QIAGEN Whole Genome Amplification REPLI-g Eliminating Sample Limitations, Potential Use for Reference Material
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1 QIAGEN Whole Genome Amplification REPLI-g Eliminating Sample Limitations, Potential Use for Reference Material MDA Technology Protocols Locus Bias Applications Kits & Service Summary
2 QIAGEN REPLI-g WGA DNA Shortage Solved MDA Technology WGA Assay Very small amounts of sample small needle biopsies Preimplantation genetic diagnosis (PGD) Limited amount of sample Precious and irretrievable samples Genome wide studies fold amplification WGA Immortalization Create more DNA in Sample Unlimited Number of Analyses Sequencing SNP genotyping Patient genotyping...
3 REPLI-g Mini / Midi Kit Protocols Sample Types & Single-Tube Protocol Alkaline Denature/ Lysis REPLI-g Mix 40 µg DNA (Midi) Neutralize Incubate at 30 C 10 µg DNA (Mini) Direct amplification 10 ng gdna 0.5 µl Blood Cell Culture Biopsy QIAamp first Blood spots Buccal swab Mouthwash Serum/Plasma STR Genotyping TaqMan SNP Assay Published with validation in Genome Research 2003 (13) 954
4 Amplification Bias? Experimental Design of Study Locus Bias ng Genomic DNA No amplification DOP PEP MDA (REPLI-g) TaqMan analysis for copy number of 8 loci DOP-PCR: PEP-PCR: MDA: Degenerate Oligonucleotide Priming Primer Extension Pre-amplification Multiple Displacement Amplification with REPLI-g
5 Amplification Bias Experimental Result with 8 TaqMan Loci Locus Bias Locus Representation (1ug WGA/1ug gdna) (%) MDA (REPLI-g) DOP-PCR PEP Risk of Locus drop-out ,3 Risk of Locus drop-out Starting material / ng Risk of Locus drop-out , ,3 Risk of Locus drop-out Data compromised Incorrect data interpretation
6 Low Amplification Bias in Many Loci, Applications and Samples Locus Bias MDA loci bias (47 loci) Locus Representation ,000x amp Patient Number many SNPs and mutations across genome analysed by TaqMan gdna, whole blood, cells, DBS on S&S 903, IsoCode, FTA STR loci in Multiplex PCR STRs analysed by GeneScan
7 Locus Bias Low Amplification Bias Low bias bemonstrated for 47 arbitrary loci on gdna samples from 44 different patients Intra locus bias is conserved over all patients Locus Representation ,1 0,01 0,001 0, Patient Number
8 Detection and Elimination of Bias TaqMan SNP Genotyping Locus Bias Ideal: Tight Clusters of Alleles Sub-optimal: Allele Scatter Allele B Allele B Risk of Heterozygote dropout Allele A Allele A If allele bias is introduced in the whole genome amplification Heterozygotes are not clustered Heterozygotes may be difficult to score as it deviate from the diagonal Risk of Heterozygote dropout => repeat with more and pure gdna, extend amplification and check again
9 Kits & Service REPLI-g Kit and Service Offering REPLI-g Mini / Midi Kits For genetic analysis and for archiving Including all buffers, primers, Polymerase. Now also includes denaturation reagent REPLI-g Screening Kit For rapid screening for mutation analysis Offers high-throughput format and procedure REPLI-g WGA-Service Various output scales available Directly from gdna and from biological samples Quantification and Quality Assessment included Customer; A A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A B B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B C C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C D D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D E E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E F F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F G G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G H
10 Summary REPLI-g WGA Summary Consistent amplification from a variety of different starting materials Highly uniform amplification across the whole genome with minimal, but detectable sequence bias Uniform and standardized DNA yield for direct use in downstream applications without quantification Significantly more DNA from limited samples have broader access to the genetic content List of published references Potential use for generation of standardized and immortal reference material where certified and approved reference material is not available
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