Construction of Small‐Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection

Jacqueline C. Pulido1, Geoffrey M. Duyk1

1 Millenium Pharmaceuticals, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.2
DOI:  10.1002/0471142905.hg0202s14
Online Posting Date:  May, 2001
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Abstract

These protocols construct a representative small‐insert genomic DNA library in a phagemid vector. First, size‐selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small‐insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single‐stranded DNA (ssDNA). Finally, this uracil‐substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer‐extension reaction are transformed into an E. coli strain maintaining wild‐type genes at the dut and ung loci. Under these conditions, uracil‐substituted ssDNA will be restricted from growing by the host‐encoded uracil‐N‐glycosylase, while the primer‐extended products are capable of replicating.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of Primary Small‐Insert Library in a Phagemid Vector
  • Basic Protocol 2: Amplification of Primary Small‐Insert Library for Single‐Stranded DNA Template Preparation
  • Basic Protocol 3: Construction of Marker‐Selected Library by Primer Extension of Single‐Stranded DNA Templates
  • Support Protocol 1: Generation of Small Genomic DNA Fragments by Restriction Digestion
  • Support Protocol 2: Generation of Small Genomic DNA Fragments by Random Shearing
  • Support Protocol 3: Large‐Scale Preparation of pJCP1 Cloning Vector
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of Primary Small‐Insert Library in a Phagemid Vector

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • ≥3 µg/µl stock of 300‐ to 900‐bp blunt‐ended genomic DNA fragments (average size 500 bp; first or second support protocol)
  • ≥6000 pmol/µl stock of 5′‐phosphorylated BstXI linker/adapters (Invitrogen; store at −20°C):
  •    5′‐PO 4‐CTTTAGAGCACA‐3′
  •      3′‐GAAATCTC‐PO 4‐5′
  • recipe10× blunt‐end‐ligation buffer (see recipe)
  • 5 mM ATP (Pharmacia Biotech or Boehringer Mannheim; dilute from 100 mM stock and store in small aliquots at −20°C)
  • 5 U/µl and 1 U/µl T4 DNA ligase (in Weiss units; Boehringer Mannheim)
  • 12× gel loading buffer ( appendix 2D)
  • 200‐ to 12,000‐bp DNA molecular size markers (e.g., GIBCO/BRL 1‐kb DNA ladder)
  • Agarose (SeaKem GTG for fragment isolation and SeaKem ME for general use; American Bioanalytical)
  • BstXI‐cut, gel‐purified plasmid pJCP1 (≥1 µg/µl stock; third support protocol)
  • 10× T4 DNA ligase buffer ( appendix 2D; add ATP to 0.5 mM and DTT to 10 mM fresh before use)
  • ∼1 × 1011 cell/ml electrocompetent JMG1 (dutungF′) bacterial strain with transformation frequency >108 cfu/µg supercoiled plasmid DNA (store at −70°C)
  • SOC medium ( appendix 2D), prewarmed to 37°C
  • recipeLB/Tc/Cb plates (see recipe) made from 100‐ or 150‐mm petri dishes
  • LB/Cb or TB/Cb medium: LB medium or terrific broth ( appendix 2D) containing recipe100 µg/ml carbenicillin (see recipe for antibiotics)
  • Appropriate restriction endonucleases (e.g., BamHI and XbaI) and buffers (Boehringer Mannheim)
  • 16°C water bath
  • Electroporator: Bio‐Rad Gene Pulser and Pulse Controller, with Gene Pulser cuvettes
  • Additional materials and reagents for agarose gel electrophoresis (unit 2.7) and alkaline lysis miniprep of plasmid DNA (unit 5.3 & CPMB UNIT )

Basic Protocol 2: Amplification of Primary Small‐Insert Library for Single‐Stranded DNA Template Preparation

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Library obtained from test electroporation (first basic protocol)
  • SOC medium ( appendix 2D), prewarmed to 37°C
  • recipeLB/Tc/Cb plates (see recipe) prepared in 243 × 243 × 18–mm Nunc bioassay dishes, prewarmed to 37°C
  • recipeTY/Tc/Cb medium (see recipe), prewarmed to 37°C
  • Glycerol, sterile
  • M13KO7 helper phage (GIBCO/BRL) with titer >1 ×1011 pfu/ml
  • recipe50 mg/ml kanamycin stock (see recipe for antibiotics)
  • 40% (w/v) PEG 8000
  • 5 M NaCl
  • recipe1× M9 salts (see recipe)
  • recipeCsCl solutions: 1.2, 1.3, 1.4, and 1.32 g/cm3 CsCl (see recipe)
  • TE buffer, pH 8.0
  • 20 mg/ml proteinase K (ultrapure; American Bioanalytical; store at −20°C)
  • 10% (w/v) Sarkosyl (ultrapure N‐lauroylsarcosine; American Bioanalytical)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2
  • 100% (ice‐cold) and 70% ethanol
  • 0.8% agarose gel
  • Electroporator: Bio‐Rad Gene Pulser and Pulse Controller, with Gene Pulser cuvettes
  • 3000‐ml Pyrex flask, sterile
  • 50‐ml conical polypropylene screw‐cap centrifuge tubes, sterile (Falcon)
  • 500‐ml Nalgene polypropylene screw‐cap centrifuge bottles
  • Sorvall RC‐5B refrigerated centrifuge (Du Pont) with GS‐3 and SS‐34 rotors and adapters (or equivalent)
  • 65° and 55°C water baths
  • Whatman no. 2 filter paper, 15‐cm
  • Two‐piece Nalgene polypropylene funnel, 15‐cm diameter
  • Two 2000‐ml Pyrex vacuum flasks with vacuum source (or equivalent), sterile
  • Beckman SW‐28 rotor and 25 × 89–mm (1 ×3.5–in.) Ultra‐Clear centrifuge tubes
  • Beckman Ti70.1 rotor and 16 × 76–mm (⅝ × 3–in., 13.5‐ml capacity)
  •  Quick‐Seal tubes
  • Beckman L8‐70 ultracentrifuge with SW‐28 and Ti70.1 rotors
  • Forceps attached to stand
  • 10‐ml disposable syringes with 16‐G needles
  • Spectra/Por dialysis membrane: MWCO 6000‐8000 (23‐mm flat width, 14.6‐mm diameter, 1.7 ml/cm) and Spectra/Por closures (Spectrum Medical)
  • 30‐ml Corex centrifuge tubes and 1.5‐ml microcentrifuge tubes, silanized (CPMB APPENDIX 3) and sterile
  • Additional materials and reagents for dialysis (CPMB APPENDIX 3) and agarose gel electrophoresis (unit 2.7)

Basic Protocol 3: Construction of Marker‐Selected Library by Primer Extension of Single‐Stranded DNA Templates

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • recipe5 pmol/µl 5′‐end‐labeled oligonucleotide primers (see recipe)
  • 2 mM 4dNTP mix ( appendix 2D; use ultrapure dNTPs)
  • 10× Mg‐free PCR buffer (Promega; store at −20°C)
  • 25 mM MgCl 2 (Promega; store at −20°C)
  • Mineral oil (PCR grade; American Bioanalytical)
  • 5 U/µl Taq DNA polymerase (AmpliTaq; Perkin‐Elmer Cetus)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol ( appendix 3C)
  • 10× T4 DNA ligase buffer ( appendix 2D); add ATP to 0.5 mM and DTT to 10 mM fresh before use
  • 1 U/µl T4 DNA ligase (in Weiss units; Boehringer Mannheim)
  • ∼1× 1011 cells/ml (store at −70°C) electrocompetent bacterial strain (dut+ung+)r K with transformation frequency >108 cfu/µg supercoiled plasmid DNA—e.g., DH10B (GIBCO/BRL), XL‐1B (New England Biolabs), or Sure (Stratagene)
  • SOC medium ( appendix 2D), prewarmed to 37°C
  • recipeLB/Cb plates (see recipe) made in 150‐mm petri dishes
  • 2× TY medium ( appendix 2D) supplemented with 10% (v/v) glycerol
  • 2×SSC/ 5% SDS ( appendix 2D)
  • 5× SSC ( appendix 2D)
  • 10 mCi/ml [γ‐32P]ATP (6000 Ci/mM; Du Pont NEN)
  • recipe100 mg/ml carbenicillin (see recipe for antibiotics)
  • Thermal cycler (Perkin‐Elmer Cetus GeneAmp PCR System 480 or 9600)
  • Thin‐walled Gene Amp reaction tubes (for primer extension; Perkin‐Elmer Cetus)
  • Electroporator: Bio‐Rad Gene Pulser and Pulse Controller, with Gene Pulser cuvettes
  • Sterile toothpicks
  • 96‐well microtiter plates
  • 96‐pin replicator (Aladin Enterprises; unit 5.6)
  • 0.45‐µm, 137‐mm positively charged nylon transfer membrane (Magna membrane; MSI)
  • Whatman 3MM filter paper
  • Additional reagents and equipment for PCR (unit 2.5), phenol/chloroform extraction and ethanol precipitation ( appendix 3C), library screening by colony hybridization (unit 2.3), and DNA sequencing (CPMB UNIT )

Support Protocol 1: Generation of Small Genomic DNA Fragments by Restriction Digestion

  Additional Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D ; for suppliers, see suppliers appendix.
  • High‐molecular‐weight genomic DNA ( appendix 3B; see also Critical Parameters)
  • AluI, HaeIII, SspI, and EcoRV restriction endonucleases and buffers (Boehringer Mannheim)
  • SeaKem GTG agarose (for fragment isolation; American Bioanalytical)
  • 5× TBE buffer ( appendix 2D)
  • Bio‐Rad Sub‐Cell DNA electrophoresis cell (15 × 33–cm; 15 × 15–cm stage) with 25 × 15 × 2.5–cm removable UV‐transparent gel tray and 20‐well, 1.5‐mm‐thick comb (or equivalent)
  • Electroeluter/concentrator tank (American Bioanalytical)
  • Electroelution cups (traps; ISCO)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), phenol/chloroform extraction and ethanol precipitation ( appendix 3C), and electroelution (CPMB UNIT )

Support Protocol 2: Generation of Small Genomic DNA Fragments by Random Shearing

  Additional Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • High‐molecular‐weight genomic DNA ( appendix 3B; see Critical Parameters)
  • 3 U/µl T4 DNA polymerase and buffer (New England Biolabs)
  • 10 U/µl T4 polynucleotide kinase and buffer (New England Biolabs)
  • 100 mM ATP (Pharmacia Biotech or Boehringer Mannheim; store at −20°C)
  • 2 mM 4dNTP mix ( appendix 2D)
  • 0.5 mg/ml BSA (dilute from 10 mg/ml stock supplied with T4 DNA polymerase and store in 500‐µl aliquots at −20°C; New England Biolabs)
  • Branson Sonifier 200 cell disruptor and 2.5‐cm‐diameter sonicator cup tuned to 20 kHz
  • Biospin P6 columns (Bio‐Rad or equivalent)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), phenol/chloroform extraction and ethanol precipitation ( appendix 3C), and electroelution from agarose gel (CPMB UNIT )

Support Protocol 3: Large‐Scale Preparation of pJCP1 Cloning Vector

  Additional Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Plasmid pJCP1 DNA (Fig. ; available from authors)
  • BstXI restriction endonuclease and buffer (Boehringer Mannheim)
  • BamHI, EcoRI, EcoRV, SacI, SpeI, XbaI, or XmaIII restriction endonuclease and buffer (Boehringer Mannheim)
  • 5× TBE buffer ( appendix 2D)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), phenol/chloroform extraction, and ethanol precipitation ( appendix 3C), and electroelution (CPMB UNIT )
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Figures

Videos

Literature Cited

Literature Cited
   Bio‐Rad Instruction Manual (#165‐2098) for Preparing Electrocompetent Cells and Electroporation. 1992.
   Brenig, B. and Brem, G. 1991. Direct cloning of sequence tagged microsatellites by DNA affinity chromatography. Nucl. Acids Res. 19:5441.
   Browne, D.L. and Litt, M. 1992. Characterization of (CA)n microsatellites with degenerate sequencing primers. Nucl. Acids Res. 20:141.
   Edwards, A., Civitello, A., Hammond, H.A., and Caskey, C.T. 1991. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49:746‐756.
   Enea, V. and Zinder, N.D. 1982. Interference resistant mutatants of phage f1. Virology 122:222‐231.
   Feener, C.A., Boyce, F.M., and Kunkel, L.M. 1991. Rapid detection of CA polymorphisms in cloned DNA: Application to the 5′ region of the dystrophin gene. Am. J. Hum. Genet. 48:621‐627.
   Holland, P., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5′ → 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88:7276‐7280.
   Kornberg, A. and Baker, T. 1992. DNA Replication, 2nd ed. W.H. Freeman & Co., New York.
   Kunkel, T.A., Bebenek, K., and McClary, J. 1991. Efficient site‐directed mutagenesis using uracil‐containing DNA. Methods Enzymol. 204:125‐139.
   Kunkel, T.A., Roberts, J.D., and Zakour, R.A. 1987. Rapid and efficient site‐specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367‐382.
   Ostrander, E.O., Jong, P.M., Rine, J., and Duyk, G. 1992. Construction of small‐insert genomic DNA libraries highly enriched for microsatellite repeat sequences. Proc. Natl. Acad. Sci. U.S.A. 89:3419‐3423.
   Richardson, C.C. and Lechner, R.L. 1983. A preformed, topologically stable replication fork. J. Biol. Chem. 258:11185‐11196.
   Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., and Anand, R. 1990. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucl. Acids Res. 18:2887‐2890.
   Sambrook, J., Fritsch, E., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
   Seed, B. 1987. LFA‐3 cDNA encodes a phospholiped‐linked membrane protein homologous to CD2. Nature 329:840‐842.
   Vieira, J. and Messing, J. 1987. Production of single‐stranded plasmid DNA. Methods Enzymol. 153:3‐11.
   Weber, J.L. 1990a. Human DNA polymorphisms based on length variations in simple sequence tandem repeats. In Genome Analysis Series (S. Tighlman and K. Davies, eds.) 1:159‐181. Cold Spring Laboratory Press, Cold Spring Harbor, N.Y.
   Weber, J.L. 1990b. Informativeness of human (dC‐dA)n (dG‐dT)n polymorphisms. Genomics 7:524‐530.
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