Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides

We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.     

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.     

Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3'-OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3'-OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3'-OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3'-modified photocleavable fluorescent nucleotide, 3'-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3'-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS.     

Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.     

A photocleavable fluorescent nucleotide for DNA sequencing and analysis

DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3alpha,4alpha-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2'-deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.     

A simple and rapid method of direct sequencing using Dynabeads

Highly specific regions of genomic DNA can now be produced in large quantities by enzymatic amplification using the polymerase chain reaction (PCR) and a thermostable Taq polymerase (Saiki et al. 1988). This has encouraged the development of direct sequencing of the amplified DNA. Apart from the obvious advantage of the avoidance of cloning of DNA, direct sequencing enables both alleles to be analysed simultaneously. The latter is particularly advantageous in heterozygous states where the mutant allele cannot be differentiated from the normal. However, direct dideoxy sequencing of PCR-amplified DNA is still not consistently satisfactory. The difficulty arises from the nature of the PCR-amplified DNA which consists of short, linear double-stranded DNA. The two strands of the DNA can rapidly reanneal during DNA sequencing, blocking or displacing the sequencing primer from the template strand, thereby decreasing the amount of specific termination products formed in the sequencing reactions.     

Genomic footprinting in mammalian cells with ultraviolet light.

A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography. By using this method we demonstrate that it is possible to footprint with ultraviolet light, in intact monkey cells, regulatory protein--DNA interactions along a single copy of a simian virus 40 viral genome integrated into the monkey genome.     

Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.     

In Vitro Synthesis of DNA Complementary to Purified Rabbit Globin mRNA

Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3'-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.     

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.     

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.     

Synthesis by the DNA primase of Drosophila melanogaster of a primer with a unique chain length.

The primase associated with the DNA polymerase alpha from embryos of Drosophila melanogaster catalyzes the synthesis of ribo-oligonucleotide primers on single-stranded M13 DNA or polydeoxythymidylate templates, which can be elongated by DNA polymerase action [Conaway, R. C. & Lehman, I. R. (1982) Proc, Natl. Acad. Sci, USA 79, 2523--2527]. The primers synthesized in a coupled primase-DNA polymerase alpha reaction with an M13 DNA template are of a unique size (15 residues); those synthesized with poly(dT) range from 8 to 15 nucleotides. Primer synthesis is initiated at multiple but nonrandom sites. Like the DNA primase of Escherichia coli and the comparable activity in intact nuclei of polyoma-infected mouse cells, the DNA primase of D. melanogaster can substitute deoxynucleotides for ribonucleotides during primer synthesis.     

General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples.

A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.     

Elongation of primed DNA templates by eukaryotic DNA polymerases.

The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template.     

DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.     

Coupling of replication to transcription in vitro

In a coupled system consisting of RNA polymerase and DNA polymerase I of Escherichia coli, the four deoxyribo- and the four ribonucleoside triphosphates, and DNA of bacteriophage f1 as template, DNA synthesis depends on the concomitant synthesis of RNA. Over a wide range of concentrations of the two polymerases, RNA synthesis was unaffected by the simultaneous synthesis of DNA, whereas the rate of DNA synthesis depended on the level of RNA synthesis. In the coupled reaction, RNA synthesis starts immediately at a high rate, which subsequently decreases, whereas DNA synthesis starts after a lag and its rate increases as the reaction proceeds. Upon addition of rifampicin, the rate of RNA synthesis falls abruptly, while that of DNA declines only gradually. The base composition of the DNA synthesized in the coupled reaction is complementary to that of f1 DNA template. It is suggested that the RNA synthesized by the RNA polymerase serves as a primer rather than as a template for the DNA polymerase.