative rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA immobilized on filters and (ii) by determination of the rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA in solution. Approximately 40% of HSV-1 and HSV-2 DNA is homologous at hybridization temperatures 25 C below the melting temperature (T(m)) of HSV DNA (liquid-filter annealing). Lowering the temperature to 34 C below the T(m) increased the extent of homology to 46% (liquid annealing). The extent of base-pairing in HSV-1-HSV-2 heteroduplex DNA was determined by thermal chromatography on hydroxyapatite. Heteroduplexes of HSV-1 and HSV-2 DNA eluted in a single peak whose midpoint (Te(50)) was 10 C below that of the homoduplex. Conspicuously absent were heteroduplexes that eluted at more than 15 C below the Te(50) of the homoduplex. The data indicate the existence of a variable region of DNA (54%) with very little, if any, homology and an invariable region (46%) with relatively good (85%) matching of base pairs.     

Sequence Homology between Chloroplast DNAs from Several Higher Plants

An estimate has been made of the amount of sequence homology present in the chloroplast DNA (ctDNA) of several higher plants by the technique of DNA-DNA hybridization. Approximately 85% of tomato, 60% of spinach, 45% of kale, and 15% of barley ctDNA sequences were found to hybridize with tobacco ctDNA under conditions in which maximum hybridization in homologous reactions reached 85%. All heteroduplexes contained significant amounts of sequence mismatch as indicated by a 3 to 9 C decrease in melting temperature as compared to homoduplex.     

Infectivity of Lambda Heteroduplex Deoxyribonucleic Acid Molecules

Heteroduplex deoxyribonucleic acid (DNA) molecules were formed in vitro by denaturing and renaturing a mixture of DNAs from a variety of lambda strains. The infectivity of these heteroduplexes was studied by using host-controlled modification and restriction to prevent infection by contaminating parental homoduplexes. Either strand was able to protect against degradation by restriction nucleases in vivo. A large proportion of progeny phage, which had been produced after infection with heteroduplexes containing noncomplementary base pairs at multiple loci, retained the genotype of one of the parental homoduplexes. The results indicate that conversion of heteroduplexes to homoduplexes in vivo by a DNA repair mechanism does not occur frequently. Molecules heterozygous for the c17 or vir operator mutations were not infectious; it is suggested that these mutations involve multiple base pairs.     

Cytosine residues influence kidney accumulations of 99mTc-labeled morpholino oligomers

Watson-Crick pairing between complementary oligomers is proving to be an effective means for rapidly directing radioisotopes specifically to the exterior surface of cancer cells in vivo. In such pretargeting applications, it is highly desirable that the excess of isotopically labeled oligomers, which do not bind to the cancer cells, be rapidly cleared from the body. In this context, understanding the influence of chain length and base sequence of the radiolabeled oligomers is critical. We had earlier determined that the kidneys are the principal targets of short-chain radiolabeled morpholino oligomers (MORFs). To explain these observations, MORFs consisting of uniform cytosines (Cs), uniform thymines (Ts), uniform adenines (As), and uniform AAG repeat were labeled with technetium-99m (99mTc) and studied in normal mice. In a limited investigation of the influence of oligomer backbone, a 20-mer MORF (MORF20) with a base sequence rich in Cs was compared with a phosphoromonothioate DNA (S-DNA20) of the same sequence. The in vivo behavior of the labeled MORFs was nearly identical in all organs, with the exception of kidneys. The kidney accumulations were about 25- to 80-fold higher for the uniform Cs relative to the other three uniform MORFs at 3 hours. The S-DNA20 rich in Cs showed only modest kidney accumulations compared with the equivalent MORF20, presumably because of preferential clearance of the S-DNA20 through the liver. Urine analysis showed no evidence of intact labeled S-DNA20 in contrast to fully intact labeled MORF20. We conclude that the high kidney levels observed by us previously for MORFs are most likely due largely to the C residues in the base sequence. In the case of S-DNAs, this phenomenon is partly disguised by the increased hepatic excretion and degradation. These results show that the base sequences of MORFs, and probably other oligomers as well, are an important determinant of biodistribution.     

Conformational properties of DNA strands containing guanine-adenine and thymine-adenine repeats.

CD spectroscopy and PAGE were used to cooperatively analyze melting conformers of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10 formed a homoduplex in neutral solutions containing physiological concentrations of salts and this homoduplex was not destabilized even in the terminal (GA)3 hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this oligonucleotide preserved the conformation adopted by (TA)10. This observation demonstrates that homoduplexes of alternating GA and TA sequences can co-exist in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them reversibly. The concentration of salt controlled the conformational switching. Hence, guanine and thymine share significant properties regarding complementarity to adenine, while the TA and GA sequences can stack in at least two mutually compatible ways within the DNA duplexes analyzed here. These properties extend our knowledge of non-canonical structures of DNA.     

Heteroduplex mobility assay as a tool for predicting phylogenetic affiliation of environmental ribosomal RNA clones

Heteroduplex mobility assay (HMA) of partial 16S rRNA gene fragments was tested as a tool for predicting bacterial phylogenetic relationships. Approximately 400-bp fragments were amplified from a selection of cloned environmental DNAs representing a range of sequence identities and phylogenetic relationships. Heteroduplexes between pairs of sequences were formed by mixing equal amounts of PCR products, denaturing and annealing. Annealed mixes were separated on 8% polyacrylamide gels and silver stained. Heteroduplexes were readily distinguished from reannealed homoduplex and unannealed fragments in all sequences where percentage identity was less than 95%. The heteroduplexes showed retarded electrophoretic migration with respect to homoduplexes. The relative retardation was strongly correlated to the percentage sequence identity between the two strands. The HMA is a useful tool for screening environmental clone libraries to systematically select clones representative of the phylogenetic diversity within the sample, or to selectively retrieve members of a particular phylogenetic group for more detailed study.     

Cooperative lectin recognition of periodical glycoclusters along DNA duplexes: alternate hybridization and full hybridization

We describe herein the construction of periodically, spatially controlled glycoclusters along DNA duplexes and their cooperative lectin recognition. Site-specifically alpha-mannosylated oligodeoxynucleotide 20-mer (Man-ODN20) was synthesized via the phosphoramidite solid-phase synthesis. Alternate hybridization of the Man-ODN20 with the half-sliding complementary ODN 20-mer (hscODN20) gave an alternately prolonged Man-cluster Man-ODN20/hscODN20. The binding of the Man-cluster to FITC-labeled ConA lectin showed sigmoidal fluorescence dependency on the concentration of Man-ODN, indicating that some mannose residues along the repeating DNA duplex were cooperatively bound to ConA (apparent affinity constant: K(af)=2.4 x 10(4)M(-1) and Hill coefficient: n=3.5). The duplex of Man-ODN20 with full complementary ODN 20-mer (fcODN20) was little bound to ConA. The binding behavior of Man-ODN20/hscODN20 is compared with that of the alternately prolonged Gal-cluster Gal-ODN20/hscODN20 previously reported. Duplexes 20-mer, 40-mer, and 60-mer presenting one, two, and three periodic galactoses were also prepared by full hybridization of 20-mer beta-galactosylated oligodeoxynucleotide (Gal-ODN20) with the periodically repeating full complementary 20-mer, 40-mer, and 60-mer ODNs. RCA(120) lectin was found to little bind the 20-mer and 40-mer duplexes and to bind weakly and non-cooperatively the 60-mer duplex (K(af)=1.1 x 10(4)M(-1)). The cooperative lectin recognition of these glycoclusters in relation with the degree of association (DA) of ODN and the numbers of glycosides along the DNA duplex is discussed.     

Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro.

The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60 degrees between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bow-shaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of chi-form Holliday junctions.     

Genetic relatedness of the genomes of equine herpesvirus types 1, 2, and 3.

Genomic DNAs of equine herpesvirus type 1 (EHV-1), EHV-2 (equine cytomegalovirus), and EHV-3 were examined by reassociation kinetic and thermal denaturation analyses to determine the extent and degree of homology among the three viral DNAs. Results of reassociation analyses indicated a limited homology among the three EHV genomes. Homologous DNA sequences equivalent to 1.8 to 3.7 megadaltons between EHV-1 and equine cytomegalovirus, 7.6 to 8.2 megadaltons between EHV-1 and EHV-3, and 1.3 to 1.9 megadaltons between equine cytomegalovirus and EHV-3 were detected. Examination by thermal denaturation of the DNA homoduplexes and heteroduplexes formed during reassociation revealed a high degree of base pairing within the duplexes, suggesting that closely related sequences may be conserved among the genomes of EHV.     

Use of phage display for detecting single-nucleotide differences in genes]

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.