Four color fret dye nucleotide terminators for DNA sequencing

The synthesis of four color set of energy transfer-dye terminators (8a-8d) starting from p-iodo-beta-phenylalanine was accomplished and their utility in the sequencing reactions has been evaluated.     

Economical sequencing of DNA with terminators

We describe several improvements of chain-termination DNA sequencing procedure of Sanger et al. For template preparation we use 0.3 ml cultures of M13 clones, grown in standard 1,5 ml polypropylene tubes. The sequencing experiment differs from the previously described by the use of deoxyNTP, labelled with phosphorus-33 (a low energy isotope with a half-life of 25 days, commercially produced in the USSR), and by a "quasi-end labelling" reaction, preceding the DNA synthesis in the presence of dideoxyNTPs. The combination of the phosphorus-33 and the quasi-end labelling produces very sharp sequencing ladders, that equal or exceed in quality those obtained with sulphur-35, and only an overnight exposure with a conventional X-ray film is required. The use of plastic tubes for bacterial growth and the 60-well microchambers for carrying out sequencing reactions results in substantial saving of time and cost in routine "middle scale" sequencing (both types of plasticware are produced in the USSR).     

Fluorescence resonance energy transfer dye nucleotide terminators: a new synthetic approach for high-throughout DNA sequencing

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.     

Two-step cycle sequencing improves base ambiguities and signal dropouts in DNA sequencing reactions using energy-transfer-based fluorescent Dye terminators

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods plays an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While dideoxy-terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity from that point on in the sequence. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrate that combining the annealing step with the extension step at one temperature (60°C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used. This modification significantly increases the number of accurately read bases in DNA sequences.     

New dye-labeled terminators for improved DNA sequencing patterns.

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.     

A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.     

Fluorescently labeled model DNA sequences for exonucleolytic sequencing

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper F?ldes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.     

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Automated sequencing and mapping of cosmid DNA with fluorescently-labeled dideoxynucleotide terminators.

We have established a method for directly sequencing cosmid DNA on an automated DNA sequencer. The major advantage of this method is that only small amounts of cosmid template DNA are needed for the sequencing reactions.     

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.     

Optimizing the cationic conjugated polymer-sensitized fluorescent signal of dye labeled oligonucleotide for biosensor applications

Methods for real time, highly selective and sensitive polynucleotide detection are of vast scientific and economic importance. Fluorescence resonance energy transfer (FRET)-based assays which take advantage of the collective response of water-soluble conjugated polymers (CPs) and the self-assembly characteristic of aqueous polyelectrolytes have been widely used for the detection of DNA, RNA, protein and small molecules. The detection sensitivity of CP-based biosensor is dependent on the signal amplification of dye emission upon excitation of CP relative to that upon direct excitation of the dye. Using cationic polyfluorene derivatives and chromophore (fluorescein or Texas Red) labeled single-stranded DNA molecules (ssDNA-C*) as donor/acceptor pairs, we show that in addition to the spectral overlap, orientation and distance between the donor and the acceptor, the energy levels and fluorescence quenching of the donor/acceptor within the polymer/DNA-C* complexes are also important factors that affect the signal output of dye emission.     

Magnetic bead purification of labeled DNA fragments for high-throughput capillary electrophoresis sequencing

We have developed an automated purification method for dye-terminator-based DNA sequencing products using a magnetic bead approach. This 384-well protocol generates sequence fragments that are essentially free of template DNA, salt, and excess dye-terminator products. In comparison with traditional ethanol precipitation protocols, this method uses no centrifugation, is rapid, completely automated, and increases the phred-20 read length by an average of 40 bases. To date, we have processed over 4 million samples with 94% averaging 641 phred-20 bases on the MegaBACE 1000 and 4000 and the ABI PRISM 3700 capillary instruments.     

A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.

We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases.     

Bis-pyrene labeled DNA aptamer as an intelligent fluorescent biosensor

The site-directed incorporation of bis-pyrenyl fluorophore into anti-ATP DNA aptamer results in a creation of an intelligent fluorescent sensor with high signal intensity and specificity for detecting the target ligand in a homogeneous system.     

A simple system of cloning in phage M13 and DNA sequencing with terminators

A compilation of techniques for DNA cloning in filamentous phage M13 based vectors for a novice in cloning is presented. It does not require either specialized microbiological facilities, or any specific knowledge in Escherichia coli genetics. The cloning strategy uses only blunt-end ligation into a vector that has been prepared once for several hundred experiments. The first part describes the isolation, preparation and checking of a blunt-ended M13 vector (with M13 mp series vectors as an example), and also the isolation of clonable fragments, transformation of competent cells and preliminary analysis of recombinants. The second part describes procedures and equipment, which enable to sequence recombinant M13 clones by the chain termination procedure of Sanger et al. It includes simplified procedures for the preparation of sequencing gels, and the rules of interpretation of the sequencing ladders. Reference material is added, which includes trouble-shooting guide, E. coli K12 strain list and polylinker sequences for use of mp-series vectors as well as a fully documented cloning and sequencing experiment.     

Negatively charged, dye labeled-dideoxynucleotides for "direct-load" DNA sequencing

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a "direct-load" DNA sequencing, obviating the laborious and time consuming post-reaction work-up.