Dideoxy DNA sequencing with end-labeled oligonucleotide primers

End-labeled oligonucleotide primers may be used effectively as the source of radiolabel in DNA sequencing by the dideoxy method. The approach is demonstrated with various end-labeled oligonucleotides, including a commercially prepared universal primer and a mixed-sequence probe. Single-stranded (M13) or denatured double-stranded template may be used. The end-labeled primers and their extension products may be stored for weeks. The method is useful for identification of clones isolated by oligonucleotide hybridization and it provides a convenient, economical alternative to the use of alpha-labeled deoxynucleoside triphosphate.     

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n 1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.     

New reagents and solid support for automated oligonucleotide synthesis

The optimal system for the rapid, efficient, convenient, and economical synthesis and purification of synthetic oligonucleotides has been advancing. By recognizing the very rapid reaction kinetics and taking advantage of an efficient, low volume delivery system, cycle times have decreased to about 5.5 minutes, without compromising synthesis performance. A new set of base protecting groups for cyanoethylphosphoramidite nucleoside monomers have been developed, which decreases the post-synthesis time requirements. A particular form of polystyrene has also been developed as a solid support for automated oligonucleotide synthesis. Typical sequencing or PCR primers (20mers) now require less than 2 hours for synthesis and 2 hours for cleavage and deprotection.     

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal extended (A, T, C or G) oligonucleotides, identical to the RAPD primer but for the single nucleotide extension. Strand-specific DNA sequence could be independently read from each of the RAPD fragments without recourse to strand separation or fragment cloning. Informative RAPD fragments could be readily converted into mapped STS or SCAR loci using this technology. The 3'-extended primers may also be used to amplify independent genomic RAPD markers.     

Fluorescent dye phosphoramidite labelling of oligonucleotides.

A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.     

Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

The use of tailed octamer primers for cycle sequencing.

Studies have been carried out on the use of octamer oligonucleotides tailed with different base analogues as primers in cycle sequencing reactions. 5-Nitroindole tails improved the performance as primers of a number of octamers. A tail length of three or four 5-nitroindole residues significantly increased the sequencing signal intensity for almost all primers. The use of incomplete libraries of tailed octamer primers for primer walking is discussed.     

Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR.

An assay based on the PCR has been developed to facilitate detection and identification of Bacillus cereus in foods. Three primers for the PCR have been designed within the sequence for cereolysin AB, a cytolytic determinant that encodes lecithin-hydrolyzing and hemolytic activities of B. cereus. With the PCR and hybridization, the specificity of the primers was tested with 39 isolates of the B. cereus group, with 17 other Bacillus spp., and with 21 non-Bacillus strains. Results demonstrate a high specificity of the three oligonucleotides for isolates of the B. cereus group. With a combined PCR-hybridization assay, the detection limit for B. cereus in artificially contaminated milk was 1 CFU/ml of milk.     

A computer program for selection of oligonucleotide primers for polymerase chain reactions.

We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.     

Enzymatic synthesis of oligonucleotides of defined sequence. Addition of short blocks of nucleotide residues to oligonucleotide primers.

Polynucleotide phosphorylase from Escherichia coli can be used to catalyse the addition of short tracts of deoxyadenylate residues to the 3'-termini of deoxyribooligonucleotides of the type pdAn-dN (where dN = dC, dT or dG) using dADP as donor. Similarly, the enzyme can also be used to catalyse the addition of short tracts of adenylate residues to the 3'-termini of ribooligonucleotides of the type An-N (where N = C, U or G) using ADP as donor. In the ribooligonucleotide series, phosphorolytic cleavage of the primer oligonucleotides is significant and results in the concommitant production of oligoadenylates lacking the N residue. Oligomers of the same length, with and without the residue N, were readily separated by thermal elution from cellulose-pdT9 columns. This latter procedure therefore provides a simple method for purification of the oligoadenylates containing an internal base substitution and it also provides a convenient assay for oligonucleotide phosphorolysis.     

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.