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Mutational Profiling of Human Disease Genes Soumya N idtha 1 , Richard R. Bennett2 Karen Poulter1, Rixun Fang 1, Robert N utter1, and Primo Baybayan1 , Applied Biosystems, Foster City, CA 944041 ; Division of Genetics, Children's H ospital, Boston, MA 2 ABSTRACT Identification of mutations in human genes to determine the genetic basis of diseases is a challenge. A combination of methods such as sequencing, Denaturing High Performance Liquid Chromatography (DHPLC) and Single Strand Conformational Polymorphism (SSCP), are used to study mutation profiles. However, even though sequencing is the most accurate and complete methodology for mutation detection it is perceived to be difficult and expensive. To eliminate the time consuming step of designing, optimizing and validating of PCR primers for resequencing human disease genes, Applied Biosystems has developed the V ariantSEQr™ Resequencing System. This system can be easily integrated into any sequencing pipeline. VariantSEQr™ takes advantage of the automated capillary electrophoresis platform, reagents and SeqScape® v2.1.1 software for mutation detection and report generation. This fully integrated and optimized system allows researchers to study: 1. Disease genes with dense mutation spectrum and polygenic diseases 2. Larger cohorts and therefore conclusions drawn may be more statistically significant We describe a new tool for mutation and SNP discovery that is easy to integrate into any sequencing laboratory and takes advantage of the high degree of data accuracy provided by DNA sequencing. INTRODUCTION The wealth of data generated during the human genome project is very valuable to elucidate the relationship between sequence variation and susceptibility to disease. M arkers such as SNPs (Single Nucleotide Polymorphisms), insertions and deletions in the human genome are useful in two ways. Firstly, the polymorphism can cause differences in the gene function or regulation that directly contribute to disease processes or serve as markers because of associations with causative mutations. However, it is still necessary to identify the type and location of the mutation in order to develop strategies for treatment. Mutations important in many diseases are found in all regions of the gene, including regulatory regions, exons and introns. A number of methods to screen for putative point mutations, insertions and deletions exist today. Some of the methods include DHPLC, SSCP, a variation of SSCP called DOVAM (Detection of Virtually All Mutations), HA (Heteroduplex Analysis) and, DGGE (Denaturing Gradient Gel Electrophoresis). Each technique assumes mutations are rare and therefore it is more economical to screen for regions where mutations occur. However, each have significant shortcomings including extensive primer design and PCR validation, allele-specific amplification due to SNPs in primer sites, inconsistent data generation and no automated data analysis. Additionally, not all mutations are reliably detected and recent publications show mutations occur, on average, once every 250-300 nucleotides in a population. This means in order to know the nature of putative mutations found by screening methods, it is necessary to design and validate new primers and sequence each region followed by very laborious analysis of the data. Figure 1. Identification of mutations of interest ultimately lead to Sequencing RESULTS In order to precisely determine the location and nature of mutations, Applied Biosystems developed the VariantSEQr™ Resequencing System which employs DNA sequencing for simultaneous discovery and genotyping of all mutations. VariantSEQr™ Resequencing System is a fully integrated system capable of quickly resequencing human genes and Mitochondrial DNA in a cost effective manner. This system consists of PCR primers of known performance. It can be fully integrated -detection of mutation variation via sequencing through data analysis using all instruments, reagents and data analysis software. DHPLC is one of the methods used to screen samples for mutations. Upon mixing, denaturing and reannealing of amplicons containing one or more mismatches characteristic peak patterns for homozygous and heterozygous samples are obtained. For example in a study on a large human gene, size~2.4million base pairs with mutations spanning all exons using 87 primers, screening known mutations first by DHPLC, followed by sequencing for a more complete mutation profile may not be most cost effective approach. Figure 3. Reliable amplification and sequence data using Universal PCR and Sequencing protocol PCR amplicons generated using optimized PCR primers that are tailed with M13 priming sites and universal protocol for easy experimental set up for projects of any size. Figure 2. Workflow for each primer set when using DHPLC for screening m utations Sequence data using universal sequencing primers and protocol. Heterozygote variants are detected and reported accurately by SeqScape® softwareV2.1.1. MATERIALS AND METHODS DHPLC: Genom ic DNA Preparation- Assessed Genomic DNA Quality. Ready -to-use Resequencing Sets for: Large set of Kinase gene fam ily -cancer studies, Muscular dy strophy , Drug metabolism Enzy mes, A utoimmune, Inflammatory, Cardiovascular, Metabolic Disorders, Mitochondrial DNA STEP 1:PCR A mplification and WAVE® sy stem for DHPLC analysis PCR primers were optimized for gene. PCR reactions were run on the validated PCR primers. Unpurified PCR amplicons from specimens were mixed in a 1:1 ratio with an aliquot of unpurified PCR amplicon from an unaffected specimen and run on WAVE system. STEP 2:Sequence analy sis PCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, V alencia, Ca.). DNA concentration was determined by spectrophotometry. Unincorporated dye and other contaminants were removed with SEQueaky Kleen 96well kit (BI O-RAD Laboratories, Hercules, Ca.) Both strands were sequenced on an ABI PRISM® 310 DNA Sequencer Data was analyzed with the SequencherTM (software (Genecodes Inc. Ann Arbor, Michigan). VariantSEQrTM Resequencing System: Genom ic DNA Preparation- Assessed Genomic DNA Quality STEP 1: PCR A mplification PCR Reactions were prepared with validated resequencing primer pair for gene of interest. PCR Reactions were run on the ABI GeneAmp® PCR System 9700. PCR products were cleaned up using Exo-SAPI T DNA Sequencing Amount of PCR product was estimated on agarose gel Sequencing Reactions were run. Extension products were purified and run on Applied Biosystems 3730 DNA Analyzer Data was analyzed using SeqScape® software V2.1.1 (Applied Biosystems, Foster City, CA) Extensive Optimization required: for each prim er set Optimizing PCR primers~1 week Optimizing conditions for amplicon hybridization~1 week Analyze DHPLC data~1week data incomplete, optimize primers for sequencing~1 week Analyze sequence data~1 week Figure 3. Workflow using VariantSEQr™ Resequencing System for a gene Order on myScienceSM Research Order on myScienceSM Research Environment-web based gene catalog: Environment-web based gene catalog: Optimized and ready to use primer pairs for Optimized and ready to use primer pairs for Resequencing hum an genes Resequencing hum an genes PCR PCR Sequencing Sequencing VariantSEQr TM employs sequencing, a well established and robust technique to screen and discover genetic variation in 1,000s of human genes. There are 3 sets offered for each gene or Resequencing Se t (RSS)Complete Coding Non-coding Universal PCR protocol for all genes/RSS. Universal sequencing prim ers and protocol for all genes/RSS. CONCLUSIONS Sequencing is the most direct and complete method for the detection of all mutation making it an efficient tool for routine resequencing projects of human genetic diseases for all types of mutations. Methods that have been developed to first screen for mutations require considerable time to design and optimize primers, optimize running conditions and analyze the data. Once putative mutations have been identified, it is still necessary to sequence every region to identify all mutations This takes time and money. VariantSEQr™ Resequencing System uses sequencing: • Verification of well known mutations and discovery of rare mutations at the same time. • No primer optimization is necessary • All primers use a universal protocol • Detection of genetic changes as well as interpretation of results is rapid and accurate. . REFERENCES 1. Detection of muta tions in t he dys troph in g en e v ia auto mate d DH PL C screen ing an d direc t seq uencing Rich ard R Bennett1 , Jo han den Dun nen2 , K ristine F O'Brien3 , Basi l T Darras4 and Louis M Kunk el1, 3, 5 BM C Gene tics 2001, 2 :17 1Division o f Genetics, Children 's Hospital, Boston , Massa chu setts, USA 2Center for Huma n an d Clinical Genetics, Leid en University Medi cal Cente r, Leiden , Ned erland 3Depa rtment of Ge netics, Har vard M edical S choo l, Boston, Massa chusetts, US A 4Depa rtment of Neuro logy, Chi ldren's Hosp ital, B oston, Massachusetts, USA 5How ard Hughes Medical Insti tute, Children 's Hospital, Ha rvard M edical Scho ol, Boston , Massachu setts, USA 2. Seq uence -Bas ed Link age A nalysis Itay Furman,1 M ark J. Rie der,2 Suz ann e da Ponte,2 Dana P. Carrington,2 Deborah A. Nickerson,2 L eonid Kruglyak ,1,3 and Kyria cos Mark iano s1 1Divisio n of Human Biology, Fred Hutch inso n Cancer Research Ce nter, a nd 2 Dep artment of Ge nome Sciences, Un iver sity of Washi ngto n, Seattle; a nd 3 How ard Hughes Medical Institute , Che vy Chase, MD 3. Meth od s in Mo lecular Biology : Sing le N ucleo tide P olymo rphisms, me tho ds an d prot oco ls. Vol 212 Edited b y Pui-Yan Kw ok ACKNOWLEDGEMENTS Data A naly sis Data A naly sis Powerful software for accurate mutation call and reporting. Thank s to Richa rd R B ennett for sharing his da ta and exp ertise on DHPLC. We w ou ld al so lik e to thank the Applie d Bi osy stems R&D tea m for help ing generate sequ ence data. TRADEMARKS/LICENSING For Research Use On ly. Not for use in d iagnosti c procedures. The PCR p roce ss is covered by patents ow ned by Roche M olecular Systems, In c. and F. Ho ffmann -La Roche, Ltd. Appl ied B iosystems, ABI PRISM , and SeqScape are registe red trademark s and AB De sign , myScience, and VariantSEQr are trad ema rks of A pplera Corporation or its subsidiaries i n the U.S. and/or certain oth er countries. TaqM an a nd Ge neAmp a re reg istered tra demarks o f Roche Mo lecular Sys tems, Inc. A ll other trad ema rks a re the property of their r espe ctive ow ners.