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.     

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.     

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.     

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.     

Novel resequencing chip customized to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis

BACKGROUND & AIMS: Inherited syndromes of intrahepatic cholestasis commonly result from mutations in the genes SERPINA1 (alpha(1)-antitrypsin deficiency), JAG1 (Alagille syndrome), ATP8B1 (progressive familial intrahepatic cholestasis type 1 [PFIC1]), ABCB11 (PFIC2), and ABCB4 (PFIC3). However, the large gene sizes and lack of mutational hotspots make it difficult to survey for disease-causing mutations in clinical practice. Here, we aimed to develop a technological tool that reads out the nucleotide sequence of these genes rapidly and accurately. METHODS: 25-mer nucleotide probes were designed to identify each base for all exons, 10 bases of intronic sequence bordering exons, 280-500 bases upstream from the first exon for each gene, and 350 bases of the second intron of the JAG1 gene and tiled using the Affymetrix resequencing platform. We then developed high-fidelity polymerase chain reactions to produce amplicons using 1 mL of blood from each subject; amplicons were hybridized to the chip, and nucleotide calls were validated by standard capillary sequencing methods. RESULTS: Hybridization of amplicons with the chip produced a high nucleotide sequence readout for all 5 genes in a single assay, with an automated call rate of 93.5% (range, 90.3%-95.7%). The accuracy of nucleotide calls was 99.99% when compared with capillary sequencing. Testing the chip on subjects with cholestatic syndromes identified disease-causing mutations in SERPINA1, JAG1, ATP8B1, ABCB11, or ABCB4. CONCLUSIONS: The resequencing chip efficiently reads SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 with a high call rate and accuracy in one assay and identifies disease-causing mutations.     

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor.

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (microCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q > or = 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The microCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of microCAE technology should make it the next generation, high performance DNA sequencing platform.     

Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing

An efficient, nanoliter-scale microfabricated bioprocessor integrating all three Sanger sequencing steps, thermal cycling, sample purification, and capillary electrophoresis, has been developed and evaluated. Hybrid glass-polydimethylsiloxane (PDMS) wafer-scale construction is used to combine 250-nl reactors, affinity-capture purification chambers, high-performance capillary electrophoresis channels, and pneumatic valves and pumps onto a single microfabricated device. Lab-on-a-chip-level integration enables complete Sanger sequencing from only 1 fmol of DNA template. Up to 556 continuous bases were sequenced with 99% accuracy, demonstrating read lengths required for de novo sequencing of human and other complex genomes. The performance of this miniaturized DNA sequencer provides a benchmark for predicting the ultimate cost and efficiency limits of Sanger sequencing.     

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.     

应用基因芯片方法检测结核分枝杆菌利福平和异烟肼的耐药性

目的 应用基因芯片方法检测结核分枝杆菌(Mycobacterium tuberculosis,Mtb)对利福平和异烟肼的耐受性,评价其临床应用价值。方法 应用聚合酶链反应（polymerase chain reaction,PCR）扩增-基因芯片杂交的方法检测经常规药敏实验证实的30株Mtb利福平和异烟肼敏感株和50株耐利福平和异烟肼分离株的rpoB基因及katG和inhA基因突变,同时以PCR-直接测序法为对照。结果 应用PCR-基因芯片与基因测序方法检测30株Mtb利福平敏感株rpoB基因和异烟肼敏感株katG基因和inhA基因均为野生型。50株Mtb利福平耐药株中,PCR-基因芯片与基因测序分析3株rpoB基因均为野生型,41株均为突变型;6株PCR-基因芯片与基因测序结果不一致。50株Mtb异烟肼耐药株中,PCR-基因芯片与基因测序分析16株katG基因和30株inhA基因均为野生型,31株katG基因均为315位密码子突变,7株inhA基因均为15位突变型,其中2株为katG和inhA双重突变;3株katG和13株inhA PCR-基因芯片与基因测序结果不一致。结论 应用PCR-基因芯片方法可快速、有效地检出大多数Mtb耐多药分离株,指导临床用药。     

Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.     

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.     

呼吸道感染致病菌16SrRNA基因V3、V6可变区序列分析及在液态芯片中应用的探索性研究

目的探讨呼吸道感染致病菌16SrRNA基因V3、V6可变区在液态芯片系统的应用价值。方法通过GenBank公布的呼吸道感染致病菌16SrRNA基因序列,设计并合成探针及引物,建立液态芯片检测系统,检测福建省立医院收集的52例呼吸道感染痰标本抽提的DNA,与测序结果比对,分析其灵敏度和特异度。结果呼吸道感染致病菌16SrRNA基因V3、V6区均存在差异,应用液态芯片可在3．5h得到检测结果,铜绿假单胞菌16SrRNAV6探针,可检测浓度大于10^2／μl标本,检测灵敏度为100．0％,特异度为100．0％;金黄色葡萄球菌16SrRNAV3探针灵敏度为66．7％,特异度为98．0％;金黄色葡萄球菌16SrRNAV6探针灵敏度为0．0％。结论呼吸道感染致病菌16SrRNA基因V3、V6可变区差异可应用于病原学诊断,但不适合检测所有的呼吸道感染致病菌。     

An arrayable flow-through microcentrifuge for high-throughput instrumentation

A compact, flow-through centrifugation system has been developed specifically for high-throughput centrifugation of large numbers of samples. The instrument is based on multiple high-speed rotors that also serve as sample holders. The small size of the rotors allows them to be arrayed in a standard 96-well microtiter plate spacing, making this device ideal for highly parallel automated instrumentation. Though initially designed for cell separation in DNA sequencing protocols, the flow-through centrifuge can be used to replace conventional centrifugation in most processes involving small samples. Techniques for recovery of both the supernatant and the pellet have been developed, as well as techniques for sample mixing, and cleaning of the reusable rotors. This paper discusses the design and performance of the flow-through centrifuge applied to cell separation and resuspension and to DNA purification and concentration.     

Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.

A nine-base DNA oligomer [d(GTATCTAAT)] was used to probe the accessibility and function of bases in the region 787-795 of Escherichia coli 16S rRNA. Hybridization of the cDNA [d(GTATCTAAT)] to 16S rRNA in situ was carried out by binding the probe to intact 30S ribosomal subunits. Nitrocellulose filter binding showed that cDNA hybridization saturated with increasing probe concentration, suggesting that the probe was binding to a discrete site or sites. RNase H digestion of the rRNA under the DNA . rRNA hybrid and sequencing of the resultant RNA fragments verified that the cDNA probe bound specifically to the 787-795 region. Hybridization experiments using the cDNA probe showed that bases in the 787-795 region of 16S rRNA are exposed on the surface of 30S subunits. The functional role of bases 787-795 was then tested by assaying various ribosomal activities with the cDNA in place. Results of these functional assays demonstrate that this 16S rRNA region is directly involved in the association of 30S and 50S subunits.     

Molecular Medicine and Molecular Pathology for Hematologists: II. Lets go Techno; Principles of the Molecular Technologies and their Applications in Haematology

Molecular techniques have a key role to play in laboratory and clinical haematology. Restriction enzymes allow nucleic acids to be reduced in size for subsequent analysis. In addition they allow selection of specific DNA or RNA sequences for cloning into bacterial plasmids. These plasmids are naturally occuring DNA molecules which reside in bacterial cells. They can be manipulated to act as vehicles or carriers for biologically and medically important genes, allowing the production of large amounts of cloned material for research purposes or to aid in the production of medically important recombinant molecules such as insulin. As acquired or inherited genetic changes are implicated in a wide range of haematological diseases, it is necessary to have highly specific and sensitive assays to detect these mutations. Most of these techniques rely on nucleic acid hybridisation, benefitting from the ability of DNA or RNA to bind tighly to complimentary bases in the nucleic acid structure. Production of artificial DNA molecules called probes permits nucleic acid hybridiation assays to be performed, using the techniques of southern blotting or dot blot analysis. In addition the base composition of any gene or region of DNA can be determined using DNA sequencing technology. The advent of the polymerase chain reaction (PCR) has revolutionised all aspects of medicine, but has particular relevance in haematology where easy access to biopsy material provides a wealth of material for analysis. PCR permits quick and reliable manipulation of sample material and its ability to be automated makes it an ideal tool for use in the haematology laboratory.     

From the Cover: Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as α-hemolysin (αHL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane β-barrel of an engineered αHL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.     

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.     

Mitochondrial DNA mutations in pancreatic cancer

Background Somatic mutations of mitochondrial DNA (mtDNA) are increasingly being recognized in many human cancers, but automated sequencing of 16.5 kb of DNA poses an onerous task. We have recently described an oligonucleotide microarray (MitoChip) for rapid and accurate sequencing of the entire mitochondrial genome (Zhou et al., J Mol Diagnostics, 8: 9_14, 2006), greatly facilitating the analysis of mtDNA mutations in cancer. In this report, we perform a comprehensive cataloging of somatic mutations in the mitochondrial genome of human pancreatic cancers using our novel array-based approach. Materials and Methods MitoChip analysis was performed on DNA isolated from 15 histologically confirmed resection specimens of pancreatic ductal adenocarcinomas. In all cases, matched nonneoplastic pancreatic tissue was obtained as germline control for mtDNA sequence. DNA was extracted from snap-frozen cryostat-embedded specimens and hybridized to the sequencing microarray after appropriate polymerase chain reaction amplification and labeling steps. The vast majority of somatic mutational analyses of mtDNA in human cancers utilize lymphocyte DNA as germline control, without excluding the potential for organ-specific polymorphisms. Therefore, we also examined a series of 15 paired samples of DNA obtained from nonneoplastic pancreata and corresponding EBV-immortalized lymphoblastoid cell lines to determine whether lymphocyte DNA provides an accurate surrogate for the mtDNA sequence of pancreatic tissue. Results We sequenced 497,070 base pairs of mtDNA in the 15 matched samples of pancreatic cancer and nonneoplastic pancreatic tissue, and 467,269 base pairs (94.0%) were assigned by the automated genotyping software. All 15 pancreatic cancers demonstrated at least one somatic mtDNA mutation compared to the control germline DNA with a range of 1–14 alterations. Of the 71 somatic mutations observed in our series, 18 were nonsynonymous coding region alterations (i.e., resulting in an amino acid change), 22 were synonymous coding region alterations, and 31 involved noncoding mtDNA segments (including ribosomal and transfer RNAs). Overall, somatic mutations in the coding region most commonly involved the ND4, COI, and CYTB genes; of note, an A–G transition at nucleotide position 841 in the 12sRNA was observed in three independent samples. In the paired analysis of nonneoplastic pancreata and lymphoblastoid cell line DNA, 14 nucleotide discrepancies were observed out of 226,876 nucleotide sequences (a concordance rate of 99.99%), with 9 samples demonstrating a perfect match across all bases assigned. Conclusions Our findings confirm that somatic mtDNA mutations are common in pancreatic cancers, and therefore, have the potential to be a clinically useful biomarker for early detection. Further, our studies confirm that lymphocyte DNA is an excellent, albeit not perfect, surrogate for nonneoplastic pancreatic tissues in terms of being utilized as a germline control. Finally, our report confirms the utility of a high-throughput array-based platform for mtDNA mutational analyses of human cancers.     

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.     

Sequencing two DNA templates in five channels by digital compression.

By applying algebraic coding methods to the Sanger dideoxynucleotide procedure, DNA sequences of two templates can be determined simultaneously in only five reactions and data channels. A 5:2 data compression is accomplished by instantaneous source coding of nucleotide sequence pairs into one set of 5-bit block codes. A general algebraic expression, 2n-1 > or = 4f, describes conditions under which f DNA templates can be sequenced using n channels. Such compression sequencing is accurate and efficient, as demonstrated by manual 35S autoradiographic detection and automated on-line analysis using fluorescent-labeled primers. Symmetric 5:2 compression is especially useful when comparing two closely related sequences.