Energy transfer cassettes for facile labeling of sequencing and PCR primers

Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the design, synthesis and evaluation of a four-color set of ET cassettes, fluorescent labeling reagents that can be quantitatively coupled to a thiol-activated target through a disulfide exchange reaction. The ET cassette consists of a sugar-phosphate spacer with a FAM donor at the 3'-end, an acceptor linked to a modified T-base at the 5'-end of the spacer and a mixed disulfide for coupling to a thiol at the 5'-end. The acceptor dye emission intensities of ET labeled primers produced in this manner are comparable to commercial ET primers. The utility of our ET cassette-labeled primers is demonstrated by performing four-color capillary electrophoresis sequencing with the M13(-21)forward primer and by generating and analyzing a set of single-nucleotide-polymorphism-specific PCR amplicons.     

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.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

Polymorphism analysis and gene detection by minisequencing on an array of gel-immobilized primers.

Two procedures, multibase and multiprimer, have been developed for single nucleotide extension of primers immobilized within polyacrylamide gel pads on a microchip. In the multibase assay, a primer is next to a polymorphic nucleotide; the nucleotide is identified by the specificity with which the primer incorporates fluorescently labeled dideoxyribo-nucleoside triphosphates. In the multiprimer assay, several primers containing different 3'-terminal nucleotides overlapping the variable nucleotide in DNA are used. The polymorphic nucleotide is identified according to the primer that is extended. The methods were compared for diagnosis of beta-thalassemia mutations. Isothermal amplification of the fluorescent signal was achieved by performing both assays at elevated temperature. Anthrax toxin genes were identified in a model system using this amplification method.     

Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension

Identification of nucleotides used for RNA chain initiation or for contacting DNA binding proteins is basic to our understanding of gene regulation. Normally, a radioactive primer is used to copy RNA or DNA. The polymerase extension stops at free ends of mRNA (as in promoter mapping) or at the position of template cleavage or modification (as in footprinting). The locations of these positions are then analyzed by polyacrylamide gel electrophoresis. These analyses have been improved using fluorescently labeled primers and commonly available DNA sequencing machines. The protocol, which we call fluorescently labeled oligonucleotide extension (FLOE), eliminates the need for handling radioactivity and polyacrylamide. The DNA sequencer delivers data as a "trace" that is ready for quantification, which eliminates the need to trace gels separately. The data analysis is further improved by new software, Scanalyze, which we present here. We demonstrate that by using promoter mapping and footprinting, FLOE shortens experimental time, extends the stretch of analyzable sequence, and simplifies quantification compared to radioactive methods and is as sensitive in terms of detecting templates.     

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.     

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase

We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA. The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel. Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms. Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity. These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers.     

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.     

A new approach to SNP genotyping with fluorescently labeled mononucleotides

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3′ end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.     

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.     

DEVELOPMENT OF HIGH THROUGHPUT SINGLE NUCLEOTIDE POLYMORPHISM GENOTYPING FOR THE ANALYSIS OF NODULARIA (CYANOBACTERIA) POPULATION GENETICS1

Any investigation of the genetic structure of populations involves the analysis of a large number of samples and therefore benefits from the use of rapid, inexpensive, and automated methods to assign individuals to a particular genotype. We developed a high throughput SNuPE (single nucleotide primer extension) assay to assess polymorphic base variations at three loci (PC‐IGS, rDNA‐ITS, and gvpA‐IGS) in the genome of the cyanobacterium Nodularia spumigena. Using a 96‐capilliary sequencer, analysis of thirteen 96‐well plates can be performed in the same electrophoretic run, allowing the throughput of 1248 samples in 75 min. The SNuPE method can be broken down into two stages. The first stage comprises amplification of a DNA fragment containing the polymorphic sequence and its purification from un‐incorporated PCR reagents. The second stage involves the annealing and extension of a third primer, the SNuPE primer, the 3′ end of which immediately precedes the variable site in the template. This primer is extended with a single fluorescently labeled dideoxy nucleotide by DNA polymerase, followed by characterization of the extended primers on a DNA sequencing instrument. The length of the extended primer is used to define the locus, and the incorporated fluorescent dideoxy nucleotide gives the identity of the nucleotide at each polymorphic site. Details of this technique and its application to study the genetic structure of Nodularia populations are described.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

Automated system for purification of dye-terminator sequencing products eliminates up-stream purification of templates.

The quality of sequencing results is to a large extent determined by the purity of the template and the purification of the sequencing products. Fragments that can act as unspecific primers and templates are removed before gel analysis, and the background of unspecific signals is highly reduced. Purification of the sequencing products is needed to remove salts, nucleotides, proteins and template DNA that can interfere with the gel separation. We have developed a product, DYNAPURE Dye Terminator Removal, that specifically isolates and purifies the sequencing products in 10 min. The method is based on biotinylated sequencing primers and super-paramagnetic streptavidin beads. A PCR product is sequenced using a biotinylated sequencing primer, and the sequencing products are then bound to streptavidin beads in a 5-min reaction. The bead-DNA complexes are magnetically separated from the rest of the solution, and the remaining buffer constituents are washed away with TE buffer or with 70% ethanol. The whole procedure can be automated on liquid-handling robots fitted with a magnet station. The method eliminates purification of templates before cycle sequencing.     

DNA sequencing using differential extension with nucleotide subsets (DENS).

Here we describe template directed enzymatic synthesis of unique primers, avoiding the chemical synthesis step in primer walking. We have termed this conceptually new technique DENS (differential extension with nucleotide subsets). DENS works by selectively extending a short primer, making it a long one at the intended site only. The procedure starts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs. The primer is extended by 6-9 bases or longer at the intended priming site, which is deliberately selected, (as is the two-dNTP set), to maximize the extension length. The subsequent termination reaction at 60-65 degrees C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. DENS allows the use of primers as long as 8mers (degenerate in two positions) which prime much more strongly than modular primers involving 5-7mers and which (unlike the latter) can be used with thermostable polymerases, thus allowing cycle-sequencing with dye-terminators compatible with Taq DNA polymerase, as well as making double-stranded DNA sequencing more robust.     

Colorimetric-detected DNA sequencing

A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described. The enzymatic incorporation of radioactive nucleotides commonly used for the band detection is replaced by biotin conjugated to the 5'-terminus of a synthetic oligonucleotide by chemical synthesis. The oligonucleotide so labeled is used as a primer for dideoxy DNA sequencing in a primer extension reaction. The products of the sequencing reactions are analyzed on a denaturing polyacrylamide gel using the direct blotting electrophoresis technique. This technique makes it possible to transfer the band pattern during the electrophoresis onto an immobilizing matrix, on which it is made visible by an enzymatic reaction in less than 3 h. This biotin-based detection method is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.