Issue 4 Summer 2002 - Applied Biosystems

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14 technical communications 15 Years in a Thin Glass Tube The Development of Capillary Electrophoresis Technology for Genetic Analysis Applications A n early publication on automated DNA sequencing raised an intriguing prospect: the move away from denaturing polyacrylamide slab gels for sequence ladder separation. The primary impact of this publication (L. Smith et al, 1986) was the use of fluorescent dye-labelled sequencing primers, together with a laser excitation and photomultiplier detection system, to create a workable prototype for the automated, real-time reading of DNA sequences via Sanger dideoxy terminator chemistry. However, the electrophoretic separation apparatus was also unusual. A 50cm tube gel, of less than 2mm diameter, was chosen to maximise the sensitivity of that prototype sequence reader. When it came to the production of a usable commercial instrument, the throughput of a single tube gel was clearly inadequate so designers turned to a scanning system utilising a conventional, denaturing polyacrylamide slab gel (C. Conell et al, 1987). Development of automated DNA sequencing and DNA fragment analysis continued with slab gel-based systems for several years, but the attractions of capillary electrophoresis (CE) remained for separation. Established as a rapid and sensitive technique for the analysis of small molecules such as peptides and oligonucleotides, CE offered fast run times, high resolution and very efficient heat dissipation from ultra-high voltage separations (up to 20 kV). Applied Biosystems developed expertise in the field of analytical CE with the design and production of the 270 instrument in the late 1980’s. Flowable polymer solutions such as hydroxy-methyl cellulose could be used to fill narrow bore capillaries for separating analytes. Ultra-violet absorption detection determined quantities and retention times of materials separated by high voltage electrophoresis. For the analysis of synthetic oligonucleotides, polymerised denaturing gel-filled capillaries were used as the separation medium. These gave excellent resolution but offered relatively short lifetimes. Thus, the major challenge in developing a functional CE-based DNA sequencing or fragment sizing system lay in the polymer chemistry. By 1994 a capillary reagent kit had been produced for the ABI 270HT analytical CE instrument, for sizing of DNA fragments from restriction enzyme digests, still using UV detection. Improvements in polymer design and the application of the laser excitation technique from Applied Biosystems, allied to a charge-coupled device camera for fluorescence detection (as first used in the 377 system), resulted in the 1995 release of the ABI PRISM ® 310 Genetic Analyzer, the first commercial CE genetic analyzer. The initial model could sequence over 400 base pairs using a 75µM internal diameter, Teflon ® lined, fused silica capillary. It also had GeneScan ® Analysis Software for DNA fragment sizing applications. The 310 system became a mainstay for low-to-medium throughput laboratories. Within 2 years of its release, improvements came with new POP (Performance Optimised Polymers) and new 50µM i.d. capillaries, allowing better resolution and heat dissipation. These capillaries were unlined, utilising dynamic coating of the walls by the improved POP polymers to achieve superior performance and greatly increased service lifetimes. Sequencing specification increased to reads over 600 base pairs with POP-6 polymer, and reliable, rapid throughput typing of microsatellite markers was achieved with POP-4 polymer, and a 96-well sample plate to supplement the earlier 48 tube arrangement. In the medium-to-high capacity field, slab gel systems continued as the workhorses of the human genome sequencing project, and major medical and forensic microsatellite based typing programmes. The 377 system finally reached 96 lane capacity, but the demand for ultra high-throughput, rapid turnaround systems continued, and the goal of complete, walk away, 24-hour plus operation (as offered by the 310 system) could not be addressed by slab gel instruments. Applied Biosystems collaborated with Hitachi Ltd, holders of key patents for analytical CE, and by late 1998 released the technical communications 96 capillary ABI PRISM ® 3700 DNA Analyzer for Genome scale projects. Capable of fully automated loading and running of samples from a series of 96- or 384-well microtitre plates, the 3700 system was a key factor driving the rapid sequencing of the human genome. Development of CE polymer chemistry continued with the release of POP-5 polymer in 2000, for enhanced sequencing capability on the 3700 system, and very recently the creation of POP-37 polymer for this instrument. This offered further increases in sequencing and fragment analysis throughput. Another joint project with Hitachi Ltd., produced the ABI PRISM ® 3100 Genetic Analyzer, a 16-capillary system designed for medium-to-high throughput laboratories and core facilities that were not so ‘production oriented’ that did not require the dedicated capacity of the 3700 system. Based on proven 50µM capillaries and POP polymers, the 3100 system has further increased the performance of CE based DNA analysis, with sophisticated laser optics giving increased sensitivity and signal to noise ratio. A range of capillary array lengths for this instrument has enabled extremely rapid Single Nucleotide Polymorphism (SNP) genotyping runs on 22cm capillaries, and 1,000 base plus sequence reads on 80cm arrays. This is in addition to more conventional applications run on 36cm and 50cm capillaries. The standard 3100 system is now joined by the new ABI PRISM ® 3100-Avant Genetic Analyzer, a 4-capillary introductory version of the full instrument. It offers the low-to-medium throughput laboratory all the versatility of the 3100 system at lower initial cost. As the needs of the laboratory grow, this instrument can be upgraded to full 16-channel capacity at a time appropriate to the workload. Applied Biosystems continues to advance the entire line of CE-based instrumentation through enhanced performance and the development of new applications. The newest capillary electrophoresis instruments to be introduced by Applied Biosystems, in collaboration with Hitachi High-Technologies Corporation, are the 3730xl and 3730 DNA Analyzers. These instruments have been specifically designed to deliver improved production capacity and lower running costs for the high-throughput production laboratory. References: 1. Smith, L.M., J.Z. Sanders, R.J. Kaiser, P. Hughes, C. Dodd, C.R. Conell, C. Heiner, S.B.H. Kent and L.E. Hood. (1986). Fluorescence detection in automated DNA sequence analysis. Nature 321: 674 - 679. 2. C.R. Conell, S. Fung, C. Heiner, J. Bridgham, V. Chakerian, E. Heron, B. Jones, S. Menchen, W. Mordan, M. Raff, M. Recknor, L. Smith, J. Springer, S. Woo and M. Hunkapiller. (1987). Automated DNA sequence analysis. BioTechniques 5: 342 - 348. For more information on: ABI PRISM family of CE Genetic Analyzers enter: No. 412 15
16 technical communications Real-Time PCR Analysis of Gene Expression Pattern in Asthma Research T he research of allergic reactions like asthma shows that not one but many parameters are altered in this disease. So far, analytical methods for quantitative RNA analysis of many samples and the required screeningmethods have not been available. With the help of Real-Time PCR even complex gene expression patterns including genes that are expressed only weakly can be analysed quantitatively and automated. This technique can be used in research of allergic diseases. Current methods for the analysis of gene expression are only detecting the formation of gene products indirectly. With these immunological methods just a couple of parameters can be detected in parallel. Furthermore, they are rather low in sensitivity and assay development is time consuming. Here, Real-Time PCR-technology leads to new ways. This very sensitive method only requires very low amounts of sample material. It even allows a direct measurement of transcripts if combined with a reverse transcription step. Thus, it gives a real view into the status of genes expressed in cells. This enables the detection of differential transcription patterns in disease related cells if compared with healthy ones. The pharmaceutical industry already uses results produced with Real-Time PCR for the development of new drugs. The goal is to identify substances that interfere selectively and specifically with altered activation or deactivation-processes in diseased cells. With Real-Time PCR, monitoring of pharmacological targets is less time-consuming, more convenient, cheaper and more sensitive – e.g. using the automated ABI PRISM ® 7900HT Sequence Detection System. Real-Time PCR not only reduces the so-called The ABI PRISM ® 7900HT Sequence Detection System random screening of big numbers of synthetic and/or natural substances. It even simplifies the validation of results in animal testing and in humans too, making this research ethically more acceptable. Dr. Andreas Pahl from the Department of Pharmacology at the University of Erlangen, Germany has already used this screening approach successfully for many years. The goal of Dr. Pahls´ research is the identification of candidate substances for the development of new drugs (leads) that selectively inhibit a sub-population of T-helper cells, the Th2-cells. The basis of this research is the so-called Th1/Th2-paradigm (see figure 1). The two sub-populations of T-helper cells produce different cytokines. While Th1-cells express the cytokines IL-2, IFN-g and TNF-ß, Th2-cells produce the cytokines IL4, -5 and -13. Figure 1. The Th1/Th2-paradigm shows how Th2-cells are involved in the development of allergic reactions. Both subpopulations of T-helper cells produce different cytokines as a result of an antigen-stimulus. Th1/Th2-Paradigm In comparison with Th1-cells, Th2-cells do not only react against typical antigenes like nematodes or protozoa’s. Th2-cells are the starting point of a reaction-cascade resulting in the release of histamines because they react on allergens, too. Thus, Dr. Pahl tries to identify substances that selectively inhibit the expression of one or more cytokines expressed by Th2-cells, but that do not interfere with the expression of Th1-cytokines. With the help of Real-Time PCR many parameters that are altered in a disease can be monitored on the same reaction plate. In his highly automated laboratory Dr. Pahl technical communications incubates cell-cultures in 384-well format with drug libraries. RNA is isolated from these cells and following reverse transcription the expression of the relevant cytokines is analysed. Real-Time PCR is performed on the 7900HT system with 24-hour unattended operation. This allows the quantitative analysis of up to 5,300 reactions per day. Finally, the results are stored in a database and analysed for a certain expression pattern (see figure 2) in order to identify potential leads for drug development. Figure 2. The results of the drug library screening are collected in a database. A pre-defined pattern is used to search for lead-substances that specifically inhibit cytokines expressed by Th2-cells. Hits are highlighted and will be studied further. Drug screening results: Pattern Searching Pattern definition : IL-2 > 0 IL-4,5,13 < -1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Conclusion Plate Well IL-2 IL-4 lL-5 lL-13 PatternMatch A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B1 B2 B3 B4 0,00 0,82 1,49 -0,51 1,78 -0,08 -0,58 0,27 0,00 -0,59 -1,07 0,00 0,00 -1,27 -1,81 -0,82 Real-Time PCR allows a higher throughput than conventional immunologically-based methods. Its very high sensitivity and large dynamic range allows the analysis of genes expressed at low levels. The basic PCR chemistry has been developed further into a very robust, easy to optimise and standardised method. Assay development is not only fast, but the assay format itself makes the analysis of many parameters on the same reaction plate very convenient too. Therefore, Real-Time PCR offers a new strategy for the quantitative analysis of gene-expression patterns. For more information on: 0,00 0,47 0,97 -0,69 1,07 -1,10 -0,63 -2,34 0,00 0,06 0,50 0,00 0,00 -1,59 -1,71 -0,38 0,00 1,39 -0,18 0,09 -0,82 1,10 1,20 -1,4 0,00 -1,40 -0,79 0,00 0,00 -1,09 -0,49 -1,33 0,00 0,73 -0,35 0,30 -0,38 -0,85 -1,19 -3,1 0,00 1,39 -0,18 0,00 0,00 1,10 1,20 -0,96 Real-Time PCR information pack enter: No. 413 X 17
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