Genotyping by Ligation Assays

Deborah A. Nickerson1, Wendy Ankener1, Claire Delahunty1, Pui‐Yan Kwok1

1 University of Washington School of Medicine, Seattle, Washington
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.6
DOI:  10.1002/0471142905.hg0206s10
Online Posting Date:  May, 2001
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This unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A describes the preparation of modified biotin‐ and digoxigenin‐labeled oligonucleotide primers.

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Table of Contents

  • Basic Protocol 1: Oligonucleotide Ligation Assay (OLA)
  • Basic Protocol 2: Ligase Chain Reaction (LCR)
  • Support Protocol 1: Preparing Modified Oligonucleotides for Ligation Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Oligonucleotide Ligation Assay (OLA)

  • recipeStreptavidin‐coated microtiter plates (see recipe)
  • Blocking buffer: 0.5% (w/v) BSA in PBS ( appendix 2D)
  • Modified oligonucleotide primers (see protocol 3): 5 µM stocks of allele‐specific primers, labeled at the 5′ end with biotin, and 5 µM stock of common reporter primer, labeled at the 3′ end with digoxigenin and phosphorylated at the 5′ end
  • recipeOLA ligation mix (see recipe)
  • Amplified target DNA (unit 7.1), 80 to 1500 bp long
  • 0.1% (v/v) Triton X‐100
  • Mineral oil
  • 5 U/µl thermostable DNA ligase (e.g., Epicentre Technologies)
  • 100 mM EDTA/0.1% (v/v) Triton X‐100
  • recipeTris/NaCl wash buffer (see recipe)
  • 0.01 M NaOH/0.05% (v/v) Tween 20
  • Alkaline phosphatase–labeled anti‐digoxigenin antibodies (Boehringer Mannheim)
  • recipeColorimetric substrate for alkaline phosphatase (see recipe)
  • 96‐well microtiter plates, flexible, round‐bottom (Falcon)
  • Thermal cycler accommodating 96‐well microtiter plate or heating block modified to accommodate a 96‐well microtiter plate
  • Microtiter plate spectrophotometer

Basic Protocol 2: Ligase Chain Reaction (LCR)

  • recipeLCR mix (see recipe)
  • recipeOligonucleotide primers for LCR: allele‐specific and 32P‐labeled joining primers complementary to both target strands (see recipe)
  • 5 U/µl thermostable ligase (e.g., Epicentre Technologies)
  • Target DNA: 10 µl of PCR product (unit 7.1) or ∼10 ng genomic DNA sample ( appendix 3B)
  • Mineral oil
  • Formamide
  • DNA molecular size markers
  • Additional reagents and equipment for labeling oligonucleotides ( appendix 3E & CPMB UNIT ), and denaturing polyacrylamide gel electrophoresis ( appendix 3F)
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Literature Cited

   Abravaya, K., Carrino, J.J., Muldoon, S., and Lee, H.H. 1995. Detection of point mutations with a modified ligase chain reaction (Gap‐LCR). Nucl. Acids Res. 23:675‐682.
   Barany, F. 1991. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Nat. Acad. Sci. U.S.A. 88:189‐193.
   Eggerding, F.A. 1995. A one‐step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. PCR Methods Appl. 4:337‐345.
   Grossman, P.D., Bloch, W., Brinson, E., Chang, C.C., Eggerding, F.A., Fung, S., Iovannisci, D.A., Woo, S., and Winn‐Deen, E.S. 1994. High density multiplex detection of nucleic acid sequences: Oligonucleotide ligation assay and sequence‐coded separation. Nucl. Acids Res. 22:4527‐4534.
   Kwok, P.Y., Gremaud, M.F., Nickerson, D.A., Hood, L., and Olson, M.V. 1992. Automatable screening of yeast‐artificial chromosome libraries based on the oligonucleotide‐ligation assay. Genomics 13:935‐941.
   Landegren, U., Kaiser, R., Sanders, J., and Hood, L. 1988. A ligase‐mediated gene detection technique. Science 241:1077‐1080.
   Nickerson, D.A., Kaiser, R., Lappin, S., Stewart, J., Hood, L., and Landegren, U. 1990. Automated DNA diagnostics using an ELISA‐based oligonucleotide ligation assay. Proc. Nat. Acad. Sci. U.S.A. 87:8923‐8927.
   Samiotaki, M., Kwiatkowski, M., Parik, J., and Landegren, U. 1994. Dual‐color detection of DNA sequence variants by ligase‐mediated analysis. Genomics 20:238‐242.
   Wiedmann, M., Wilson, W.J., Czajka, J., Luo, J., Barany, F., and Batt, C.A. 1994. Ligase chain reaction (LCR)—Overview and applications. PCR Methods Appl. 3:S51‐S64.
   Winn‐Deen, E.S. and Iovannisci, D.M. 1991. Sensitive fluorescence method for detecting DNA ligation amplification products. Clin. Chem. 37:1522‐1523.
   Wu, D.Y. and Wallace, R.B. 1989a. Specificity of the nick closing activity of bacteriophage T4 ligase. Gene 76:245‐254.
   Wu, D.Y. and Wallace, R.B. 1989b. The ligation amplification reaction (LAR): Amplification of specific DNA sequences using sequential rounds of template‐dependent ligation. Genomics 4:560‐569.
Key References
   Nickerson et al., 1990. See above.
  Describes the PCR/OLA method and the application of robotic workstations in processing these reactions.
   Wiedmann et al., 1994. See above
  Describes the basic method for the ligase chain reaction, the utility of a thermostable enzyme in sample genotyping, and variations on the basic procedure.
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