QIAGEN Whole Genome Amplification REPLI-g Eliminating Sample Limitations, Potential Use for Reference Material MDA Technology Protocols Locus Bias Applications Kits & Service Summary
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 10.000 fold amplification WGA Immortalization Create more DNA in Sample Unlimited Number of Analyses Sequencing SNP genotyping Patient genotyping...
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
Amplification Bias? Experimental Design of Study Locus Bias 0.3 300 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
Amplification Bias Experimental Result with 8 TaqMan Loci Locus Bias Locus Representation (1ug WGA/1ug gdna) (%) 10000 1000 100 10 1 0.1 0.01 MDA (REPLI-g) DOP-PCR PEP Risk of Locus drop-out 0 300 30 3 0,3 Risk of Locus drop-out Starting material / ng Risk of Locus drop-out 300 30 3 0,3 300 30 3 0,3 Risk of Locus drop-out Data compromised Incorrect data interpretation
Low Amplification Bias in Many Loci, Applications and Samples Locus Bias MDA loci bias (47 loci) 10000 1000 100 Locus Representation 10 1 0.1 0.01 0.001 10,000x amp 0.0001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 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
Locus Bias Low Amplification Bias 10000 Low bias bemonstrated for 47 arbitrary loci on gdna samples from 44 different patients Intra locus bias is conserved over all patients 1000 100 Locus Representation 10 1 0,1 0,01 0,001 0,0001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Patient Number
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
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; 011155 1 2 3 4 5 6 7 8 9 10 11 12 A A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 551 120 97 119 71 72 82 107 64 69 90 72 697 758 830 841 831 831 881 844 791 785 716 769 B B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 150 70 82 106 89 69 81 73 72 162 77 97 606 812 902 857 827 823 858 806 834 749 818 781 C C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 68 83 92 91 77 93 61 72 81 130 75 68 796 816 883 823 778 813 786 850 800 815 858 806 D D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 64 135 94 80 107 180 86 66 55 86 75 126 807 754 825 732 681 732 793 775 767 769 787 780 E E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 71 57 96 92 68 85 113 82 65 69 86 139 816 865 882 817 711 798 684 818 839 837 599 814 F F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 117 58 87 88 104 73 112 77 83 88 78 45 815 735 884 801 710 850 838 844 801 853 773 737 G G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 55 75 89 92 78 68 143 63 132 103 65 85 733 846 895 808 709 849 792 886 847 861 787 768 H
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 http://www.qiagen.com/refdb/search_adv.asp Potential use for generation of standardized and immortal reference material where certified and approved reference material is not available