Non-radioactive automated sequencing of oligonucleotides by chemical degradation

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.     

Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers

Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.     

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.     

Human telomeric DNA sequences are a major target for the antitumour drug bleomycin

The DNA sequence specificity of the cancer chemotherapeutic agent bleomycin was examined in a human telomeric DNA sequence and compared with that of non-telomeric sequences. The target DNA sequence contained 17 repeats of the human telomeric sequence and other primary sites of bleomycin cleavage. The 377-base-pair target DNA was fluorescently labelled at the 3′-end, damaged with bleomycin and electrophoresed in an ABI 3730 automated capillary sequencer to determine the intensity and sequence specificity of bleomycin damage. The results revealed that bleomycin cleaved primarily at 5′-GT in the telomeric sequence 5′-GGGTTA. Maxam–Gilbert chemical sequencing reactions were utilised as DNA size markers to determine the precise sites of bleomycin cleavage. The telomeric region contained strong sites of bleomycin cleavage and constituted 57% of the 30 most intense bleomycin damage sites in the DNA sequence examined. These data indicated that telomeric DNA sequences are a major site for bleomycin damage.     

Automated Sanger DNA sequencing with one label in less than four lanes on gel

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.     

New Labelling Technology for Molecular Probes Applied to the Ligation Detection Reaction–Universal Array System

The ligation detection reaction (LDR) associated with universal arrays (UA) uses a fluorescently labelled probe (DP) and a Zip Code-extended probe to detect single nucleotide polymorphisms in DNA target sequences. When used for genotyping, the LDR-UA technique uses two DPs, each specific to an allele and labelled with a different fluorophore. The fluorescent signals are processed to calculate the genotype. The uneven decay of fluorophores due to ageing and freezing/thawing cycles and the consequent unequal fluoresce level can lead to erroneous genotype calls. To circumvent this problem, an indirect labelling strategy was developed based on the substitution of the fluorophore with allele-specific 22 bp universal labelling sequences (ULS). Labelling is achieved with fluorescently labelled oligos complementary to the ULS (cULS). The strategy improved the uniformity in probe labelling, and generated results comparable to those using direct-labelled probes, as shown by genotyping 22 polymorphic sites in 70 samples with both strategies. This method can be easily implemented in the routine screening with LDR-UA or other techniques. Moreover, the approach results in a significant cost reduction over traditional direct labelling, and offers the possibility to interchange fluorophores and to increase the fluorescent signal by using multiple-labelled cULS.     

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.     

Oligonucleotide facilitators enhance the catalytic activity of RNA-cleaving DNA enzymes.

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.     

Enzymatic multiplex DNA sequencing.

The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.     

Sequence-specific oxidative cleavage of DNA by a designed metalloprotein, Ni(II).GGH(Hin139-190).

A 55-residue protein containing the DNA binding domain of Hin recombinase, residues 139-190, with the tripeptide Gly-Gly-His (GGH) at the NH2 terminus was synthesized by stepwise solid-phase methods. GGH(Hin139-190) binds sequence specifically to DNA at four 13 base pair sites (termed hixL and secondary) and, in the presence of Ni(OAc)2 and monoperoxyphthalic acid, reacts predominantly at a single deoxyribose position on one strand of each binding site [Mack, D.P., & Dervan, P.B. (1990) J. Am. Chem. Soc. 112, 4604]. We find that, upon treatment with n-butylamine, the DNA termini at the cleavage site are 3'- and 5'-phosphate, consistent with oxidative degradation of the deoxyribose backbone. The nickel-mediated oxidation can be activated with peracid, iodosylbenzene, or hydrogen peroxide. The sequence specificity of the reaction is not dependent on oxidant, but the rates of cleavage differ, decreasing in the order peracid greater than iodosylbenzene greater than hydrogen peroxide. Optimal cleavage conditions for a 1 microM concentration of protein are 50 microM peracid, pH 8.0, and 1 equiv of Ni(OAc)2. The preferential cleavage at a single base pair position on one strand of the minor groove indicates a nondiffusible oxidizing species. A change of absolute configuration in the GGH metal binding domain from L-His to D-His [Ni(II).GG-(-D-)H(Hin139-190)] affords cleavage at similar base pair locations but opposite with regard to strand specificity.     

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.     

Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements

A recent publication reported on measurements of Exonuclease I activity using a real-time fluorescence method that measures the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. The observed fluorescence-intensity curves were interpreted as a sign of strong heterogeneity of the activity of Exonuclease I. Here, I propose a different model, which assumes that Exonuclease I activity is nucleotide-dependent, and that a fluorescent label bound to a nucleotide significantly slows its cleavage rate. The presented model fits the observed data equally well, but can be used to make specific predictions upon observable sequence dependence of measured fluorescence-intensity curves.     

A method for sequencing restriction fragments with dideoxynucleoside triphosphates.

A rapid enzymatic approach is described for the sequence analysis of a 5' terminally labelled restriction fragment. It involves limited nicking of the strands of the molecule throughout the sequence by pancreatic DNAase I. The 3' hydroxyl groups exposed by each nick are then used to prime chain extension by DNA polymerase I in four separate reactions. Each reaction uses one of the four chain terminating dideoxynucleoside triphosphates (ddNT-PSs), together with the four deoxynucleoside triphosphates (dNTPs). In a single reaction all the 3' ends are terminated in positions of the same base, which is different for each of the four reactions. When the products of these reactions are resolved by gel electrophoresis according to size, a sequence can be deduced from the pattern of radioactive bands. Sequences can be determined onwards from 10-20 residues from the 5' labelled end. The length of sequence which can be determined is only limited by the resolution of the gel.     

In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.

A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.     

Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.     

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.     

Single gene retrieval from thermally degraded DNA

To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent. By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval of single gene from ancient remains.     

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

LAGLIDADG homing endonucleases (LHEs) cleave 18–24bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.     

Adenine specific DNA chemical sequencing reaction.

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions.