Nucleic Acid Analysis - Analytical Chemistry (ACS Publications)

Anal. Chem. 1999, 71, 425R-438R

Review

Nucleic Acid Analysis Theodore K. Christopoulos*

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada The rapid progress in the Human Genome Project has stimulated the development of diverse analytical methods for mutation detection, elucidation of complex biological problems, molecular diagnosis and prognosis of disease, and assessment of treatment. This review will discuss recent advances in nucleic acid analysis as reported in articles that appeared in Chemical Abstracts between October 1, 1996 and October 1, 1998. Major areas of nucleic acid analysis covered in this review include purification methods, hybridization, in situ hybridization, DNA sequencing, amplification techniques, oligonucleotide arrays, miniaturization of nucleic acid analysis, and nucleic acid sensors. GENERAL REVIEWS Several articles discussing the general subject of nucleic acid analysis as applied to diagnosis and monitoring of disease were published during the period of this review. Narayanan reviewed the basic concepts, principles, and applications of molecular biology techniques in clinical biochemistry (1). A review on the principles of current methods for genetic diagnosis and detection of mutations was presented (2). Polyak and Gretch reviewed the nucleic acid analysis methods used for detection and characterization of various hepatitis viruses and their application to the clinical management of chronic viral hepatitis (3). The genotyping of hepatitis C virus, the detection/determination of viral RNA, and the need for standardization were examined by Conry-Cantilena (4). Rezuke and co-workers reviewed the application of nucleic acid analysis techniques, such as Southern transfer and PCR, to the diagnosis of B- and T-cell lymphomas (5). The use of nucleic acid analysis in the diagnosis of lymphoma and the detection of minimal residual disease were also reviewed by Veronese and coworkers (6). Finke discussed the principles of molecular diagnosis as applied to thyroid disease (7). Boerwinkle et al. reviewed the genetic analysis of atherosclerosis (8). The use of DNA as a marker for detection of various targets was reviewed by Sano et al. (9). NUCLEIC ACID PURIFICATION METHODS The success of nucleic acid analysis depends heavily on the method used for purification of nucleic acids from clinical specimens. Klein et al. compared various methods for DNA extraction from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences (10). A cDNA bank for human colorectal cancer and surrounding normal tissues was constructed from biopsy specimens by using a unique mRNA assay system (11). Mauhay et al. demonstrated that cells from the oral cavity can be used as a source for DNA analysis (12). Morgan et al. used a * Tel.: 519-253-4232 ext. 3550. Fax: 519-973-7098. E-mail: [email protected]. 10.1021/a19900161 CCC: $18.00 Published on Web 09/15/1999

© 1999 American Chemical Society

microwave-based method and magnetic beads to isolate DNA suitable for PCR from archival, formaldehyde-fixed paraffin waxembedded human tissue (13). A simple protocol for the extraction of genomic DNA from archived human serum, plasma, and paraffin-embedded human tissue was reported (14). Ng et al. developed a high-throughput plasmid isolation system using a 96well plate format (15). NUCLEIC ACID HYBRIDIZATION Dangler edited a book that covers the principles and strategies of hybridization, the generation and labeling of nucleic acid probes, and the applications of nucleic acid probe techniques in human and veterinary medicine, food technology, agriculture, and environmental sciences (16). A book edited by Ross describes widely used techniques in hybridization and discusses their advantages and disadvantages. Topics covered include labeling and detection methods, solution hybridization, gene mapping by fluorescence in situ hybridization (FISH), filter hybridization, and specialized techniques for in situ hybridization (17). The use of lanthanide ions as probes in fluorescence nucleic acid hybridization assays and immunoassays was reviewed (18). Silin and Plant discussed the applications of surface plasmon resonance in biotechnology. This technique allows direct, realtime kinetic measurements of the interactions of unlabeled biomolecules on surfaces (19). Forozan et al. reviewed the comparative genomic hybridization technique for genome-wide scanning of differences in DNA sequence copy number (20). The application of comparative genomic hybridization for analysis of chromosomal imbalances in solid tumors and hematologic malignancies was also reviewed (21). Ermolaeva and Sverdlov discussed the advantages and disadvantages of the genomic subtractive hybridization technique (22). The structure and DNA mimicking properties of peptide nucleic acid (PNA) were reviewed by Nielsen (23). The kinetics of hybridization and thermally induced dehybridization of immobilized single-stranded DNA were studied by using two-color surface plasmon resonance spectroscopy to measure both the dielectric constant and the thickness of the immobilized DNA film (24). Lane et al. examined the thermodynamics of “stacking hybridization” reactions and compared the melting behavior of nicked and gapped DNA duplexes. It was found that a nick was energetically favored over a gap by at least 1.4 kcal/ mol (25). Malygin et al. demonstrated that hybridization of two oligonucleotides to both strands of a RNA hairpin increases the efficiency of RNA-DNA duplex formation (26). Asymmetric PCR was optimized for generation of single-stranded probes (1000 bases) (27). Analytical Chemistry, Vol. 71, No. 18, September 15, 1999 425R

Fisher et al. introduced nuclease P1 as a label in DNA hybridization assays. The enzyme catalyzes the dephosphorylation of flavin adenine dinucleotide 3′-phosphate (FADP) to give FAD. FAD activates apoglucosidase to glucosidase, which catalyzes the oxidation of glucose to gluconolactone with generation of H2O2. Then, H2O2 is used as a substrate in a chromogenic reaction catalyzed by horseradish peroxidase (28). The bispyrenyl alcohol was synthesized as a potential label for oligonucleotide probes (29). A luminescent complex composed of bathophenanthroline disulfonate and europium(III) was proposed for reversible staining of proteins and nucleic acids immobilized on solid supports. Membranes were illuminated at 302 nm and the bands were quantitated by a CCD camera (30). Rule et al. used dye-containing liposomes as labels in a sandwich hybridization assay. The liposome stability was studied under conditions of increasing stringency (temperature, formamide and salt concentration) required for hybridization (31). Fujita et al. synthesized new fluorogenic substrates for alkaline phosphatase and horseradish peroxidase suitable for detection of hybrids on Southern blots (32). Proudnikov and Mirzabekov reported chemical methods for fluorescent labeling of DNA and RNA. Aldehyde groups were introduced to DNA, by partial depurination, and to RNA, by periodate oxidation of the 3′-terminal ribonucleoside. These groups were then reacted with fluorescent labels carrying a hydrazine moiety. Alternatively, the aldehyde group was converted to a primary amino group and reacted with isothiocyanate or succinimide derivatives of fluorescent dyes (33). Oligodeoxynucleotides were immobilized on microparticles and hybridized to complementary Eu3+-labeled probes. Eu3+ was then released in enhancement solution and determined by time-resolved fluorometry of single microparticles (34, 35). Heinonen et al. combined the polymerase chain reaction with a triple-label (Eu3+, Tb3+, and Sm3+) time-resolved fluorescence hybridization assay for detection of seven cystic fibrosis mutations (36). Manganese meso-tetraphenylporphine/oligodeoxynucleotide conjugates were synthesized for hybridization assays. The label catalyzes the chemiluminescent oxidation of luminol (37). Galvan and Christopoulos developed sensitive bioluminescence hybridization assays in microtiter wells by using the photoprotein aequorin as a reporter molecule. In combination with reverse transcriptase-polymerase chain reaction, the assay could detect prostate-specific antigen mRNA from a single cancer cell amidst 1 million normal cells (38). Nucleic acid probes were conjugated to 6-aminoglucose homo- and copolymers and applied to the quantification of hepatitis B virus (39). During slot blot analysis of adeno-associated virus it was found that Mg2+ inhibits the binding of viral DNA, released from disrupted virions, to nylon and nitrocellulose membranes (40). An improved method for DNA immobilization on microtiter wells was developed. A genomic DNA solution was added to the wells, evaporated overnight at 37 °C, and heated to dryness at 60 °C. Hybridization efficiency increased 3-5 times (41). An acetylaminofluorene-modified dGTP was synthesized and used for labeling of oligonucleotide probes with terminal transferase for hybridization assays. The hybrids were measured by reacting with alkaline phosphatase-labeled anti-acetylaminofluorene antibody (42). A simple method for 3′-labeling of RNA with [R-32P]dATP was developed by annealing a short DNA template at the 3′-end of 426R

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RNA with a two-nucleotide overhang and extending with the Klenow fragment of DNA polymerase I (43). Fluorescence polarization allows for homogeneous (nonseparation) detection of DNA sequences through hybridization with fluorescent probes. Kumke et al. studied the effects of temperature and collisional quenching on polarization detection of DNA hybridization by measuring the fluorescence intensity, anisotropy, lifetime, and anisotropy decay of the single-stranded and doublestranded DNA (44). A homogeneous hybridization assay using pyrene as a label was developed based on the decrease in the fluorescence intensity upon hybridization (45). Castro and Williams developed a homogeneous method for detection of a specific nucleic acid sequence in a background of unrelated sequences without amplification. A rhodamine-6G-labeled and a bodipy-TRlabeled peptide nucleic acid probe were hybridized to adjacent sites on the same strand of target DNA. The sample was then analyzed by a laser-excited fluorescence system capable of detecting single fluorescent molecules at two wavelength channels simultaneously. Coincident detection of both dyes signified the presence of target DNA (46). A homogeneous hybridization assay was also developed based on the decrease of emission intensity of fluorescein attached to single-stranded DNA when it forms double strands (47). The interaction of homopyrimidine peptide nucleic acid with single-stranded DNA targets was studied by thermal dissociation experiments (48). The kinetics of hybridization of a pentadecamer peptide nucleic acid to DNA and RNA and the effect of mismatches were studied by Jensen et al. using the biospecific interaction analysis technique (49). An alternative to the Southern hybridization method was developed in which a labeled peptide nucleic acid oligomer was hybridized to heat-denatured target DNA at low ionic strength (pregel hybridization) and the mixture was then separated by slab-gel electrophoresis or capillary electrophoresis. Because of the neutral backbone of peptide nucleic acid, its hybridization to complementary DNA was independent of ionic strength. The low ionic strength ensured slow reannealing of the DNA strands which otherwise would expell the much shorter bound probe. High specificity was obtained with single base pair discrimination (50). Su et al. used subtraction hybridization to identify and clone the progression elevated gene-3 (PEG-3), a gene associated with transformation progression in virus- and oncogene-transformed rat embryo cells. PEG-3 has sequence homology and DNA damage-inducible properties similar to gadd34 and MyD116 (51). Shuber et al. developed a multiplex allele-specific diagnostic assay (MASDA) which allows for parallel analysis of a large number of samples for a large number of known mutations. The target DNA was immobilized on a membrane (dot-blot) and then hybridized with a pool of allele-specific oligonucleotide probes. Following removal of the unbound probes, specific band patterns were obtained by chemical or enzymic sequencing of the hybridized oligonucleotides (52). Phosphodiester and phosphorothioate antisense oligonucleotides were determined in mouse plasma by competitive hybridization to immobilized sense oligonucleotides using a 5′-biotinylated antisense oligonucleotide as a tracer. The bound tracer was determined by reacting with streptavidinacetylcholinesterase. The limit of quantification was 900 pM (53). Guo et al. inserted artificial mismatches into oligonucleotide

probes, by using the base analogue 3-nitropyrrole, to enhance the thermal stability differences and therefore improve the discrimination of single-nucleotide polymorphisms in DNA hybridization (54). Zehnder et al. developed a cross-linking hybridization assay for direct detection of factor V Leiden mutation (most common inherited risk factor in thrombosis). The target DNA was hybridized with biotinylated allele-specific capture probes and fluoresceinlabeled detection probes. The hybrids were photo-cross-linked, to enhance stability, captured on streptavidin-coated beads, and detected with an alkaline phosphatase-antifluorescein antibody conjugate and a fluorogenic substrate (55). IN SITU HYBRIDIZATION The unique advantage of in situ hybridization is that it provides information on the topology of specific nucleic acid sequences. Consequently, this technique is applied to answer questions about genome organization, structure, and function and plays an important role in molecular diagnosis. Recent advances in this area include improvements in probe labeling, microscopy, and digital imaging. RNA in situ hybridization was reviewed by Dirks (56). Carter discussed various aspects of FISH such as probe labeling and detection, fluorescence microscopy, and digital imaging (57). Nonradioactive mRNA in situ hybridization techniques were reviewed by Houggaard et al. (58). Chevalier et al. examined the use of biotin and digoxigenin as labels for in situ hybridization (59). Hacker reviewed the protocols and applications of using a streptavidin-nanogold-silver staining for localization of viral DNA or RNA sequences (60). The localization of mRNA in cells and tissues by in situ hybridization suitable for electron microscopy was reviewed (61). The principles and applications of an oligonucleotide-primed in situ labeling technique were discussed (62). Werner et al. reviewed the principles and application of FISH to the detection of chromosomal abnormalities (numerical changes and structural aberrations) in interphase cells (63). Cuneo et al. discussed the use of FISH for detection of numerical aberrations in hematologic neoplasias (64) while Siebert et al. discussed the application of FISH for diagnosis of lymphatic neoplasms (65). McCarthy also reviewed the application of nucleic acid analysis techniques, especially PCR and FISH, to molecular diagnosis of lymphomas and discussed the advantages and limitations of each technique (66). The use of FISH to investigate the origin of radiation-induced chromosome aberrations was reviewed (67). Kontogeorgos discussed the application of FISH in detecting chromosomal alterations associated with endocrine diseases (68), and Miyazaki examined the application of in situ hybridization to the analysis of gene expression in renal biopsy specimens (69). The molecular cytogenetic analysis of bone and soft tissue tumors was reviewed by van Kessel (70). Luke et al. reviewed cytogenetics as applied to the detection of gene amplification, translocation, and deletion in a variety of tissue specimens (71). Harris et al. developed a high-volume microtiter plate-based in situ hybridization assay using radioactive probes (72). The integration sites of HIV-1 were mapped in two model cell lines by using FISH. This application allowed the analysis of the HIV status at the level of a single cell (73). FISH, using digoxigenin-labeled cosmid probes (119 kbp), was applied to the detection of human cytomegalovirus DNA in peripheral blood leukocytes (74). Insitu hybridization on skeletal tissues was performed by using

oligonucleotide probes end-labeled with digoxigenin (75). Sinclair et al. used a triple-probe/three-color approach for detection of Philadelphia translocation by FISH (76). FISH was also used in the detection of female Duchenne muscular dystrophy carriers (77). A multiprobe FISH method was developed to detect chromosome abnormalities (such as terminal duplications or deletions in chromosome 1p, aneuploidy involving chromosome 1 or 8, and diploidy) in sperm of healthy men (78). A photostable alkaline phosphatase (ELF-97) substrate with a large Stokes shift was used in FISH. Upon dephosphorylation, a fluorescent precipitate was formed (79). The peroxidase-catalyzed deposition of hapten- and fluorochrome-labeled tyramides was shown to improve the sensitivity of immunofluorescence and FISH techniques (80). It was demonstrated that single-copy DNA and RNA can be detected by FISH and catalyzed reporter deposition using Cy 3.29 tyramides (81). A similar method was described for detecting chromosome-specific repeat sequences (82). Differentially fluorochrome-labeled tyramides were used in multicolor FISH for catalyzed reporter deposition (83). Heiskanen et al. reported an optimized protocol for the fiber FISH technique on agaroseembedded yeast cells (84). Vrolijk et al. examined various aspects of image acquisition, processing, and analysis for DNA mapping by fiber FISH (85). A quantitative DNA fiber mapping method was reported that consists of preparation of DNA fibers, hybridization of nonisotopically labeled probes, and determination of the relative mapping position by fluorescence image analysis (86). Veldman et al. developed a novel method, named spectral karyotyping, based on the hybridization of 24 fluorescently labeled chromosome painting probes. The method allowed the simultaneous and differential color display of all human chromosomes (87). Spectral karyotyping was also applied to the simultaneous visualization of all mouse chromosomes, and the potential of the technique to identify complex chromosomal aberrations in mouse models of human carcinogenesis was demonstrated (88). FISH, with rRNA targeted probes, was combined with immunofluorescence for the flow-cytometric identification of bacteria (89). FISH was compared to flow cytometry for detecting DNA aberrations in urinary bladder cancer (90). A chemiluminescence in situ hybridization assay was developed for detection of human papillomavirus DNA in biopsy specimens. Hapten-labeled probes were used and anti-hapten antibodies conjugated to alkaline phosphatase. Dioxetane phosphate was the substrate (91). DNA SEQUENCING A book edited by Ansorge, Voss, and Zimmermann discussed the sequencing strategies and cloning protocols, preparation of templates, DNA sequencing protocols, selection, synthesis and purification of primers, preparation of DNA sequencing gels, electrophoresis, and computer analysis (92). Rapley edited a book on polymerase chain reaction (PCR) sequencing protocols. Topics covered were related to the preparation and analysis of sequencing gels, purification of PCR products, enzymatic, fluorescence, and biotin labeling of primers, sequencing by using sequenase, sequencing by thermal asymmetric PCR, rapid sequencing of cDNA clones, sequencing using chemiluminescence detection, sequencing using magnetic beads, affinity capture and solid-phase sequencing of PCR products, solid-phase minisequencing, sequencing using digoxigenin, silver sequencing, sequencing with DNA binding proteins, sequencing with the aid of detergents, Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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sequencing with degenerate and inosine-containing primers, determination of unknown genomic sequences without cloning, sequencing by the chemical method, sequencing with denaturants, PCR production of single-stranded DNA sequencing templates, preparation and direct automated cycle sequencing, solid-phase automated sequencing, cloning, and sequencing of PCR products in M13 vectors, genome amplification with transcript sequencing, DNA rescue by vectorette method and sequencing of cloned mixed PCR products from microbial populations (93). Rowen et al. reviewed the challenges for sequencing the human genome (94), while Cantor et al. discussed the future of DNA sequencing after completion of the Human Genome Project (95). Dovichi et al. discussed the application of capillary electrophoresis to largescale DNA sequencing (96). The instrumentation and applications of capillary electrophoresis to DNA separation and sequencing were also reviewed (97). Monforte and Becker discussed the highthroughput analysis of DNA by time-of-flight (TOF) mass spectrometry (98). Weber and Myers reviewed the shot-gun sequencing of human genome (99). Fluorescently labeled and partially double-stranded DNA probes were used to capture and sequence double-stranded DNA targets and to serve as primers in conventional solid-state Sanger sequencing. PCR products end-labeled with biotin were captured on magnetic beads, dephosphorylated, ligated to the probe, and subjected to sequencing by strand displacement (100). Nguyen et al. described a strategy to obtain DNA duplexes with thermal stability independent of their base content for sequencing by hybridization (101). The performance of automated laser-induced fluorescence DNA sequencing was improved and a more equalized sequencing peak profile was obtained by optimizing the ddNTP/ dNTP ratio, the acrylamide concentration, and the electrical settings for electrophoresis (102). A system was developed for automated parallel loading of >200 lanes on a 30-cm-wide gel in automated DNA sequencing. The technique is applicable to horizontal or vertical systems with standard or ultrathin gels (103). A high-speed automated DNA sequencing system was described consisting of a horizontal gel electrophoresis cell, a laser and optical components to provide a line of fluorescence excitation across the gel, a charge-coupled device, and a computer system (104). A method was described for simultaneous PCR amplification and sequencing of DNA using boron-modified nucleotides. Sequencing was based on the resistance of boranophosphate linkages to exonuclease digestion (105). A method was developed for making in vivo nested deletions on a large scale and it was applied to the sequencing of 300 kbp of human amyloid precursor protein locus on chromosome 21 (106). Target amplification was combined with sequence determination by using two DNA polymerases with differential incorporation rates for dideoxyribonucleotides (107). A near-infrared time-correlated single-photoncounting instrument was developed for dynamic lifetime measurements in DNA sequencing applications (108). Johnson et al. reported a fluorescence-based dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded DNA binding protein (109). Ronaghi developed a method for real-time DNA sequencing without electrophoresis. The method relied on the detection of DNA polymerase activity by measuring the pyrophosphate released with a fluorometric enzymic assay (110). Arlinghaus et al. 428R

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described two DNA sequencing methods based on DNA hybridization on biosensor chips. One technique involved labeling of free DNA with enriched stable tin isotopes and hybridization to immobilized oligodeoxyribonucleotides. In the second method, unlabeled DNA target hybridized to immobilized peptide nucleic acid. In both cases, sputter-initiated resonance ionization microprobe analysis was used for detection (111). Flow cytometric DNA sequencing analysis was performed by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes (112). Mercurated derivatives of dUTP were synthesized and they were incorporated into full-length single-stranded M13 DNA for using in image-based DNA sequencing (113). Cyanine dyes were exploited as donor chromophores with fluorescein or rhodamine as acceptors in fluorescent energy-transfer primers for DNA sequencing (114). A series of energy-transfer fluorescent dye-labeled primers was synthesized and their spectroscopic properties were studied (115). The on-line coupling of the dyelabeled terminator cycle sequencing reaction to capillary electrophoresis was achieved. The sequencing reaction was carried out in a fused-silica capillary and the ladder was injected directly to a gel filtration column, for purification from fluorescently labeled dideoxynucleotides, followed by on-line injection to a capillary for electrophoresis. Electropherograms were obtained by using onewavelength excitation and dual-wavelength detection (116). The design, construction, and operation of a four-color capillary array electrophoresis scanner was described. Energy-transfer primers were used. Data were collected from up to 25 capillaries in parallel (117). Figeys et al. reported a method that allows loading of a 3-µL sample in capillary electrophoresis for DNA sequencing. The method used low ionic strength formamide to resuspend DNA after ethanol precipitation. Excessive band broadening was thus avoided, and the electric current passing through the sample was carried mostly by the DNA fragments (118). Visible fluorescent dyes were used in a four-decay detection scheme for DNA sequencing based on capillary electrophoresis (119). Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at 55-60 °C was used to extend the separation of DNA sequencing fragments to greater than 800 bases (120). A highthroughput DNA sequencing system based on capillary array electrophoresis and capable of sequencing 48 DNA samples simultaneously was reported (121). Capillary electrophoresis with laser-induced fluorescence was used to detect known point mutations using the method of single-nucleotide primer extension (122). Koester et al. developed two methods for solid-phase Sanger DNA sequencing followed by MALDI-TOF mass spectrometry. The high speed and superior resolution of mass spectrometry were demonstrated (123). A method was described for the covalent attachment of DNA to silicon wafers at high density for hybridization detection by MALDI-TOF mass spectrometry (124). DNA sequences were recovered from immobilized biotin-streptavidin complexes by treatment with NH4OH as a purification step prior to sequencing by MALDI-TOF mass spectrometry (125). A MALDI mass spectrometric method was reported for sequencing of single- and double-stranded DNA (126). Oligonucleotide segments up to 35 mer were sequenced by fragmentation in MALDITOF mass spectrometry (127). MALDI-TOF mass spectrometry

was applied to the detection of mutations (including substitutions and deletions) involving single nucleotides (128). A generally applicable algorithm was developed to allow base composition of PCR products to be determined from molecular weights measured by mass spectrometry (129). A new sequence-by-hybridization method was reported in which oligonucleotide probes labeled with stable isotopes were hybridized with DNA immobilized on a nylon membrane. The hybrids were detected by time-of-flight resonance ionization mass spectrometry. By using isotopically enriched tin labels, up to 10 probes could be examined in a single hybridization to the DNA matrix (130). A dideoxy sequencing method was developed using electrophore (compound that ionizes in the gas phase)-labeled DNA oligonucleotide primers and detection by mass spectrometry (131). A method for multiplex detection of mutations by solid-phase minisequencing was reported. The mutations were detected by extending immobilized primers (in an array format), which anneal to their template sequences immediately adjacent to the mutant nucleotide positions, with single-labeled ddNTP using DNA polymerase (132). Solid-phase minisequencing was applied to the sequencing of seven point mutations of the CYP21 gene encoding 21-hydroxylase (133). A multiplex solid-phase minisequencing method was developed for detection of single nucleotide polymorphisms in an undivided sample (134). A method for preimplantation diagnosis was developed which involves whole genome amplification followed by locus-specific amplification and mutation detection by solid-phase minisequencing (135). AMPLIFICATION TECHNIQUES White discussed the impact of PCR on molecular biology, on the human genome project, and on the molecular diagnosis of disease (136). Mitsuhashi reviewed the critical factors for designing successful PCR primer sequences (137). The various inhibitors of the polymerase chain reaction and a series of approaches to overcome these factors in clinical, food, and environmental microbiology were reviewed (138). A review was published on amplification and subtractive methods used for isolation of novel viruses that cause human disease (139). The role of nucleic acid amplification techniques in screening for virus-infected blood products was reviewed. PCR, nucleic acid sequence-based amplification, and branched DNA were discussed (140). The advantages and limitations of nucleic acid amplification techniques as applied to routine diagnostic bacteriology were examined (141). Keilholz et al. reviewed the application of PCR to the detection of circulating tumor cells with emphasis on the standardization and quality control (142). The application of nucleic acid amplification techniques in the diagnosis of respiratory tract infections was discussed (143). Lo reviewed the use of PCR for fetal cell detection in maternal blood (144). A review was published on the application of capillary electrophoresis in the analysis of PCR products, RFLP analysis, single-strand conformation polymorphism analysis, variable number of tandem repeat analysis, microsatellite analysis, hybridization, and monitoring of DNA-based drugs (145). The application of capillary electrophoresis to the analysis of PCR amplification products from clinically relevant DNA sequences was reviewed. Applications in the areas of genetics, microbiology/ virology, forensic medicine, and therapeutic DNA were discussed (146). Krafft reviewed the various methods for isolation and PCR amplification of RNA from formalin-fixed, paraffin-embedded

tissues (147). The self-sustained sequence replication reaction (3SR) for isothermal RNA amplification and the in situ 3SR were reviewed by Mueller (148). A book edited by Micheli and Bova describes various strategies and detailed protocols for fingerprinting based on arbitrarily primed PCR. Specific applications such as gene mapping, detection of somatic mutations abnormally expressed genes in tumors or differentially expressed genes were covered (149). Polymerase Chain Reaction. A fusion protein comprising Taq DNA polymerase and a serum albumin binding protein was immobilized on a solid support coated with human serum albumin. Immobilized Taq showed low catalytic activity. The enzyme however was released and reactivated at the high temperatures required for extension, thus allowing for hot-start PCR with improved performance (150). Oligonucleotides with a high affinity for Thermus aquaticus DNA polymerase were selected from a random sequence library. The enzymic activity was inhibited at ambient temperature but not at temperatures above 40 °C. It was proposed that these oligonucleotides may eliminate the need for hot-start PCR (151). Brownie et al. described a method for elimination of primer dimers in PCR (152). A guanidine thiocyanate-impregnated filter paper (GT-903) was evaluated as a DNA collection device from whole blood for subsequent PCR. It was found that GT-903 retained most of the PCR inhibitors (153). Human genomic DNA was purified from whole blood by using a biotinylated peptide nucleic acid that forms a triple helix with A7 sequence motifs in the target DNA. The complexes were captured on streptavidin-coated particles and used directly for PCR (154). A microdissection and genomic DNA extraction protocol from histological tissue sections was reported (155). A tripeptide that binds to the minor groove of DNA was conjugated to the 5′-end of 8-10 mers. It was shown that the conjugates can be used as PCR primers with high effciency and specificity. The reducedlength primers may find applications to the amplification of viral sequences that possess a high degree of variability or for techniques such as gene hunting and differential display, which amplify multiple sequences using short primers (156). Jadhav et al. reported the synthesis of PCR primers containing a novel fluorescent nucleobase in which fluorescein is linked to 2′deoxyuridine at C-5 (157). It was shown that DNA template crosslinked to an uncharged nylon membrane could be used repetitively for PCR amplification. The method may be useful in cases of limited availability of DNA such as DNA isolated from biopsies and archival tissue (158). A 30-min procedure for preparing PCRready DNA using magnetic beads was described (159). It was demonstrated that the addition of betaine in the PCR mixture improves the amplification of GC-rich DNA sequences (160). It was found that Taq-amplified DNA fragments may appear as doublets of bands in denatured gradient gel electrophoresis leading to misinterpretation. The problem was solved by optimizing the PCR buffer (161). Herman et al. developed a PCR assay (methylation-specific PCR) for rapid analysis of the methylation status of CpG islands. Primers were designed to distinguish methylated from unmethylated DNA in bisulfite-modified DNA. The method is more sensitive than Southern analysis, it does not rely on restriction enzymes, and it allows for confirmation of the DNA integrity (162). De Francesco developed the replication synchrony PCR for assessing the cell replication status in human Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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tissues and tumors (163). It was shown that rational design of primers improved the ability of differential display PCR to identify relevant genes (164). A method for removal of mutant sequences that arise during PCR amplification was developed. After denaturation and reannealing of the PCR product, the mismatched base pairs were cleaved on both strands via the MutS-, MutL-, and ATPdependent activation of MutH-associated endonuclease (165). Roy used a primer covalently linked to an infrared fluorescent dye for detection of PCR products (166). Lockley et al. performed PCR with primers attached to a solid phase, thus leading to generation of immobilized and labeled amplification products ready for detection (167). Cheung et al. used degenerate oligonucleotide primers for whole genome amplification of less than 1 ng of human genomic DNA for genotyping with microsatellite repeat markers (168). A nonradioactive method for determination of PCR products was reported which involves labeling of the DNA with biotin during PCR, electrophoretic separation, transfer to membrane, and detection with streptavidin-alkaline phosphatase (169). A nonradioactive method for determination of PCR products was reported which involves labeling of the DNA with biotin during PCR, electrophoretic separation, transfer to membrane, and detection with streptavidin-alkaline phosphatase (170). Christopherson et al. studied the effects of internal primertemplate mismaches on the efficiency of RT-PCR and the accuracy of target RNA determination (171). Barragan-Gonzalez et al. compared two reverse transcriptases, the avian myelomatosis virus and the murine moloney leukemia virus, which are commonly used in RT-PCR (172). RT-PCR studies on partially degraded RNA samples showed that various mRNAs were degraded to the same extent, thus enabling the determination of a particular mRNA, even in degraded samples, by normalizing to the mRNA of a constitutively expressed gene such as glyceraldehyde phosphate dehydrogenase (173). Su et al. developed a high-throughput multiplex RT-PCR assay for analysis of medium- to low-copy cellular RNA transcripts from small numbers of cells (104). RNA extraction and RT-PCR were performed in microplates followed by electrophoresis and staining with SYBR Green I. The method was used in the investigation of differential mRNA expression levels of TNFR and IL-1β in lipopolysaccharide-stimulated THP-1 cells and the identification of specific IL-1β transcriptional inhibitors (174). A method was developed for PCR amplification of genes, single RNA transcripts, and cDNA molecules from a single cell without cloning. Genes were amplified from isolated single nuclei. Single cDNA molecules were isolated by limited dilution prior to amplification (175). A RT-PCR method was described for analyzing the expression of several mRNAs from single cells. Total RNA from a single cell was reverse transcribed, the cDNA was tailed with poly(dA) and amplified (176). Wittwer et al. described a fluorometer with microvolume multisample capabilities and temperature control for real-time monitoring of PCR. Three techniques were compared for determination of the amplification product: i.e., reaction with the dsDNA-selective dye SYBR green; determination by fluorescence energy transfer using two probes that hybridize to adjacent segments on the amplified DNA; a technique based on the 5′exonuclease activity of Taq polymerase. Complete amplification and analysis required only 10-15 min (177). Swerdlow et al. designed a fully automated instrument for PCR amplification and 430R

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analysis. The sample was automatically loaded, sealed, cycled in a rapid air thermal cycler, and injected onto a gel filtration HPLC column for removal of salt and dNTPs followed by analysis of amplified DNA by capillary electrophoresis. A non-cross-linked polymer-filled capillary was employed with automatic refilling. Detection was accomplished by laser-induced fluorescence. The amplification and analysis were completed within 20 min (178). Kalinina et al. carried out PCR in a volume of 10 nL in microcapillaries with single-molecule fluorescence detection using the TaqMan system (179). A capillary electrophoresis method was developed for high-throughput sizing of PCR products (180). Capillary electrophoresis followed by laser-induced fluorescence was applied to the determination of RT-PCR products of basic fibroblast growth factor mRNA (181). A mutation detection method was developed which involved PCR amplification by using a primer labeled with the cyanine dye Cy5. The products were digested with a restriction enzyme that recognized the mutated sequence and then analyzed by capillary electrophoresis and determined by laser-induced fluorescence (182). A thermocycler was constructed that includes, optionally, a 96-channel or 960channel glass fiber fluorometer combined with a cooled charge coupled device camera. The instrument was used in monitoring amplification reactions based on PCR and Q-β replicase (183). A system for high-throughput setup of polymerase chain reactions was developed (184). Electrospray ionization mass spectrometry was used for detection of one-base substitution from 52-bp PCRamplified human DNA (185). A new protocol for purification of PCR products for analysis by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry was reported. PCR products were bound to a silica resin, eluted, and subjected to microdialysis thus allowing for removal of dNTPs, primers, and salt. Single-base substitutions were readily detected (186). The multiplex genotyping of PCR products by MALDI-TOF mass spectrometry was described (187). Stolovitzky et al. developed a kinetic model for PCR which provides the probability of replication of a DNA molecule as a function of the physical parameters of the system, i.e., the rate constants of the different reactions. The model was used for the design of a method for quantitative PCR (188). The effect of heteroduplex formation on the accuracy of competitive quantitative PCR was studied and various approaches to minimize this effect were proposed (189). Competitive quantitative PCR protocols for evaluating the abundance of mRNA targeted for ribozymemediated destruction were developed (190). A technique was reported for production of PCR mimics for use in semiquantitative PCR (191). A competitive RT-PCR was developed for the study of in vivo regulation of gene expression in small tissue samples (192). A method was described for preparation of double-labeled fluorescent probes for 5′-nucleace assays (193). The Taqman method was used for the semiquantitative analysis of the Vβ repertoire of antigen-reactive T-cell populations. Fluorescencedetected amplification was linear within 2-3 orders of magnitude. The sensitivity of Taqman PCR was comparable to conventional detection of PCR products by agarose gel staining, while processing time was reduced (194). A novel method for real-time quantitative competitive RT-PCR was developed based on the 5′nuclease activity of DNA polymerase. An internal standard sharing the same primer binding sites with the target RNA was used, and

two specific probes were labeled with different fluorescent dyes (195). Mutations of the p53 gene are an important feature of neoplastic progression in humans. Katsuragi et al. developed a multiple fluorescence-based symmetric PCR single-strand conformation polymorphism analysis method for the detection of mutations in the p53 gene. Exons 5-8, which contain 86% of the mutations, were amplified simultaneously in a single tube with four-color fluorescence-labeled sense and antisense primers. The products were heat denatured and analyzed electrophoretically in a DNA sequencer. The authors analyzed lung cancer tissue specimens (196). Martincic and Whitlock compared SSCP and dideoxy-fingerprinting with sequencing for detection of mutations in exons 5-8 of the p53 tumor suppressor gene (197). An automated fluorescence-based PCR-SSCP capillary electrophoresis system was developed and applied to the analysis of p53 gene mutations (198). Ishioka et al. developed a novel PCR-based method for detection of protein-truncating mutations in the BRCA1 gene in patients with early onset of breast cancer and in the APC gene in patients with familial adenomatous polyposis (199). Rohlfs applied PCR-mediated site-directed mutagenesis to the detection of mutations in the breast and ovarian cancer susceptibility gene (200). Lopez-Crapez et al. developed a large-scale method for detection of K-ras gene mutations in tumors. DNA was amplified by asymmetric PCR and labeled with dinitrophenyl. The amplification products were hybridized with oligonucleotide probes immobilized on a solid support. The perfectly matched hybrids were detected with 125I-labeled anti-dinitrophenyl antibody. The method was applied to the analysis of K-ras codon 12 mutations in human colorectal cancers (201). A method that combines PCR with electrospray ionization mass spectrometry was developed for detection of a five-base deletion in APC gene (202). Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients was accomplished by immunomagnetic isolation of epithelial tumor cells from blood followed by RT-PCR of cytokeratin mRNA (203). Bishop et al. developed a quantitative RT-PCR method for tumor necrosis factor R mRNA in liver biopsy specimens. The PCR products were determined by dot-blot hybridization with radioactive probes. It was found that the variation in the efficiency of cDNA synthesis was as high as the variation in the amplification efficiency (204). A competitivedifferential PCR method was described for gene dosage estimation of erb-1, erb-2, and erb-3 oncogenes (205). The c-erb-2 oncogene amplification in tumor specimens was determined by a 5′-nucleasebased quantitative PCR assay (206). Competitive PCR was used in the study of c-erb-2 oncogene amplification in transitional cell bladder carcinoma (207). Competitive RT-PCR with a fluorescently labeled primer was applied to the determination of c-erb-2 expression in breast cancer. The products were separated by denaturing polyacrylamide gel electrophoresis and measured in a DNA sequencer (208). A microtiter well-based time-resolved fluorometric hybridization assay was developed for determination of RT-PCR products from BCR-ABL mRNA of chronic myelogenous leukemia (209). A quantitative RT-PCR assay was also developed for the BCR-ABL mRNA. The target RNA was coamplified with a constant amount of internal standard; the products were hybridized in microtiter wells with specific probes and measured by enzyme-amplified time-resolved fluorometry of Tb3+

chelates (210). Using RT-PCR, Henke et al. showed that the prostate-specific antigen (PSA) mRNA is not specific for prostate cancer cells but it is also present in normal tissues of healthy individuals (211). They also found that as the sensitivity of nested RT-PCR for PSA mRNA in whole blood increased, the diagnostic specificity decreased. It was therefore concluded that a quantitative PCR assay was necessary for discrimination between the prostatic and nonprostatic origin of PSA mRNA (212). A quantitative RTPCR assay for PSA mRNA based on a time-resolved fluorometric hybridization assay was reported (213). In another publication, PCR was compared with flow cytometry for analysis of prostatespecific antigen-positive cells in peripheral blood of prostate cancer patients (214). A multiplex RT-PCR assay was developed for studying the expression of the mismatch repair genes in human gliomas by measuring simultaneously the relative levels of the transcripts. The β-actin gene was used as an internal control (215). The immunobead-PCR technique was developed, which involves immunomagnetic isolation of carcinoma cells in whole blood followed by RT-PCR (216). Wong and Lam showed that hair follicles and cheek cells can be used for quantitative PCR screening of mutant mitochondrial DNA in affected and asymptomatic family members (217). Fortina et al. used entangled solution capillary electrophoresis with laserinduced fluorescence detection in quantitative analysis of multiplex PCR products for determination of carrier status of Duchenne/ Becker muscular dystrophy (218). Warner et al. described a PCR amplification method, using a fluorescently labeled primer, for detection of CAG repeats in myotonic dystrophy patients (219). PCR followed by DNA enzyme immunoassay technique was used for detection of the eight most common β-thalassemia mutations in the Mediterranean population (220). The nested PCR amplification refractory mutation system was combined with capillary zone electrophoresis for detection of the 21-hydroxylase deficiency mutations. The products were stained with SYBR Green I and determined by laser-induced fluorescence (221). Angrist discussed the pros and cons of using an automated system for SSCP analysis of gene mutations (222). Microsatellites are polymorphic, short oligonucleotide repeats scattered throughout the genome and used as genetic markers detectable by PCR. A method was described for resolution of the overlapping patterns causing problems in the analysis of microsatellite data (223). Fetal nucleated erythrocytes in maternal circulation are potentially useful for noninvasive prenatal diagnosis, but their small number and the uncertainty as to whether they are of fetal or maternal origin impose problems with their analysis. Von Eggeling developed a method for noninvasive prenatal diagnosis, in which nucleated erythrocytes were isolated by density gradient centrifugation followed by enrichment through binding to monoclonal antibody. Random PCR performed on single nucleated cells allows for genetic diagnosis (224). PCR was also applied to the detection of fetal cells in maternal peripheral blood in normal and aneuploid pregnancies (225). A quantitative PCR was developed for sex determination in fetal cells (226). PCR-single-strand conformation polymorphism was applied to the prenatal diagnosis of 21hydroxylase deficiency (227). Metherell et al. showed that microbial identification can be accomplished by PCR amplification of endonuclease class II target sequences (228). Competitive PCR was applied to quantification Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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of 16S-rRNA by using an electrochemiluminescence hybridization assay (229). A 5′-nuclease-based quantitative PCR assay was developed for determination of the number of human papillomavirus genotypes associated with cervical cancer (230). De Vos et al. reported a multiplex PCR test based on the simultaneous amplification of two genes for detection of pseodomonas in clinical specimens (231). A method for the detection of low numbers of hepatitis A viral particles was described. The procedure involves capture of the virus on magnetic beads coated with anti-HAV antibody, washing, lysis of the virus, and subsequent RT-PCR amplification of the RNA released (232). Three methods were compared for quantification of hepatitis B virus in serum: a hybridization technique without amplification, a signal amplification assay based on branched DNA, and a PCR-based assay (233). Jurinke et al. developed a nested PCR-based method for detection of hepatitis B virus DNA in serum samples. The products were captured on streptavidin-coated magnetic beads, denatured, and analyzed by MALDI-TOF mass spectrometry. The detection limit was 100 molecules in 1 mL of serum (234). Park reported the detection of hepatitis C virus in formalin-fixed paraffin-embedded liver tissues by ligation-dependent PCR (235). Competitive and noncompetitive RT-PCR were compared for the quantification of hepatitis C virus RNA (236). Various reports compared the branched DNA hybridization assay with a RT-PCR method for detection or quantitation of hepatitis C virus RNA in serum or plasma (237-239). A competitive PCR assay was developed for quantitation of parvovirus B19 sequences in serum samples (240). A 5′-nuclease activity-based PCR assay was developed for the detection of single-base polymorphism in orthopoxvirus. Two different reporter dyes and the same quencher dye were used (241). Mycobacterium ulcerans was detected in clinical specimens by PCR and oligonucleotide-specific hybridization on microtiter plates (242). Tseng reported a homogeneous assay that combines PCR amplification with direct fluorescence detection of the amplification products for identification of Salmonella. The fluorescence signal was generated by the nucleic acid dye YO-PRO-1 (243). Patel described a method for identification of mycobacteria that combines RT-PCR of the characteristic 16S rRNA gene and detection by an enzyme-linked immunosorbent assay (244). PCRsingle-strand conformation polymorphism analysis was compared to PCR-restriction fragment length polymorphism for detection of a point mutation in the catalase-peroxidase gene of Mycobacterium tuberculosis, which has been associated with isoniazid resistance (245). Merkelbach et al. compared four DNA extraction methods for their ability to provide DNA for PCR amplification of viral sequences from paraffin-embedded human tissue samples (246). The “touchdown” PCR method was used in order to avoid mispriming in the detection of Helicobacter pylori (247). Crotchfelt described a PCR-based method for detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples by coamplification in a single tube (248). A microtiter well-based test was developed for detection of Mycoplasma pneumoniae in bronchoalveolar fluid specimens using PCR (249). Campylobacter rRNA was amplified by PCR, and the products were hybridized in microtiter wells and detected with anti-RNA-DNA hybrid antibodies (250). A method was described for detection and typing of human enteroviruses which involves capture of the viral particles with specific antibodies, release of 432R

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the RNA by heating, and RT-PCR amplification (251). Quiros et al. developed a method for detection of enteroviral RNA using a closed-tube seminested RT-PCR followed by hybridization on a solid phase (252). Kubota et al. developed a methylation-specific PCR method for imprinting analysis. Treatment of the DNA with bisulfite resulted in conversion of cytosine to uracil except when cytosine was methylated. PCR primers specific for the methylated and unmethylated versions of the CpG islands were used (253). A complementary strand analysis method was developed for separating allleles in complex polyallelic systems. The method involves PCR amplification of a locus using a biotinylated primer, capture of the products on streptavidin-coated beads, release of the antisense strand, hybridization with a sense reference strand, and analysis by nondenaturing polyacrylamide gel electrophoresis (254). Pokorny et al. modified the SSCP technique in order to improve resolution. The DNA was first amplified by symmetric PCR and the product was purified before an asymmetric PCR amplification using only one primer (255). Fluorescent dNTPs were incorporated in the PCR products followed by denaturation and SSCP analysis using a nondenaturing polyacrylamide gel on an automated DNA sequencer (256). Guldberg et al. described a method for mutation scanning of the entire coding sequence and the splice junction of the p53 gene. The method is based on a combination of PCR and analysis of amplification fragments by denaturing gradient gel electrophoresis (257). In situ RT-PCR and hybridization were combined with computerized image analysis for determining the levels of vitamin D receptor mRNA in human kidney and bone cells (258). Zhang and Wadler developed a micropreparative method for in situ RT-PCR, which is based on culturing the cells directly on the glass slide that is used for RTPCR. The effects of drugs on gene expression in cultured cells was assessed (259). A method for the direct PCR amplification of paraffin-embedded tissue DNA that eliminates the timeconsuming deparaffinization and DNA isolation steps was reported (260). Chen and Warner developed a competitive RT-PCR method to measure inducible nitric oxide gene expression in human and rat (261). Immuno-PCR is an immunoassay in which a DNA fragment is used as a label. After completion of the immunoreaction, the label is subjected to PCR amplification. Niemeyer et al. compared various methods for determination of the amplification products (262). Immuno-PCR was used for highly sensitive detection of various antigens (263) including R human atrial natriuretic peptide (264). Other Amplification Techniques. Reyes et al. developed a ligase chain reaction-based assay for detection of mutations in the β-globin gene in sickle cell anemia. The amplification products were captured on microwells with specific oligonucleotide probes and detected through biotin-streptavidin (265). Muth et al. used high-speed capillary electrophoresis with laser-induced fluorescence to detect ligase chain reaction products stained with ethidium bromide. The method was applied to the detection of three-point mutations in human mitochondrial DNA responsible for Leber’s hereditary optic neuropathy (266). An oligonucleotide ligation assay with colorimetric detection was developed for K-ras point mutations commonly associated with colorectal cancer (267). The oligonucleotide ligation assay was also adapted to the genotyping of the N-arylamine-acetyltransferase gene using 96-

well plates and robotic stations for high-throughput analysis (268). Harris et al. applied the branched-DNA technique to the detection of Trypanosoma brucei in human blood (269). Collins et al. improved the detection limit of the branched DNA hybridization assay for HIV to about 50 target molecules/mL by including the novel nucleotides isoC and isoG to prevent nonspecific hybridization. HIV patients whose RNA titers were lower than 500 molecules/mL were monitored (270). Nilsen et al. reported a physical-mathematical model for the construction of dendrimers (highly branched nucleic acid structures) from nucleic acid monomers by sequential hybridization (271). Vogelbacker et al. designed dendrimers with specificity to the HIV-1 virus LTR and the FSH receptor gene and applied to conventional Southern and dot-blot assays. A 100-fold signal amplification was observed (272). Romano et al. applied the nucleic acid sequence-based amplification (NASBA) technique for detection of HTLV I RNA and quantification of HIV-1 RNA (273). Bruisten et al. examined the influence of different storage temperatures and anticoagulants on the determination of HIV-1 RNA load by NASBA (274). The NASBA reaction was combined with fluorescence correlation spectroscopy for on-line detection of HIV-1 RNA. Fluorescence correlation spectroscopy measures the increase of the diffusion time of a fluorescent probe caused by its hybridization to the gag1 sequence and its subsequent extension during the cyclic phase of NASBA. Study of the hybridization/extension kinetics allowed estimation of the initial HIV-1 RNA concentration in the sample prior to amplification (275, 276). Smith et al. reported the development and clinical evaluation of a fully automated Q-βreplicase-amplified probe assay for direct detection of Mycobacterium tuberculosis in sputum (277). A novel strategy was investigated for selective degradation of the unhybridized probe in the Q-β-replicase amplification system. An oligonucleotide sequence/molecular switch was incorporated in the probe. In the unhybridized probe, this sequence forms the recognition site for Escherichia coli ribonuclease III (278). Thermophilic strand displacement amplification was used to amplify the Chlamydia trachomatis and the Mycobacterium tuberculosis DNA. The amplification products were measured in a homogeneous format by fluorescence polarization using a fluorescent oligonucleotide probe (279, 280). Ehricht et al. developed a self-sustained sequence replication-based cooperatively coupled isothermal amplification system (CATCH, cooperative amplification of templates by cross hybridization) and fluorescence detection methods for the monitoring of CATCH (281, 282). OLIGONUCLEOTIDE ARRAYS Lockhart et al. synthesized high-density oligonucleotide arrays using photolithography and applied them to the direct monitoring of a large number of mRNAs in parallel (283). Schena et al. used microarrays containing over 1000 human cDNAs from a peripheral blood library for monitoring of the differential expression of the cognate genes using a two-color fluorescence hybridization assay. Novel genes expressed by T cells were identified upon heat shock and protein kinase C activation (284). cDNA microarray technology was also used for the analysis of genes related to rheumatoid arthritis and inflammatory bowel disease (285). A pancreatic cancer-specific expression profile was derived by using arrays of pancreatic cancer cDNA libraries and differential hybridization (286). A method for covalent attachment of thiol-modified DNA

oligomers to self-assembled aminosilane monolayer films on fused silica or oxidized silicon by using heterobifunctional cross-linkers was described (287). A systematic study of the various steric factors affecting the hybridization of nucleic acids to oligonucleotide arrays was reported. It was found that the length of the spacer between the oligonucleotide and the solid support had the largest effect on hybridization (288). Oligonucleotide arrays were prepared by immobilizing 10 mers in polyacrylamide gel fixed on the glass slide of a microchip. Fluorescently labeled target RNA sequences were then analyzed by hybridization to the array (289). Oligonucleotides immobilized in polyacrylamide gel on a glass slide were used for hybridization analysis of rRNA sequences from environmental bacteria (290). A fiber-optic DNA biosensor microarray was described for analysis of multiple DNA sequences simultaneously. Different oligonucleotide probes were immobilized on the fibers and hybridized with fluorescently labeled target DNA (291). A new approach for constructing oligonucleotide arrays was described by combining solid-phase oligonucleotide synthesis with polymeric photoresist films (292). MINIATURIZATION OF NUCLEIC ACID ANALYSIS (CHIPS) Burke et al. reviewed microfabrication technologies for nucleic acid analysis (293). The use of microfabrication and array technologies for DNA sequencing and molecular diagnosis were also reviewed (294). Duke et al. investigated the feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing. The devise was able to resolve rapidly oligonucleotides containing several hundred bases (295). PCR and capillary electrophoresis were functionally integrated on a single microdevice. The sample DNA was amplified in a microfabricated PCR chamber and then it was injected electrophoretically (no manual transfer) to a capillary electrophoresis chip. The total analysis times, including PCR and electrophoresis, for a β-globin gene target and a Salmonella genomic DNA target were 20 and 45 min, respectively (296). Woolley et al. microfabricated capillary array electrophoresis chips that allow DNA sizing of 12 samples in parallel with a resolution of 10 bp and a higher throughput than conventional techniques. A laser-excited confocal fluorescence scanner was used for detection. The performance of the chip was tested by genotyping HLA-H, a marker gene for hereditary hemochromatosis. Following PCR and digestion, the sample DNA was mixed with a DNA ladder and sized in the chip. A two-color fluorescence detection system facilitated the detection of standard and unknown in the same channel (297). A microfabricated gel electrophoresis device was developed for ultrafast genotyping by short tandem repeat analysis. The device can perform repeated analyses without replacement of the sieving matrix (298). The entire process of cell lysis, multiplex PCR, and electrophoretic analysis were integrated on a single microchip. Cell lysis and PCR were carried out by thermally cycling the entire microchip in a commercial thermal cycler (299). A microfabricated electrochemiluminescence cell for determination of PCR products was reported (300). Poser et al. constructed silicon-based miniaturized thermocyclers with 1, 2, and 10 chambers for microliter volumes (301). NUCLEIC ACID SENSORS Zhai et al. reviewed the DNA-based biosensors that rely on nucleic acid hybridization. Electrochemical, acoustic, and piezoelectric biosensors were covered (302). Wang et al. reviewed the Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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construction of DNA electrochemical biosensors for environmental analysis. The coupling of a nucleic acid recognition layer with the electrochemical transducer and the use of the sensor for detection of sequences specific to various pathogens or for monitoring of pollutants interacting with the recognition layer were discussed (303). Caruso studied the immobilization of a biotinylated and a mercaptohexyl-labeled oligonucleotide onto modified gold surfaces using a quartz crystal microbalance and surface plasmon resonsnce (304, 305). Nilsson et al. employed real-time biospecific interaction analysis for screening PCR products for mutations. Biotinylated PCR products or biotinylated probes were immobilized onto a sensor chip (306). Adsorptive stripping potentiometry was applied to the reductive detection of nucleic acids at mercury electrodes (307). A chronopotentiometric biosensor was developed using an immobilized 17-mer peptide nucleic acid as the recognition molecule for detecting a specific mutation at the p53 gene (308). The formation of double- and triple-labeled complexes between homopolyribonucleotides in solution and at surfaces of the hanging mercury drop and carbon paste electrodes was studied by voltametric and chronopotentiometric methods (309). A screen-printed chronopotentiometric hybridization biosensor was described for detection of E. coli DNA sequences (310). Liu et al. immobilized a single-stranded DNA oligomer onto a graphite electrode and hybridized it with complementary peptide nucleic acid in the presence of ethidium bromide. The ethidium bound to the hybrids was determined by cyclic voltametry (311). An antibody-based submicrometer biosensor for benzo[a]pyrene DNA adducts was developed (312). Cai et al. studied various types of carbon electrodes for adsorptive stripping analysis of nucleic acids (313). An electrochemical sensor was constructed in which an oligonucleotide immobilized on a gold electrode and a ferrocene-modified oligonucleotide form a sandwich complex with the target DNA to give a redox current (314). Healey et al. developed a fiber-optic DNA sensor array for simultaneous monitoring of multiple hybridization events and examined its ability to detect point mutations. Oligonucleotide probes were immobilized on the sensor and hybridized with fluorescein-labeled target oligonucleotides (315). Napier et al. reported an electrochemical sensor based on the oxidation of guanine by a rutheniumbipyridine complex. The hybridization was monitored by cyclic voltametry of the complex (316). Evanescent wave optical sensors were developed for real-time monitoring of hybridization (317, 318). An electrochemical sensor was prepared by immobilizing DNA on a porous carbon electrode. The sensor was used to study the interaction between DNA and an osmium complex as a sequence-recognizing reagent (319). Various linkers were tested for the covalent immobilization of single-stranded DNA onto optical fibers for the development of biosensors (320). Theodore K. Christopoulos is Professor of Chemistry and Biochemistry at the University of Windsor, Ontario, Canada. He received his B.S. in pharmacy in 1982 and his Ph.D. in analytical chemistry in 1987 from the University of Athens, Greece. From 1989 to 1992 he was a Postdoctoral Fellow at the Department of Clinical Biochemistry, University of Toronto, Canada. He is a Fellow of the Canadian Academy of Clinical Chemists and a Diplomate of the American Board of Clinical Chemistry. His research interests are in the area of bioanalytical chemistry with focus on the development of highly sensitive nonradioactive and automatable methods for analysis of disease-related nucleic acid sequences and proteins. LITERATURE CITED (1) Narayanan, S. Adv. Clin. Chem. 1996, 32, 1-38. 434R

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