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Peer-reviewed

Affymetrix Genome-wide Human SNP Assay

Affymetrix全基因组人单核苷酸多态性试验

AB Alexis Bosman

发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1806 浏览次数: 7997

评审: Francesco Dal GrandeIrit AdiniAnonymous reviewer(s)

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Abstract

To assess genomic variation, it is possible to identify the single nucleotide polymorphisms (SNP) which an individual carries. Using the Affymetrix Genome-wide Human SNP Assay, it is possible to assess 906,600 SNPs on a single array. This protocol, the next iteration of the GeneChip Mapping 500K array set, is based directly on the manufacturers’ protocol and shows steps which are highly similar to that which is found here: http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf.

Keywords: Single nucleotide polymorphisms (单核苷酸多态性) search Array (阵列) search Human cells (人体细胞) search

Materials and Reagents

  1. 96-well V-bottom plates, elution catch (Greiner Bio-One GmbH, catalog number: 651101 )
  2. Greiner UV star transparent, 96-well (Greiner Bio-One GmbH, catalog number: 82050-788 )
    Note: Currently, it is “VWR International, catalog number: 82050-788”.
  3. Multiplate 96-well unskirted PCR plate (Bio-Rad Laboratories, catalog number: MLP-9601 )
  4. Multiscreen deep well plate (Merck Millipore Corporation, catalog number: MDRLN0410 )
  5. 2.4 ml deep well storage plate/pooling plate (Greiner Bio-One GmbH, catalog number: 780280 )
  6. 100 ml solution basin (VWR International, LabcorTM, catalog number: 730-014 )
  7. 55 ml solution basin (VWR International, LabcorTM, catalog number: 730-004 )
  8. 15 ml centrifuge tubes (VWR International, catalog number: 20171-020 )
  9. 50 ml centrifuge tubes (VWR International, catalog number: 21008-178 )
  10. 2.0 ml Eppendorf tubes (VWR International, catalog number: 20901-540 )
  11. 0.2 ml 12-strip tubes (VWR International, catalog number: 21008-940 )
  12. Lab tape
  13. Tough-Spots®
  14. Microseal ‘B’ adhesive seal (Bio-Rad Laboratories, model: MSB1001 )
  15. MicroAmp® clear adhesive film applied (Biosystems, catalog number: 4306311 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4306311”.
  16. Kimwipes (source as desired)
  17. Pipet tips as appropriate for pipettes (source as desired)
  18. AccuGENE® water, molecular biology-grade (Affymetrix, catalog number: 71786 )
  19. GenElute Mammalian Genomic DNA Miniprep (Sigma-Aldrich, catalog number: G1N70 )
  20. Affymetrix SNP 6 Core Reagent Kit, 100 reactions (Affymetrix, catalog number: 901706 )
    1. 10x Sty I buffer/NE buffer 3 (New England Biolabs)
    2. T4 DNA Ligase (400 U/μl) (New England Biolabs)
    3. T4 DNA Ligase Buffer (10x) (New England Biolabs)
    4. Adaptor, sty (50 μM)
    5. PCR Primer 002 (100 μM)
    6. 10x Nsp I buffer/NE buffer 2 (New England Biolabs)
    7. NspI (10 U/μl) (New England Biolabs)
    8. Adaptor, nsp (50 μM)
    9. Elution buffer (Buffer EB)
    10. Fragmentation buffer (10x)
    11. Fragmentation reagent (DNase I)
    12. DNA Labeling reagent
    13. Terminal deoxynucleotidyl transferase (TdT) (30 U/μl)
    14. Terminal deoxynucleotidyl transferase buffer (TdT buffer) (5x)
    15. Oligo control reagent (OCR), 0100
    16. 10 mg/ml BSA (100x)
    17. Hybridization Master Mix
  21. DNA marker (Bionexus, catalog number: BN2050 )
  22. 2% TBE gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: G8008-02 )
  23. Gel loading solution (Faster Better Media, catalog number: SB5N-8 )
  24. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  25. 50 ml magnetic beads (AMPure, catalog number: A63881 )
    Note: It is named “Agencourt AMPure XP-PCR Purification” on Beckman Coulter website.
  26. 4% TBE gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: G8008-04 )
  27. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
  28. MES sodium salt (Sigma-Aldrich, catalog number: M3885 )
    Note: All catalog numbers are listed in the Affymetrix datasheet (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf).
  29. Clontech TITANIUMTM DNA Amplification Kit (Clontech, catalog number: 639240 ) (see Recipes)
  30. 12x MES Stock Solution (see Recipes)

Equipment

  1. Applied Biosystems units
    1. 2720 Thermal Cycler
    2. GeneAmp® PCR System 9700
    3. GeneAmp® PCR System 9700 (silver block or gold-plated silver block)
  2. 48 Genome-Wide Human SNP Array 6.0 (one array per sample)
  3. GeneChip® Hybridization Oven 640
  4. Jitterbug
  5. Spectrophotometer plate reader
  6. Vacuum Manifold (Merck Millipore Corporation)
  7. Vortexer
  8. Cooler (chilled to -20 °C)
  9. Cooling chamber (chilled to 4 °C) (do not freeze)
  10. Cooling chamber (double, chilled to 4 °C) (do not freeze)
  11. Ice bucket, filled with ice
  12. Marker, fine point, permanent
  13. Microcentrifuge
  14. Pipet, 12-channel P1200
  15. Pipet, 12-channel P200
  16. Pipet, 12-channel P20 (accurate to within ± 5%)
  17. Pipet, serological
  18. Pipet, single channel P1000
  19. Pipet, single channel P200
  20. Plate centrifuge with deep-well capacity (54 mmH x 160 g)
  21. Optical plates
  22. Centrifuge plate holders
    Notes:
    1. Use only the PCR plate, adhesive film and thermal cyclers listed here.
    2. All relevant equipment numbers are listed in the Affymetrix datasheet.
    (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf)

Procedure

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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Bosman, A. (2016). Affymetrix Genome-wide Human SNP Assay. Bio-protocol 6(10): e1806. DOI: 10.21769/BioProtoc.1806.

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分类

分子生物学 > DNA > 微阵列/基因芯片

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