European Journal of Human Genetics (1998) 6, 417–429 © 1998 Stockton Press All rights reserved 1018–4813/98 $12.00 t http://www.stockton-press.co.uk/ejhg ORIGINAL PAPER Parallel molecular genetic analysis Steven E McKenzie1,2, Elaine Mansfield3, Eric Rappaport4, Saul Surrey1 and Paolo Fortina4 1Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA and the du Pont Hospital for Children, Wilmington, DE 2Department of Chemical Engineering, University of Pennsylvania School of Engineering and Applied Science 3DiaDexus, 3303 Octavius Drive, Sante Clara, CA, USA 4Department of Pediatrics, University of Pennsylvania School of Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA We describe recent progress in parallel molecular genetic analyses using DNA microarrays, gel-based systems, and capillary electrophoresis and utilization of these approaches in a variety of molecular biology assays. These applications include use of polymorphic markers for mapping of genes and disease-associated loci and carrier detection for genetic diseases. Application of these technologies in molecular diagnostics as well as fluorescent technologies in DNA analysis using immobilized oligonucleotide arrays on silicon or glass microchips are discussed. The array-based assays include sequencing by hybridization, cDNA expression profiling, comparative genome hybridization and genetic linkage analysis. Developments in non microarray- based, parallel analyses of mutations and gene expression profiles are reviewed. The promise of and recent progress in capillary array electro- phoresis for parallel DNA sequence analysis and genotyping is summarized. Finally, a framework for decision making in selecting available technology options for specific molecular genetic analyses is presented. Keywords: chip DNA; microarray; mRNA level; capillary array electrophoresis; single nucleotide polymorphism; mutation detection; genetic analysis Introduction mutations. Knowledge of genomes of multiple other eukaryotic and prokaryotic organisms has progressed Progress from the Human Genome Initiative and as well. Increasing attention has been devoted to related efforts will soon result in the generation of the parallel molecular genetic analysis methods in order to sequence of all expressed human genes and the identity facilitate the search for multiple genetic alterations at of many more disease-causing or disease-predisposing the same time and to advance functional genomics (assessment of gene expression in specific tissues, Correspondence: Paolo Fortina, MD, The Children’s Hospital during development, or in disease states). This review of Philadelphia, 310-C Abramson Pediatric Research Center, summarizes recent developments in parallel molecular 34th Street and Civic Center Boulevard, Philadelphia, PA genetic analysis of interest to human genetics. 19104-1804, USA. Tel: (215) 590 3318; Fax: (215) 590-3660; Functional genomics studies increasingly necessitate Email: [email protected] Received 10 November 1997; revised 2 March 1998; accepted obtaining the results of multiple molecular genetic 31 March 1998 analyses at the same time. For example, genes which Parallel Molecular Genetic Analysis t SE McKenzie et al 418 cause human diseases may contain many different DNA Microarrays mutations linked to the phenotype, and efficient strate- gies to scan for these mutations are needed. Fur- DNA microarrays have been variously called DNA thermore, thousands of genes are expressed in any chips and DNA biochips. The common denominator is given cell. Differences in gene expression between cells a solid platform with a set of immobilized nucleic acid accompany both normal development and develop- species that participates in a solid-fluid interfacial ment of the malignant cell phenotype. Methods for interaction with a solution of complementary nucleic rapid identification of differentially expressed genes acid targets. Uses of the DNA microarray include will lead to increased understanding of cell develop- mutation and polymorphism detection, definition of gene expression profiles, genotyping, defining gene ment and perhaps better therapy for cancer. Parallel organization and mapping, and DNA sequence analysis molecular genetic analysis provides simultaneous infor- of previously uncharacterized regions, among oth- mation about many genes. Parallel analyses can also ers.16–30 Since a high density of genes can be studied in provide large amounts of information from small parallel, only a small amount of sample is needed. This amounts of starting cellular material. The concept of technology is mostly robotic driven; consequently it can parallel is distinct from but related to that of high easily be introduced into many research and clinical throughput, which results in the generation of large laboratories. amounts of molecular genetic information per unit time. It is possible to have a parallel process which is Mutation and Polymorphism Detection not high throughput if it takes a long time to obtain Microchips consisting of ordered arrays of oligonucleo- information about multiple genes. Conversely, most tide probes have been applied to hybridization-based high-throughput processes are parallel, because serial mutation detection schemes.16–39 This approach repre- molecular genetic analyses will tend to increase the sents in some sense a miniaturization of the use of overall time needed. Methods in which multiple wet immobilized oligonucleotides in 96 well plates with biochemical steps within a single tube or microwell for several important technical and conceptual differences. each molecular analysis are followed by gel electro- With glass or silicon chips, a high degree of parallel phoresis of radiolabeled products and then develop- array formation increases the information content. ment of a gel image by autoradiography have been With a high number of closely related oligos on a standard in molecular genetic analysis, but such meth- surface, information about partial matches may also be ods are intrinsically low throughput. In this review we obtained when perfect match duplex values are examine several methods of parallel molecular genetic obtained with fluorescence. Furthermore, the PCR step analysis which are of increasing importance in a which is often used to generate reagents for annealing number of fields.1–5 DNA microarrays, capillary array to immobilized probes can now be done in a silicon chip 40–42 electrophoresis, mass spectrometry, homogeneous solu- microenvironment. tion assays and hybrids of these methods are all The DNA microarray technique has been used to 24 particularly promising technologies. analyze the entire human mitochondrial DNA, to We begin with DNA microarrays with their myriad detect polymorphisms in the HIV-1 clade B protease 25 26,36 uses, then move to parallel mutation detection methods gene, and to detect mutations in BRCA1, the cystic fibrosis transmembrane receptor gene and and gene expression profiling methods that do not use 23,34 microarrays, and finally capillary array electrophoresis p53. In an extension of DNA microarray technol- approaches to DNA sequence analysis and micro- ogy, Pastinen and colleagues reported allele-specific satellite-based genotype analysis. Generalizable high- detection of 12 common disease-causing mutations in the Finnish population using a microarray and single- throughput analytic approaches accessible to any labo- nucleotide extension.43 Detection of beta-globin gene ratory with molecular genetics expertise will be alleles and mutations has also been reported by several highlighted. Areas which will not be discussed and for groups.28,44,45 which the reader is directed to recent reviews or references include gene expression profiling in situ,6 Gene Expression Profiling protein expression profiling using 2D gels and mass The potential for this type of gene expression chip is spectrometry analysis,7–9 parallel cellular and nucleic enormous. There are at least three things one wants to acid sample preparation,10–12 and bio-informatics know about an mRNA in a cell. Is it present or absent? approaches to data processing and interpretation.13–15 What is its quantitative level of abundance? What Parallel Molecular Genetic Analysis SE McKenzie et al t 419 difference is there in this level between two different the end user, after an initial investment, may meet the cellular samples? Parallel mRNA expression monitor- needs of many research laboratories. The Molecular ing by DNA microarray methods has been reported by Dynamics scanner employs confocal microscopy and a several different groups with different approaches. Two photomultiplier tube and is sensitive to 0.1 attomole of papers in the peer-reviewed literature summarize the fluorescent dye per square micron.52 Affymetrix experience in the gene expression area.46,47 For microarray methods for mRNA detection and The work demonstrates detection of transcripts at the 1 quantitation, gene discovery efforts may benefit from in 300 000 level, equivalent to 1 copy per cell, with a arraying probes for potentially all the expressed genes dynamic range between 1000 and 10 000. Fragmented in the human genome. Even before the whole human RNA target preparation appears advantageous kinet- genome is sequenced, the huge repertoire of expressed ically, in that information was available with hybrid- sequence tagged sites (ESTs) can be arrayed and 48,53 ization times as short as 2 hours. For the less abundant studied. Such a chip can be used to study gene transcripts, hybridization