Single‐Sperm Typing

Sigbjorn Lien1, Joanna Szyda2, Esther P. Leeflang3, Rene Hubert3, Lin Zhang3, Karin Schmitt3, Norman Arnheim3

1 Agricultural University of Norway, Aas, Norway, 2 Wroclaw Agricultural University, Wroclaw, Poland, 3 University of Southern California, Los Angeles, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 1.6
DOI:  10.1002/0471142905.hg0106s32
Online Posting Date:  May, 2002
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This unit presents protocols for sperm isolation using two different methods, amplification of simple sequence‐length polymorphisms (SSLP) and/or single nucleotide polymorphisms (SNP) from single cells or whole genome‐amplified single cells using primer extension preamplification (PEP), and discusses the statistical analysis of sperm‐typing recombination data. Newer methods for studying recombination over very short distances (a few kilobases) using total sperm DNA and allele‐specific PCR are also discussed.

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Table of Contents

  • Basic Protocol 1: PCR Amplification of Genetic Markers from Single‐Sperm Cells
  • Support Protocol 1: Isolation of Single Sperm Cells from Agarose Films
  • Support Protocol 2: Isolation of Lysed Single Sperm Cells by FACS
  • Support Protocol 3: Multiplex Amplification from Single Sperm Cells
  • Support Protocol 4: Whole‐Genome Amplification of DNA from Single Cells by Primer‐Extension Preamplification (PEP)
  • Basic Protocol 2: Sperm‐Typing Data Analysis
  • Basic Protocol 3: Segregation Distortion Based on Haplotype Transmission
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: PCR Amplification of Genetic Markers from Single‐Sperm Cells

  • Lysed sperm (see Support Protocols protocol 21 or protocol 32) or PEP product (see protocol 5)
  • recipeNeutralization buffer (see recipe)
  • recipe10× amplification buffer with and without potassium (see recipe)
  • 2 mM 4dNTP mix ( appendix 2D)
  • 100 µM first‐round primers (see protocol 4; store at −20°C): forward and reverse
  • 5 U/µl AmpliTaq DNA polymerase (Perkin‐Elmer, Promega)
  • Light mineral oil
  • 100 µM second‐round primers (see protocol 3; store at −20°C): hemi‐nested or full nested
  • Thermal cycler accommodating samples in a 96‐well format (e.g., PTC‐200; MJ Research) and appropriate plates or tubes
  • Additional reagents and equipment for nondenaturing PAGE (unit 7.4)
CAUTION: Ethidium bromide is a carcinogen. Gloves must be worn when handling gels and solutions containing ethidium bromide.NOTE: All solutions should be prepared using ultrapure water.

Support Protocol 1: Isolation of Single Sperm Cells from Agarose Films

  • Semen
  • 0.5% (w/v) low‐melting‐point agarose
  • recipeCell lysis solution (see recipe)
  • recipeNeutralization buffer (see recipe)
  • Minigel casting and electrophoresis apparatus (e.g., Hoefer 200 series)
  • Inverted phase‐contrast microscope with 100× objective
  • Paragon no. 11 scalpel blade (Maersk Medical)
  • PCR tube
  • Stereomicroscope with 4.6× objective
NOTE: To reduce contamination, as much work as possible should be carried out in a laminar‐flow hood. Standard sterile procedures should be used.

Support Protocol 2: Isolation of Lysed Single Sperm Cells by FACS

  • Semen from genotyped donor (see Critical Parameters)
  • 5 µg/ml Hoechst 33342
  • 70% (w/v) sucrose
  • Hoechst 33342‐conjugated fluorescent beads (Flow Cytometry Standards)
  • 10% (v/v) bleach (sodium hypochlorite; e.g., Chlorox)
  • recipeCell lysis solution (see recipe)
  • recipeNeutralization buffer (see recipe)
  • Biosonik III sonicator with 4‐mm probe (Brownwill Scientific)
  • Phase‐contrast and fluorescence microscopes
  • FACStar Plus fluorescence‐activated cell sorter (Becton Dickinson)
  • Flexible 96‐well microtiter plate (e.g., Falcon) or 96‐tube system (e.g., GeneAmp PCR System 9600, Perkin‐Elmer Cetus)
  • 65°C water bath or thermal cycler compatible with microtiter plates or 96‐tube system
NOTE: To reduce contamination, as much work as possible should be carried out in a laminar‐flow hood. Standard sterile procedures should be used.

Support Protocol 3: Multiplex Amplification from Single Sperm Cells

  • 400 mM Poly N random 15‐base primers (Operon)
  • Sorted, lysed, and neutralized single sperm cells frozen in a 96‐well microtiter plate (see protocol 2)
NOTE: All solutions should be prepared using ultrapure water.
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Literature Cited

Literature Cited
   Arnheim, N. and Shibata, D. 1997. DNA mismatch repair in mammals: Role in disease and meiosis. Curr. Opin. Genet. Dev. 7:364‐370.
   Arnheim, N., Li, H., and Cui, X. 1990. PCR analysis of DNA sequences insingle cells: Single sperm mapping and genetic disease diagnosis. Genomics 8:415‐419.
   Boehnke, M., Arnheim, N., Li, H., and Collins, F.S. 1989. Fine‐structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm: Experimental design considerations. Am. J. Hum. Genet. 45:21‐32.
   Brock, G.J., Anderson, N.H., and Monckton, D.G. 1999. Cis‐acting modifiers of expanded CAG/CTG triplet repeat expandability: Associations with flanking GC content and proximity to CpG islands. Hum. Mol. Genet. 8:1061‐1067.
   Brown, G.M., Leversha, M., Hulten, M., Ferguson‐Smith, M.A., and Affara, N.A. 1998. Genetic analysis of meiotic recombination in humans by use of sperm typing: Reduced recombination within a heterozygous paracentric inversion of chromosome 9q32‐q34.3. Am. J. Hum. Genet. 62:1484‐1492.
   Carrington, M. 1999. Recombination within the human MHC. Immunol. Rev. 167:245‐256.
   Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre‐PCR mispriming and primer dimerization improves low copy number amplifications. Nucl. Acids Res. 20:1717‐1723.
   Cui, X. and Li, H. 1997. Discriminating between allelic and interlocus differences among human immunoglobulin VH4 sequences by analyzing single spermatozoa. Hum. Genet. 100:96‐100.
   Cui, X. and Li, H. 1998. Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa. Proc. Natl. Acad. Sci. U.S.A. 95:10791‐10796.
   Cui, X., Li, H., Goradia, T., Lange, D., Kazazian, H.H., Galas, D., and Arnheim, N. 1989. Single‐sperm tying: Determination of genetic distance between the G‐gamma globin and parathyroid hormone loci by using the polymerase chain reaction and allele‐specific oligomers. Proc. Natl. Acad. Sci. U.S.A. 86:9389‐9393.
   Cui, X., Gerwin, J., Navidi, W., Li, H., Kuehn, M., and Arnheim, N. 1992. Gene‐centromere mapping by PCR analysis of individual oocytes. Genomics 13:713‐717.
   Da, Y., Jarrell, V.L., Wang, T., Fernando, R.L., and Wheeler, M.B. 1995. Multilocus analysis for gene‐centromere mapping using first polar bodies and secondary oocytes. Genetics 139:1091‐1097.
   Day, C.E., Schmitt, K., and Robinson, M.A. 1994. Frequent recombination in Frequent recombination in the human T‐cell receptor beta gene complex. Immunogenetics 39:335‐342.
   Dear, P.H. and Cook, P.R. 1993. Happy mapping: Linkage mapping using a physical analogue of meiosis. Nucl. Acids Res. 21:13‐20.
   Furlong, R.A., Goudie, D.R., Carter, N.P., Lyall, J.E.W., Affara, N.A., and Ferguson‐Smith, M.A. 1993. Analysis of four microsatellite markers on the long arm of chromosome 9 by meiotic recombination in flow‐sorted single sperm. Am. J. Hum. Genet. 52:1191‐1199.
   Girardet, A., Lien, S., Leeflang, E.P., Beaufrere, L., and Tuffery, S. 1999. Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing. Eur. J. Hum. Genet. 7:239‐242.
   Girardet, A., McPeek, M.S., Leeflang, E.P., Munier, F., and Arnheim, N. 2000. Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single‐sperm typing. Am. J. Hum. Genet. 66:167‐175.
   Goradia, T.M. and Lange, K. 1990. Multilocus ordering strategies based on sperm typing. Ann. Hum. Genet. 54:49‐77.
   Goradia, T.M., Stanton, V.P., Cui, X., Aburatani, H., Li, H., Lange, K., Housman, D.E., and Arnheim, N. 1991. Ordering three DNA polymorphisms on human chromosome 3 by sperm typing. Genomics 10:748‐755.
   Grewal, R.P., Cancel, G., Leeflang, E.P., Durr, A., and McPeek, M.S. 1999. French Machado‐Joseph disease patients do not exhibit gametic segregation distortion: A sperm typing analysis. Hum. Mol. Genet. 8:1779‐1784.
   Han, L.L., Keller, M.P., Navidi, W., Chance, P.F., and Arnheim, N. 2000. Unequal exchange at the charcot‐marie‐tooth disease type 1A recombination hot‐spot is not elevated above the genome average rate. Hum. Mol. Genet. 9:1881‐1889.
   Haugland, R.P. 1994. Nucleic acid stains. In Molecular Probes. Handbook of Fluorescent Probes and Research Chemicals, 5th Edition (K.D. Larison, ed.) pp. 221‐229. Molecular Probes, Eugene, Or.
   Hubert, R., Stanton, V.P. Jr., Aburatani, H., Warren, J., and Li, H. 1992a. Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3. Genomics 12:683‐687.
   Hubert, R., Weber, J., Schmitt, K., Zhang, L., and Arnheim, N. 1992b. A new source of polymorphic DNA markers for sperm typing: Analysis of microsatellite repeats in single cells. Am. J. Hum. Genet. 51:985‐991.
   Hubert, R., MacDonald, M., Gusella, J., and Arnheim, N. 1994. A sperm typing approach for high resolution localization of meiotic recombination hot spots:Analysis of the 4p16.3 region near the Huntington disease locus. Nature Genet. 7:420‐424.
   Jarrell, V.L., Lewin, H.A., Da, Y., and Wheeler, M.B. 1995. Gene‐centromere mapping of bovine DYA, DRB3, and PRL using secondary oocytes and first polar bodies: Evidence for four‐strand double crossovers between DYA and DRB3. Genomics 27:33‐39.
   Jeffreys, A.J., Ritchie, A., and Neumann, R. 2000. High resolution analysis of haplotype diversity and meiotic crossover in the human TAP2 recombination hotspot. Hum. Mol. Genet. 9:725‐733.
   Klungland, H., Gomez‐Raya, L., Howard, C.J., Collins, R.A., and Rogne, S. 1997. Mapping of bovine FcgammaR (FCGR) genes by sperm typing allows extended use of human map information. Mamm. Genome 8:573‐577.
   Lange, K., Weeks, D., and Boehnke, M. 1988. Programs for pedigree analysis: MENDEL, FISHER, and dGENE. Genet. Epidemiol. 5:471‐472.
   Lazzeroni, L.C., Arnheim, N., Schmitt, K., and Lange, K. 1994. Multipoint mapping calculations for sperm typing data. Am. J. Hum. Genet. 55:431‐436.
   Leeflang, E.P., Zhang, L., Tavare, S., Hubert, R., Srinidhi, J., MacDonald, M.E., Myers, R.M., de Young, M., Wexler, N.S., Gusella, J.F., and Arnheim, N. 1995. Single sperm analysis of the trinucleotide repeats in the Huntington's disease gene. Hum. Mol. Genet. 4:1519‐1526.
   Leeflang, E.P., McPeek, M.S., and Arnheim, N. 1996. Analysis of meiotic segregation, using single‐sperm typing: Meiotic drive at the myotonic dystrophy locus. Am. J. Hum. Genet. 59:896‐904.
   Leeflang, E.P., Tavare, S., Marjoram, P., Grewal, R., Neal, C.O.S., and Arnheim, N. 1998. Human germline mutation analysis by single genome PCR: Application to dynamic mutations. In Genetic Instabilities and Hereditary Neurological Diseases (S.T. Warren and R.D. Wells, eds.) pp. 543‐558. Academic Press, New York.
   Leeflang, E.P., Tavare, S., Marjoram, P., Neal, C.O.S., Srinidhi, I., Macfarlane, H., Macdonald, M.E., Gusella, J.F., De Young, M., Wexler, N.S., and Arnheim, N. 1999. Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism. Hum. Mol. Genet. 8:173‐183.
   Lewin, H.A., Schmitt, K., Hubert, R., van Eijk, M.J.T., and Arnheim, N. 1992. Close linkage between bovine prolactin and Bola‐DRB3 genes: Genetic mapping in cattle by single sperm typing. Genomics 13:44‐48.
   Li, H., Gyllensten, U., Cui, X., Saiki, R., Erlich, H., and Arnheim, N. 1988. Amplification and analysis of DNA sequences in singles human sperm. Nature 335:414‐417.
   Li, H., Cui, X., and Arnheim, N. 1990. Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm using PCR. Proc. Natl. Acad. Sci. U.S.A. 87:4580‐4584.
   Li, H., Cui, X., and Arnheim, N. 1991. Analysis of DNA sequence variation in single cells. Methods 2:49‐59.
   Lien, S., Kaminski, S., Alestrom, P., and Rogne, S. 1993. A simple and powerful method for linkage analysis by amplification of DNA from single sperm cells. Genomics 16:41‐44.
   Lien, S., Cockett, N.E., Klungland, H., Arnheim, N., and Georges, M. 1999. High‐resolution gametic map of the sheep callipyge region: Linkage heterogeneity among rams detected by sperm typing. Anim. Genet. 30:42‐46.
   Lien, S., Szyda, J., Schechinger, B., Rappold, G., and Arnheim, N. 2000. Evidence for heterogeneity in recombination in the human pseudoautosomal region: High resolution analysis by sperm typing and radiation‐hybrid mapping. Am. J. Hum. Genet. 66:557‐566.
   Lin, Z., Cui, X., and Li, H. 1996. Multiplex genotype determination at a large number of gene loci. Proc. Natl. Acad. Sci. U.S.A. 96:2582‐2587.
   Matsunaga, S., Schutze, K., Donnison, I.S., Grant, S.R., Kuroiwa, T., and Kawano, S. 1999. Technical advance: Single pollen typing combined with laser‐mediated manipulation. Plant J. 20:371‐378.
   McPeek, M.S. 1999. SPERMSEG: Analysis of segregation distortion in single‐sperm data. Am. J. Hum. Genet. 65:1195‐1197.
   Navidi, W. and Arnheim, N. 1994. Analysis of genetic data from the polymerase chain reaction. Stat. Sci. 9:320‐333.
   Park, C., Russ, I., Da, Y., and Lewin, H.A. 1995. Genetic mapping of F13A to BTA23 by sperm typing: Difference in recombination rate between bulls in the DYA‐PRL interval. Genomics 27:113‐118.
   Park, C., Frank, M.T., and Lewin, H.A. 1999. Fine‐mapping of a region of variation in recombination rate on BTA23 to the D23S22‐D23S23 interval using sperm typing and meiotic breakpoint analysis. Genomics 59:143‐149.
   Schmitt, K. and Arnheim, N. 1994. Genetic recombination analysis using sperm typing. In Genetic Engineering, Principles and Methods (J.K. Setlow, ed.) pp. 157‐165. Plenum, New York.
   Schmitt, K., Lazzeroni, L.C., Foote, S., Vollrath, D., and Fisher, E.M. 1994. Multipoint linkage map of the human pseudoautosomal region, based on single‐sperm typing: Do double crossovers occur during male meiosis? Am. J. Hum. Genet. 55:423‐430.
   Schutze, K. and Clement‐Sengewald, A. 1994. Catch and move‐cut or fuse. Nature 368:667‐669.
   Shi, Q. and Martin, R.H. 2001. Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men. Reproduction 121:655‐666.
   Shi, Q., Spriggs, E., Field, L.L., Ko, E., and Barclay, L. 2001. Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploid 24,XY human sperm. Am. J. Med. Genet. 99:34‐38.
   Simianer, H., Szyda, J., Ramon, G., and Lien, S. 1997. Evidence for individual and between‐family variability of the recombination rate in cattle. Mamm. Genome 8:830‐835.
   Stephens, J.C., Rogers, J., and Ruano, G. 1990. Theoretical underpinning of the Single‐Molecule‐Dilution (SMD) method of direct haplotype resolution. Am. J. Hum. Genet. 46:1149‐1155.
   Sun, F., Arnheim, N., and Waterman, M.S. 1995. Whole genome amplification of single cells: Mathematical analysis of PEP and tagged PCR. Nucl. Acids Res. 23:3034‐3040.
   Szyda, J., Simianer, H., and Lien, S. 2000. Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region. Genet. Res. 75:53‐59.
   Tanke, H.J. and van der Keur, M. 1993. Selection of defined cell types by flow‐cytometric cell sorting. Tibtech 11:55‐62.
   Tusie‐Luna, M.T. and White, P.C. 1995. Gene conversions and unequal crossovers between CYP21 (steroid 21‐ hydroxylase gene) and CYP21P involve different mechanisms. Proc. Natl. Acad. Sci. U.S.A. 92:10796‐10800.
   van Eijk, M.J.T., Russ, I., and Lewin, H.A. 1993. Order of bovine DRB3, DYA and PRL determined by sperm typing. Mamm. Genome 4:133‐138.
   Williams, C., Davies, D., and Williamson, R. 1993. Segregation of ΔF508 and normal CFTR alleles in human sperm. Hum. Mol. Genet. 2:445‐448.
   Windemuth, C., Simianer, H., and Lien, S. 1998. Fitting genetic mapping functions based on sperm typing: Results for three chromosomal segments in cattle. Anim. Genet. 29:425‐434.
   Yu, J., Lazzeroni, L., Qin, J., Huang, M.M., and Navidi, W. 1996. Individual variation in recombination among human males [see comments]. Am. J. Hum. Genet. 59:1186‐1192.
   Zhang, L., Cui, X., Schmitt, K., Hubert, R., Havidi, W., and Arnheim, N. 1992. Whole genome amplification from a single cell: Implications for genetic analysis. Proc. Natl. Acad. Sci. U.S.A. 89:5847‐5851.
   Zhang, L., Leeflang, E.P., Yu, J., and Arnheim, N. 1994. Studying human mutations by sperm typing: Instability of CAG trinucleotide repeats in the human androgen receptor gene. Nature Genet. 7:531‐535.
   Zhao, S., Li, K., Yu, M., and Peng, Z. 2000. Ordering three microsatellites on porcine chromosome 12 by single sperm typing. Anim. Biotechnol. 11:45‐49.
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