Single‐Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis

Samuel S. Chong1, Robert E. Gore‐Langton1, Mark R. Hughes1, Stanislawa Weremowicz2

1 Georgetown University Medical Center, Washington, D.C., 2 Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.10
DOI:  10.1002/0471142905.hg0910s64
Online Posting Date:  January, 2010
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Abstract

Preimplantation genetic testing, which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS), is a form of a very early prenatal testing. The goal of this method is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole‐genome amplification by primer‐extension preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single‐cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with support protocols for probe preparation and probe validation, which is recommended as a preliminary step before performing any PGD or PGS FISH analysis. Curr. Protoc. Hum. Genet. 64:9.10.1‐9.10.39. © 2010 by John Wiley & Sons, Inc.

Keywords: PGD; PGS; blastomere biopsy; mutation detection; FISH; probe validation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Specific Gene Loci in Single Diploid Cells
  • Support Protocol 1: Whole‐Genome Amplification of Single Diploid Cells by Primer‐Extension Preamplification (PEP)
  • Support Protocol 2: Preimplantation Embryo Biopsy
  • Support Protocol 3: Isolation of Blastomeres from Affected Embryos for Further Investigation
  • Support Protocol 4: Isolation of Single Lymphocytes/Lymphoblastoid Cells
  • Basic Protocol 2: FISH Analysis of Single Blastomeres
  • Support Protocol 5: Preparation of DNA Probes for FISH
  • Support Protocol 6: Probe Validation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Specific Gene Loci in Single Diploid Cells

  Materials
  • Single cells or blastomeres in 0.5‐ml tubes, lysed and neutralized with oil or wax overlay (see Support Protocols protocol 32, protocol 43, and protocol 54) and corresponding control tubes
  • 10× potassium‐free amplification buffer (unit 1.6)
  • 25 mM 4dNTP mix (Boehringer Mannheim; appendix 2D)
  • 20 µM forward and reverse primers (Table 9.10.1)
  • 5 U/µl Taq DNA polymerase (AmpliTaq from Perkin‐Elmer)
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • Light mineral oil (Perkin‐Elmer)
  • 20 µM forward and reverse inner (nested) primers for secondary PCR (Table 9.10.2)
  • DNA fragments from known homozygous CFTR ΔF508 mutant genomic DNA or HEXA exon 11 +TATC mutant genomic DNA and from homozygous normal DNA, PCR‐amplified using the inner primers for the appropriate locus (see Table 9.10.2)
  • 10× TBE buffer ( appendix 2D)
  • DNA gel loading buffer ( appendix 2D)
  • Size marker: ϕX‐174 plasmid digested with HaeIII
  • 0.1 µg/ml ethidium bromide ( appendix 2D)
  • DdeI digestion buffer (see recipe)
  • Restriction enzymes: DdeI, BclI, and HaeIII
  • 10× One‐Phor‐All Buffer Plus (Pharmacia Biotech)
  • 0.5‐ml clarified polypropylene PCR reaction tubes (Out Patient Services)
  • Thermal cycler (PTC‐100 or ‐200 from MJ Research; DNA Thermal Cycler or DNA Thermal Cycler 480 from Perkin‐Elmer)
  • PAGE minigel casting and electrophoresis apparatus (MiniProtean II, Bio‐Rad)
  • Additional reagents and equipment for primer‐extension preamplification (PEP; protocol 2 and unit 1.6; optional), heteroduplex analysis (unit 7.3), nondenaturing PAGE (unit 7.4), ethidium bromide staining ( appendix 2D), and agarose gel electrophoresis (unit 2.7)
    Table 9.0.1   MaterialsPrimer Sequences and Concentrations for Primary PCRPrimer Sequences for Secondary (Nested or Hemi‐Nested) PCR

    Locus Primer Sequence (5′ to 3′) a Final concentration b (µM) Volume b for 10 reactions (µl) Product size (bp)
    CFTRΔF508 1F GACTTCACTTCTAATGATGAT 0.8 20 193/190
    1R CTCTTCTAGTTGGCATGC 0.8 20
    DMD exon 4 1F TTGTCGGTCTCCTGCTGGTCAGTG 0.25 6.25 196
    1R CAAAGCCCTCACTCAAACATGAAGC 0.25 6.25
    DMD exon 8 1F GTCCTTTACACACTTTACCTGTTGAG 0.25 6.25 360
    1R GGCCTCATTCTCATGTTCTAATTAG 0.25 6.25
    DMD exon 12 1F GATAGTGGGCTTTACTTACATCCTTC 0.25 6.25 331
    1R GAAAGCACGCAACATAAGATACACCT 0.25 6.25
    DMD exon 45 1F AAACATGGAACATCCTTGTGGGGAC 0.25 6.25 547
    1R CATTCCTATTAGATCTGTCGCCCTAC 0.25 6.25
    DMD exon 48 1F TTGAATACATTGGTTAAATCCCAACATG 0.25 6.25 506
    1R CCTGAATAAAGTCTTCCTTACCACAC 0.25 6.25
    FVIII intron 18 HA1F CTACCTGGCTTAGTAATGGCTC 0.8 20 429
    HA1R AAAGGAATAAATTCCTTTTCCC 0.8 20
    HEXA exons 11 & 12 TS‐F GGTGTGGCGAGAGGATATTCCA 0.8 20 580/584
    TS‐R TCTCTCAGGCCTGAAAGAAGGG 0.8 20
    ZFX & ZFY Z868F ACCRCTGTACTGACTGTGATTACAC 0.4 10 495
    Z1338R GCACYTCTTTGGTATCYGAGAAAGT 0.4 10
    Locus Primer Sequence (5′ to 3′) c Final concentration (µM) d Volume for 10 reactions (µl) d Product size (bp)
    CFTRΔF508 2F TGGGAGAACTGGAGCCTT 0.8 20 154/151 e
    2R GCTTTGATGACGCTTCTGTAT 0.8 20
    DMD exon 4 1F TTGTCGGTCTCCTGCTGGTCAGTG 0.25 6.25 168
    2R CTGTGTCACAGCATCCAGACCTTGT 0.25 6.25
    DMD exon 8 2F TCATGGACAATTCACTGTTCATTAA 0.25 6.25 321
    1R GGCCTCATTCTCATGTTCTAATTAG 0.25 6.25
    DMD exon 12 1F GATAGTGGGCTTTACTTACATCCTTC 0.25 6.25 301
    2R TATGTTGTTGTACTTGGCGTTTTAG 0.25 6.25
    DMD exon 45 2F GCTCTTGAAAAGGTTTCCAACTAAT 0.25 6.25 506
    1R CATTCCTATTAGATCTGTCGCCCTAC 0.25 6.25
    DMD exon 48 1F TTGAATACATTGGTTAAATCCCAACATG 0.25 6.25 444
    2R AATGAGAAAATTCAGTGATATTGCC 0.25 6.25
    FVIII intron 18 HA2F ATCAAAGGATTCGATGGTATCT 0.8 20 339
    HA2R TTTCCTTTTTAGCAATTTTTCT 0.8 20
    HEXA exon 11 TS11F AACTGGTCACCAAGGCCGGCTT 0.8 20 118/122 f
    TS11R CCTTCAAATGCCAGGGGTTCCA 0.8 20
    HEXA exon 12 TS12F CAGGTACCCCTGAGCAGAAGGC 0.8 20 176
    TS12R GGTGGCTAGATGGGATTGGGTC 0.8 20
    ZFX & ZFY Z916F AYAACCACCTGGAGAGCCACAAGCT 0.4 10 344
    Z1233R TGCAGACCTATATTCRCAGTACTGGCA 0.4 10

     aR = A or G; Y = C or T.
     bAlso see protocol 1, steps 2a and 2b.
    Table 9.0.2   MaterialsPrimer Sequences and Concentrations for Primary PCRPrimer Sequences for Secondary (Nested or Hemi‐Nested) PCR

    Locus Primer Sequence (5′ to 3′) a Final concentration b (µM) Volume b for 10 reactions (µl) Product size (bp)
    CFTRΔF508 1F GACTTCACTTCTAATGATGAT 0.8 20 193/190
    1R CTCTTCTAGTTGGCATGC 0.8 20
    DMD exon 4 1F TTGTCGGTCTCCTGCTGGTCAGTG 0.25 6.25 196
    1R CAAAGCCCTCACTCAAACATGAAGC 0.25 6.25
    DMD exon 8 1F GTCCTTTACACACTTTACCTGTTGAG 0.25 6.25 360
    1R GGCCTCATTCTCATGTTCTAATTAG 0.25 6.25
    DMD exon 12 1F GATAGTGGGCTTTACTTACATCCTTC 0.25 6.25 331
    1R GAAAGCACGCAACATAAGATACACCT 0.25 6.25
    DMD exon 45 1F AAACATGGAACATCCTTGTGGGGAC 0.25 6.25 547
    1R CATTCCTATTAGATCTGTCGCCCTAC 0.25 6.25
    DMD exon 48 1F TTGAATACATTGGTTAAATCCCAACATG 0.25 6.25 506
    1R CCTGAATAAAGTCTTCCTTACCACAC 0.25 6.25
    FVIII intron 18 HA1F CTACCTGGCTTAGTAATGGCTC 0.8 20 429
    HA1R AAAGGAATAAATTCCTTTTCCC 0.8 20
    HEXA exons 11 & 12 TS‐F GGTGTGGCGAGAGGATATTCCA 0.8 20 580/584
    TS‐R TCTCTCAGGCCTGAAAGAAGGG 0.8 20
    ZFX & ZFY Z868F ACCRCTGTACTGACTGTGATTACAC 0.4 10 495
    Z1338R GCACYTCTTTGGTATCYGAGAAAGT 0.4 10
    Locus Primer Sequence (5′ to 3′) c Final concentration (µM) d Volume for 10 reactions (µl) d Product size (bp)
    CFTRΔF508 2F TGGGAGAACTGGAGCCTT 0.8 20 154/151 e
    2R GCTTTGATGACGCTTCTGTAT 0.8 20
    DMD exon 4 1F TTGTCGGTCTCCTGCTGGTCAGTG 0.25 6.25 168
    2R CTGTGTCACAGCATCCAGACCTTGT 0.25 6.25
    DMD exon 8 2F TCATGGACAATTCACTGTTCATTAA 0.25 6.25 321
    1R GGCCTCATTCTCATGTTCTAATTAG 0.25 6.25
    DMD exon 12 1F GATAGTGGGCTTTACTTACATCCTTC 0.25 6.25 301
    2R TATGTTGTTGTACTTGGCGTTTTAG 0.25 6.25
    DMD exon 45 2F GCTCTTGAAAAGGTTTCCAACTAAT 0.25 6.25 506
    1R CATTCCTATTAGATCTGTCGCCCTAC 0.25 6.25
    DMD exon 48 1F TTGAATACATTGGTTAAATCCCAACATG 0.25 6.25 444
    2R AATGAGAAAATTCAGTGATATTGCC 0.25 6.25
    FVIII intron 18 HA2F ATCAAAGGATTCGATGGTATCT 0.8 20 339
    HA2R TTTCCTTTTTAGCAATTTTTCT 0.8 20
    HEXA exon 11 TS11F AACTGGTCACCAAGGCCGGCTT 0.8 20 118/122 f
    TS11R CCTTCAAATGCCAGGGGTTCCA 0.8 20
    HEXA exon 12 TS12F CAGGTACCCCTGAGCAGAAGGC 0.8 20 176
    TS12R GGTGGCTAGATGGGATTGGGTC 0.8 20
    ZFX & ZFY Z916F AYAACCACCTGGAGAGCCACAAGCT 0.4 10 344
    Z1233R TGCAGACCTATATTCRCAGTACTGGCA 0.4 10

     cR = A or G; Y = C or T.
     dAlso see protocol 1, step 5.
     eFragment with ΔF508 deletion mutation is 3 bp shorter.
     fFragment with +TATC insertion mutation is 4 bp longer.

Support Protocol 1: Whole‐Genome Amplification of Single Diploid Cells by Primer‐Extension Preamplification (PEP)

  Materials
  • Calcium‐ and magnesium‐free medium (EB‐10, Vitrolife)
  • Acid Tyrodes solution (see recipe)
  • Embryo culture and wash medium (see recipe for culture and micromanipulation media)
  • Light paraffin oil for cultures (EM Science), filter sterilized, then washed and equilibrated with culture medium
  • Biopsy medium (see recipe for culture and micromanipulation media)
  • Blastomere wash medium (see recipe for culture and micromanipulation media)
  • Human embryos
  • Cell lysis buffer: 200 mM KOH/50 mM DTT (prepare fresh)
  • Light mineral oil for PCR (Perkin‐Elmer) or Chill‐Out 14 liquid wax (MJ Research)
  • Neutralization buffer (unit 1.6)
  • Micropipet puller (Sutter Instruments P‐97 or Narishige PB‐7)
  • Microforge (Narishige MF‐9 or Research Instruments MF42)
  • Glass microcapillaries (0.7‐mm i.d. × 0.87‐mm o.d., Pyrex glass from Vitrocom or 0.75‐mm i.d. × 1.0‐mm o.d., borosilicate glass from Sutter Instruments), dry‐sterilized 3 hr at 120°C
  • Tissue culture plates (Falcon):
    • 60 × 15–mm
    • 50 × 9–mm
    • 35 × 10–mm
  • Humidified 37°C, 5% CO 2 incubator (e.g., Forma)
  • 37°C warm plate
  • Olympus IX70 inverted microscope with Hoffman modulation contrast condenser (40‐mm WD) and objectives (HMC EF10×, HMC 20× LWD), mechanical X‐Y stage and Brook stage warmer (video camera, monitor, and videocassette recorder are optional)
  • Diamond pen
  • Antivibration table (Newport)
  • Two dual instrument holders (Narishige)
  • Drilling and biopsy micropipet controllers consisting of:
    • Micrometer drive
    • Gastight syringe (Hamilton)
    • Support base (base, micrometer drive and syringe are available preassembled from Stoelting)
    • Luer adapters
    • Polyethylene tubing (PE90, Intramedic)
  • Holding micropipet controller consisting of:
    • Gastight threaded‐plunger syringe (Hamilton)
    • Luer adapters
    • Polyethylene tubing (PE90, Intramedic)
    • Stand clamp
  • Two upright joystick‐type micromanipulators (Narishige; electronic coarse control and hydraulic fine control)
  • Two stereoscopic zoom microscopes with transmitted‐light base and (preferably) wide‐field oculars
  • 0.5‐ml clarified polypropylene PCR reaction tubes (Out Patient Services), irradiated 15 min in a UV cross‐linker at maximum energy
  • Micropipet controller consisting of mouthpiece (Fisher) connected through a 0.22‐µm Millex‐GV filter (Millipore) or equivalent to one end of a 60‐cm length of Tygon tubing with the other end fitted to either a microinstrument holder (Leica) or microcapillary pipet holder (Microcap, Drummond)
  • Laminar‐flow hood
  • Heating block

Support Protocol 2: Preimplantation Embryo Biopsy

  Materials
  • Human embryos (affected with a genetic disease of interest; see protocol 1) in drops of culture medium (see protocol 3)
  • Acid Tyrodes solution (see recipe)
  • Embryo culture and wash medium (see recipe for culture and micromanipulation media)
  • Blastomere wash medium (see recipe for culture and micromanipulation media)
  • Cell lysis buffer: 200 mM KOH/50 mM DTT (prepare fresh)
  • Light mineral oil for PCR (Perkins‐Elmer) or Chill‐Out 14 liquid wax (MJ Research)
  • Embryo handling pipets (see protocol 3, step 2)
  • Two stereoscopic zoom microscopes with transmitted‐light base and (preferably) wide‐field oculars
  • 35 × 10–mm tissue culture plates (Falcon)
  • Cell transfer pipets (50‐ to 60‐µm o.d.; see protocol 3, step 1)
  • Micropipet controller consisting of a mouthpiece (Fisher) connected through a 0.22‐µm Millex‐GV filter (Millipore) or equivalent to one end of a 60‐cm length of Tygon tubing with the other end fitted to either a microinstrument holder (Leica) or microcapillary pipet holder (Microcap, Drummond)
  • 0.5‐ml clarified polypropylene PCR reaction tubes (Out Patient Services), irradiated 15 min in a UV cross‐linker at maximum energy

Support Protocol 3: Isolation of Blastomeres from Affected Embryos for Further Investigation

  Materials
  • Primary lymphocytes or EBV‐transformed lymphoblastoid cell lines prepared from male and female gamete donors of each case under investigation
  • RPMI 1640 medium with HEPES (Sigma), without serum or protein supplement
  • Cell lysis buffer: 200 mM KOH/50 mM DTT (prepare fresh)
  • Light mineral oil for PCR (Perkin‐Elmer) or Chill‐Out 14 liquid wax (MJ Research)
  • Neutralization buffer (unit 1.6)
  • Micropipet puller (Sutter Instruments P‐97 or Narishige PB‐7)
  • Microforge (Narishige MF‐9 or Research Instruments MF42)
  • Glass microcapillaries (0.7‐mm i.d. × 0.87‐mm o.d., Pyrex glass from Vitrocom or 0.75‐mm i.d. × 1.0‐mm o.d., borosilicate glass from Sutter Instruments).
  • IEC Clinical centrifuge or equivalent
  • 0.5‐ml clarified polypropylene PCR reaction tubes (Out Patient Services), irradiated 15 min in a UV cross‐linker at maximum energy
  • 35 × 10–mm tissue culture plates (Falcon)
  • Two stereoscopic zoom microscopes with transmitted‐light base and (preferably) wide‐field oculars (dark‐field illumination may also be used)
  • Micropipet controller consisting of a mouthpiece (Fisher) connected through a 0.22‐µm Millex‐GV filter (Millipore) or equivalent to one end of a 60‐cm length of Tygon tubing with the other end fitted to either a microinstrument holder (Leica) or microcapillary pipet holder (Microcap, Drummond)
  • Laminar‐flow hood
  • Heating block
  • Additional reagents and equipment for determining cell viability by trypan blue dye exclusion ( appendix 3G)
CAUTION: Universal precautions should be observed in handling all biological materials of human origin. Caution should be exercised when pipetting cells potentially contaminated with Epstein‐Barr virus (EBV) or other pathogenic viruses.

Support Protocol 4: Isolation of Single Lymphocytes/Lymphoblastoid Cells

  Materials
  • Biopsy medium (see recipe for culture and micromanipulation media)
  • Embryo culture and wash medium (see recipe for culture and micromanipulation media)
  • Hypotonic/partial fixative solution (see recipe)
  • 3:1 fixative solution (see recipe)
  • Hybridization and probe mixture for PGD FISH ( protocol 7)
  • 70%, 80%, 90%, and 100% (v/v) ethanol
  • Rubber cement
  • Wash solutions (0.4× SSC, pH 7.0/0.3%Tween 20 and 2× SSC, pH 7.0/0.1% Tween 20; see reciperecipes)
  • 1× PBD buffer (MC Biomedicals)
  • DAPI II (Abbott Molecular)
  • MultiVysion probe mixture for PGS FISH ( protocol 7)
  • Antifade (Abbott Molecular)
  • D15Z4, DXZ1, and DYZ1 probe mixture for PGS FISH ( protocol 7)
  • Hybridization buffer for PGS or PGD (e.g., LSI and CEP hybridization buffer; Abbott Molecular)
  • 2× SSC, pH 7.0 ( appendix 2D)
  • 70% formamide solution (see recipe), 73°C
  • 20‐µl, 200‐µl, and 1‐ml pipettors (Gilson)
  • Micropipettor (Eppendorf)
  • Micropipet (80‐µm bore size)
  • 37°C warm plate
  • 2‐well Nunc dishes
  • Clay Adams glass slides with etched rings
  • Phase‐contrast microscope
  • Pencil
  • 37°C and 73°C water baths
  • Coplin jars
  • Microcentrifuge (e.g., Accu‐Tech‐Optical)
  • Microcentrifuge tubes (USA/Scientific Plastics)
  • 18‐mm2 glass coverslips (Fisher)
  • HyBrite hot plate (Abbott Molecular)
  • Moist chamber (Fig. 4.3.3)
  • 24 × 50–mm no. 1 glass coverslips (Corning)
  • Paper towels
  • Fluorescence microscope
  • Filter set (e.g., FITC, set no. 41001; TRITC, set no. 31002; DAPI, set no. 31000v2 from Chroma Technology, and single bandpass filters: Yellow, Blue, and Aqua from Abbott Molecular)
  • Field finder (Fisher)
  • 12‐mm2 glass coverslips (Fisher)
  • Parafilm
  • Additional reagents and equipment for embryo biopsy (see protocol 3)
CAUTION: Formamide and DAPI are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 2: FISH Analysis of Single Blastomeres

  Materials
  • Peripheral blood lymphocytes from each member of couple
  • Probes (Abbott Molecular) used in protocol 6
  • Chorionic villus cells (unit 8.3) from control specimen
  • Additional reagents and equipment for culture and metaphase harvest of peripheral blood (unit 4.1), in situ hybridization of probes to metaphase and interphase chromosomes (unit 4.3), and chromosome banding (unit 4.2)
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Literature Cited

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Key References
   ESHRE, 1999. See above.
  Presents the largest dataset to date compiled from multiple centers performing PGD. The data presented is comprehensive and includes information on clinical indications, reproductive histories, genetic analyses, pregnancies, and liveborn children.
   Scriven et al., 1998. See above.
  Provides a thorough review of meiotic segregation of reciprocal translocations and describes practical strategies for choosing probes to detect the possible unbalanced products of conception in a preimplantation setting.
Internet Resources
  http://www.acmg.net
  American College of Medical Genetics. Standard and Guidelines for Clinical Genetic Laboratories.
  http://www.phppo.cdc.gov/clia/regs/toc.aspx.
  Clinical Laboratory Improvement Amendments. CLIA:42 CFR Part 493‐1253.
  http://www.cap.org
  College of American Pathologists Laboratory Accreditation Checklist.
  www.fda.gov/cder/guidance/pv.htm
  Food and Drug Administration. Guidelines on General Principle of Process Validation.
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