Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols

Martin Ingelsson1, Youngah Shin1, Michael C. Irizarry1, Bradley T. Hyman1, Lena Lilius2, Charlotte Forsell2, Caroline Graff2

1 Harvard Medical School/Massachusetts General Hospital, Charlestown, Massachusetts, 2 Karolinska Institutet, Stockholm
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.14
DOI:  10.1002/0471142905.hg0914s38
Online Posting Date:  November, 2003
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Abstract

Disease‐associated gene polymorphisms provide both scientific insight into pathophysiological mechanisms and clinical information regarding risk and progression. Of special interest is the ɛ4 allele of the apolipoprotein E gene, which has emerged as a substantial risk factor for late‐onset forms of Alzheimer disease and also influences the risk of cardiovascular disease. Genotyping of apolipoprotein E can be performed by several methods; presented here are a quality and cost‐benefit analysis of four different protocols on a cohort of 42 clinical samples is included in the unit. Each method resulted in genotyping with a high sensitivity and specificity. The newer microtiter‐plate‐based high‐throughput techniques, fluorescence polarization and SNaPshot analysis, were as reliable as the traditional techniques of restriction fragment length polymorphism analysis and reverse hybridization. The reverse hybridization method tends to be more cost‐ and time‐effective when the number of analyses is limited, although economy of scale favors fluorescence polarization or SNaPshot analysis in larger studies. The latter approaches also provide the flexibility to investigate other polymorphic disease markers.

Keywords: neurodegenerative diseases; apolipoprotein E; single nucleotide polymorphisms; genotyping; high‐throughput analysis

     
 
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Table of Contents

  • Basic Protocol 1: APOE Genotyping by RFLP Analysis
  • Alternate Protocol 1: APOE Genotyping by Reverse Hybridization
  • Alternate Protocol 2: APOE Genotyping by Fluorescence Polarization (FP)
  • Alternate Protocol 3: APOE Genotyping by SNaPshot Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: APOE Genotyping by RFLP Analysis

  Materials
  • 3 to 5 ng/µl genomic DNA (isolated from blood, see unit 14.4) in TE buffer, pH 7.4 ( appendix 2D)
  • APOE oligonucleotide primers:
    • Forward: 5′‐TAA GCT TGG CAC GGC TGT CCA AGG A‐3′
    • Reverse: 5′‐ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC‐3′
  • 100 mM 4dNTP mix (Roche; 25 mM each dNTP; also see appendix 2D)
  • 5 U/µl Taq DNA polymerase (Roche) Dimethylsulfoxide (DMSO)
  • 10× PCR amplification buffer containing 15 mM MgCl 2 (Roche)
  • 10 U/µl HhaI restriction endonuclease (Promega)
  • 10× buffer C (Promega)
  • Low‐melting agarose (e.g., Fisher) NuSieve agarose (FMC Bioproducts)
  • 10× TBE buffer ( appendix 2D)
  • 10 mg/µl ethidium bromide ( appendix 2D)
  • 6× gel loading buffer ( appendix 2D)
  • Additional reagents and equipment for PCR (unit 7.1) and agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: APOE Genotyping by Reverse Hybridization

  Materials
  • 3 to 5 ng/µl genomic DNA (isolated from blood, see unit 14.4) in TE buffer, pH 7.4 ( appendix 2D)
  • INNO‐LiPA ApoE kit (Innogenetics) including:
    • ApoE amplification buffer
    • ApoE primer mix
    • MgCl 2 solution
    • Glycerol solution
    • Membrane strips
    • Hybridization solution
    • Stringent wash solution
    • Denaturation solution
    • Conjugate, substrate
    • Conjugate diluent
    • Substrate solution
    • Substrate buffer
    • Rinse solution
    • Test troughs
    • Incubation trays (all provided in the kit)
  • Taq DNA polymerase (Roche)
  • 45°C water bath with shaking platform
  • Calibrated thermometer
  • Additional reagents and equipment for PCR (unit 7.1)

Alternate Protocol 2: APOE Genotyping by Fluorescence Polarization (FP)

  Materials
  • 3 to 5 ng/µl genomic DNA (isolated from blood, see unit 14.4) in TE buffer, pH 7.4 ( appendix 2D)
  • APOE oligonucleotide primers for PCR:
    • Forward: 5′‐TAA GCT TGG CAC GGC TGT CCA AGG A‐3′
    • Reverse: 5′‐ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC‐3′
  • 40 mM 4dNTP mix (Roche; 10 mM each dNTP; also see appendix 2D)
  • 5 U/µl Taq DNA polymerase (Roche)
  • Dimethylsulfoxide (DMSO)
  • 10× PCR amplification buffer containing 15 mM MgCl 2 (Roche)
  • Exo‐SAP‐IT (USB)
  • Oligonucleotide primers for SNPs (elongation reaction):
    • 5 ′‐GGC GCG GAC ATG GAG GAC GTG‐3′ (APOE 112)
    • 5 ′‐CGG CCT GGT ACA CTG CCA GGC‐3′ (APOE 158)
  • AcycloPrime‐FP SNP Detection Kit (Perkin‐Elmer Life Sciences) including:
    • 10× reaction buffer
    • AcycloTerminator mix
    • AcycloPol DNA polymerase
  • 96‐well PCR plates (MJ Research)
  • Thermal cycler accommodating 96‐well PCR plates
  • 80 °C water bath
  • 96‐well plate reader capable of fluorescence polarization measurements (e.g., Victor series from Perkin‐Elmer Life Sciences)
  • Additional reagents and equipment for PCR (unit 7.1)

Alternate Protocol 3: APOE Genotyping by SNaPshot Analysis

  Materials
  • 3 to 5 ng/µl genomic DNA (isolated from blood, see unit 14.4) in TE buffer, pH 7.4 ( appendix 2D)
  • APOE oligonucleotide primers:
    • Forward: 5′‐CCA AGG AGC TGC AGG CGG CGC A‐3′
    • Reverse: 5′‐GCC CCG GCC TGG TAG ACT GCC A‐3′
  • 100 mM 4dNTP mix (Roche; 25 mM each dNTP; also see appendix 2D)
  • Taq DNA polymerase (Roche)
  • Dimethylsulfoxide (DMSO)
  • 10× PCR amplification buffer containing 15 mM MgCl 2 (Roche)
  • QiaQuick DNA purification kit (Qiagen)
  • Oligonucleotide primers for minisequencing:
    • 5 ′‐CGG ACA TGG AGG ACG TG‐3′ (APOE 112)
    • 5′‐TTT TTT TTT TCC GAT GAC CTG CAG AAG‐3′ (APOE 158)
  • SNaPshot mix (containing fluorescent [F]ddNTPs; Applied Biosystems)
  • Calf intestinal phosphatase (CIP; New England Biolabs)
  • DNA size standard ladder (GeneScan‐120 LIZ, Applied Biosystems)
  • Hi‐Di formamide (Applied Biosystems)
  • 96‐ or 384‐well PCR plates (MJ Research)
  • 75°C water bath
  • ABI PRISM 3100 Genetic Analyzer (Applied Biosystems)
  • GeneMapper 2.0 or GeneScan software (Applied Biosystems)
  • Additional reagents and equipment for PCR (unit 7.1)
NOTE: The amplification primers used in the SNaPshot procedure are different from those used in the Basic Protocol, since the protocols were developed in different laboratories.
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Figures

Videos

Literature Cited

Literature Cited
   Chen, X., Levine, L., and Kwok, P.‐Y. 1999. Fluorescence polarization in homogenous nucleic acid analysis. Genome Res. 9:492‐498.
   Corder, E.H., Saunders, A.M., Risch, N.J., Strittmat‐ter, W.J., Schmechel, D.E., Gaskell, P.C.J., Rimmler, J.B., Locke, P.A., Conneally, P.M., and Schmader, K.E. 1994. Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat. Genet. 7:180‐184.
   Eichner, J.E., Terence Dunn, S., Perveen, G., Thompson, D.M., Stewart, K.E., and Stroehla, B.C. 2002. Apolipoprotein E polymorphism and cardiovascular disease: A HuGE review. Am. J. Epidemiol. 487‐495.
   Gibson, N., Gillard, H., Whitcombe, D., Ferrie, R., Newton, C., and Little, S. 1997. A homogeneous method for genotyping with fluorescence polarization. Clin. Chem. 43:1336‐1341.
   Hallman, D., Boerwinkle, E., Saha, N., Sandholzer, C., Menzel, H., Csazar, A., and Utermann, G. 1991. The apolipoprotein E polymorphism: A comparison of allele frequencies and effects in nine populations. Am. J. Hum. Genet. 49:338‐349.
   Hixson, J. and Verniery, D. 1990. Restriction isotyp‐ing of human apolipoprotein E by gene amplification and cleavage by HhaI. J. Lipid. Res. 31:545‐548.
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   Nishimura, T., Takeda, M., Shinosaki, K., Nishikawa, T., Nakamura, Y., Yoshida, Y., Sasaki, H., Arai, H., Hirai, S., Shouji, M., K, I., Tanaka, K., Hamamoto, M., Yamamoto, H., Mat‐subayashi, T., Nakashima, K., Urakami, K., Adachi, Y., Nakamura, S., Toji, H., and Yoshida, H. 1998. Basic and clinical studies on APOE gene typing by line probe assay (LiPA) as a biological marker for Alzheimer's disease and related disorders: Multicenter study in Japan. Meth. Find Exp. Clin. Pharmacol. 20:793‐799.
   Rebeck, G., Reiter, J., Strickland, D., and Hyman, B. 1993. Apolipoprotein E in sporadic Alzhe‐imer's disease: Allelic variation and receptor interactions. Neuron 11:575‐580.
   Saiki, R., Walsh, P., Levenson, C., and Ehrlich, H. 1989. Genetic analysis of amplified DNA with immobilized sequence specific oligonucleotide probes. Proc. Natl. Acad. Sci. U.S.A. 86:6230‐6234.
   Saunders, A.M., Strittmatter, W.J., Schmechel, D., George‐Hyslop, P.H., Pericak‐Vance, M.A., Joo, S.H., Rosi, B.L., Gusella, J.F., Crapper‐MacLachlan, D.R., and Alberts, M.J. 1993. Association of apolipoprotein E allele epsilon 4 with late‐onset familial and sporadic Alzhe‐imer's disease. Neurology 43:1467‐1472.
   Strittmatter, W.J., Saunders, A.M., Schmechel, D., Pericak‐Vance, M., Enghild, J., Salvesen, G.S., and Roses, A.D. 1993. Apolipoprotein E: High‐avidity binding to β‐amyloid and increased frequency of type 4 allele in late‐onset familial Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 90:1977‐1981.
   West, H., Rebeck, G., and Hyman, B. 1994. Frequency of the apolipoprotein E epsilon 2 allele is diminished in sporadic Alzheimer disease. Neurosci. Lett. 175:46‐48.
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