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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Abstract

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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Materials

Basic Protocol 1: PCR Amplification of Genetic Markers from Single‐Sperm Cells

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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