Deriving Probes From Large‐Insert Clones by PCR Methods

Hans Albertsen1, Andrew Thliveris1, John H. Riley2, David J. Munroe3, Paul Watkins3, Craig T. Basson4

1 University of Utah, Salt Lake City, Utah, 2 Glaxo Wellcome Medicines Research Centre, Herts, United Kingdom, 3 Sequana Therapeutics, La Jolla, California, 4 Cornell University Medical College, New York, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.9
DOI:  10.1002/0471142905.hg0509s17
Online Posting Date:  May, 2001
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Abstract

This unit describes several polymerase chain reaction (PCR)‐based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.

     
 
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Table of Contents

  • Basic Protocol 1: Inter‐Irs PCR to Generate Complex Probes
  • Alternate Protocol 1: IRS‐Bubble PCR to Generate Complex Probes
  • Support Protocol 1: Construction of T Vector for Cloning of PCR Products
  • Basic Protocol 2: IRS‐Vector PCR to Isolate YAC Extremities
  • Basic Protocol 3: Degenerate Primer‐Vector PCR to Determine Rescue Ends of P1 Clones
  • Alternate Protocol 2: Inverse PCR to Isolate YAC Extremities
  • Alternate Protocol 3: Bubble‐Vector PCR to Isolate YAC Extremities
  • Basic Protocol 4: Generation of ALU‐PCR Products from YAC Clones for Fluorescence in Situ Hybridization
  • Alternate Protocol 4: Scrambling ALU Elements within YACS to Generate Fluorescence In Situ Hybridization Probes
  • Support Protocol 2: Isolating Total DNA from YAC‐Bearing Yeast by Spheroplast Method
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Inter‐Irs PCR to Generate Complex Probes

  Materials
  • Sample DNA: genomic DNA from somatic cell hybrid (units 3.1& 3.2), genomic DNA in cosmid or λ phage vector, or total yeast DNA from YAC‐bearing strain (see 5.9)
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 2.5 U/µl Taq DNA polymerase
  • Mineral oil
  • recipe20 µM interspersed repetitive sequence (IRS) primers: Alu‐S/Alu‐J [used as a 4:1 (w/w) mixture], and/or Alu ‐end, 47‐23, and L1Hs (see recipe; Fig. )
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 0.5‐ml microcentrifuge tube
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: IRS‐Bubble PCR to Generate Complex Probes

  • AluI, HaeII, or RsaI restriction endonuclease and 10× buffer
  • 20 µM “bubble‐bottom” oligonucleotide (Fig. )
  • 20 µM “bubble‐top” oligonucleotide (Fig. )
  • 20× SSC ( appendix 2D)
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2D)
  • 400,000 U/ml T4 DNA ligase (in cohesive‐end units) and 10× T4 DNA ligase buffer ( appendix 2D)
  • 0.25× TE buffer (1/4 dilution of TE buffer, appendix 2D)
  • 20 µM bubble primer (Fig. )
  • 100°C water bath

Support Protocol 1: Construction of T Vector for Cloning of PCR Products

  Materials
  • Plasmid DNA (e.g., Stratagene pBluescript KS or SK)
  • Blunt end–forming restriction endonuclease (e.g., EcoRV or SmaI) and 10× buffer
  • 70% ethanol, ice‐cold
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 1.25 mM dTTP ( appendix 2D)
  • 5 U/µl Taq DNA polymerase
  • 0.25× TE buffer (1/4 dilution of TE buffer, appendix 2D)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA ( appendix 3C)

Basic Protocol 2: IRS‐Vector PCR to Isolate YAC Extremities

  Materials
  • Sample DNA: genomic DNA from somatic cell hybrid (units 3.1 & 3.2), genomic DNA in cosmid or λ phage vector, or total yeast DNA from YAC‐bearing strain (see 5.9)
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 20 µM vector primers:
    • YAC4L: 5′‐GCC AAG TTG GTT TAA GGC GCA AGA C‐3′ and
    • YAC4R: 5′‐GGC GAG TCG AAC GCC CGA TCT CAA G‐3′ (Fig. ) or
    • T7A (5′‐AAT ACG ACT CAC TAT AGG G‐3′) and
    • T3A (5′ATT AAC CCT CAC TAA AGG G‐3′)
  • recipe20 µM interspersed repetitive sequence (IRS) primer: Alu‐S/Alu‐J [used as a 4:1 (w/w) mixture], Alu‐end, 47‐23, or L1Hs (see recipe; Fig. )
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 2.5 U/µl Taq DNA polymerase
  • Mineral oil
  • 3 M sodium acetate
  • Isopropanol
  • 70% ethanol, ice cold
  • 0.25× TE buffer (1/4 dilution of TE buffer, appendix 2D)
  • Appropriate restriction endonuclease and 10× buffer
  • Thermal cycler, preheated to 94°C
  • 0.5‐ml microcentrifuge tube
  • Additional reagents and equipment for isopropanol precipitation and chloroform extraction of DNA ( appendix 3C), agarose gel electrophoresis (unit 2.7), and isolation of DNA fragments from agarose gel (e.g.,CPMB UNIT )

Basic Protocol 3: Degenerate Primer‐Vector PCR to Determine Rescue Ends of P1 Clones

  Materials
  • Sample DNA: genomic DNA from P1, cosmid, or λ phage vector; or total yeast DNA from YAC‐bearing strain (see protocol 10)
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 25 ng/µl NS degenerate primer: 5′‐GTC AGT CAG TCA GAN NNN GAG‐3′
  • 100 ng/µl vector primers:
  •   P1T7: 5′‐TTT GCG GCC GCT AAT ACG ACT CAC TAT AG‐3′
  •   P1SP6: 5′‐CCG TCG ACA TTT AGG TGA CAC TAT A‐3′ (or other vector‐specific primer; see annotation to step )
  • 5 U/µl Taq DNA polymerase
  • Glycerol
  • TE buffer, pH 8.0 ( appendix 2D)
  • 0.5 M EDTA, pH 8.0 ( appendix 2D)
  • Bromphenol blue
  • NuSieve agarose (FMC BioProducts)
  • Thermal cycler, oil free
  • 200‐µl PCR tubes
  • Additional reagents and equipment for agarose gel electrophoresis and ethidium bromide staining (e.g., unit 2.7) and for isolation of DNA fragments from agarose gel (e.g., QiaQuick kit, or see CPMB UNIT )

Alternate Protocol 2: Inverse PCR to Isolate YAC Extremities

  • YAC DNA: total yeast DNA from 2 × 108 cells (e.g., 20 µl of wet cell pellet) containing a pYAC4‐derived YAC, prepared in a 50‐µl agarose plug (unit 5.1)
  • Restriction endonuclease—HaeIII, HhaI, HpaII, Sau3A, or TaqI (see Fig. )—and appropriate 1× restriction buffer
  • Tris/EDTA: 10 mM Tris·Cl (pH 8.0)/0.1 mM EDTA
  • 1 Weiss U/µl T4 DNA ligase and 10× T4 DNA ligase buffer ( appendix 2D)
  • 40 pmol/µl vector primers (see Fig. ):
  •  YAK3‐R: 5′‐TGT AAA ACG ACG GCC AGT CCC GAT CTC AAG ATT ACG GAA T‐3′
  •  YAK3‐L: 5′‐CAG GAA ACA GCT ATG ACC CAT TCA CTT CCC AGA CTT CGA A‐3′
  •  YAK5‐R: 5′‐CAG GAA ACA GCT ATG ACC TGA AGA AAG AGT ATA CTA CAT  AAC A‐3′
  •  YAK5‐L: 5′‐TGT AAA ACG ACG GCC AGT GTT GGT TTA AGG CGC AAG ACT T‐3′
  • 5 U/µl Taq DNA polymerase
  • Mineral oil
  • Low gelling/melting agarose (SeaKem GTG; FMC Bioproducts)
  • 40 µM M13 universal primer: 5′‐TGTAAAACGACGGCCAGT‐3′
  • 40 µM M13 reverse primer: 5′‐CAGGAAACAGCTATGACC‐3′
  • 14‐ml tube (Falcon)
  • Centricon 100 concentrator (Amicon)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA ( appendix 3C), agarose gel electrophoresis (unit 2.7), isolation of DNA fragments from agarose gels (e.g.,CPMB UNIT ), and direct sequencing of PCR products (e.g., CPMB UNIT )

Alternate Protocol 3: Bubble‐Vector PCR to Isolate YAC Extremities

  • Vectorette top and bottom oligonucleotides (Fig. A; available from Genosys Biotechnology)
  • 10 mM Tris⋅Cl (pH 7.5)/15 mM MgCl 2
  • YAC DNA: total yeast DNA from a strain bearing a pYAC4‐derived YAC prepared in 25‐µl agarose plug (unit 5.1)
  • TE buffer ( appendix 2D)
  • Restriction endonucleases—EcoRV, RsaI, AluI, and PvuII (see Fig. )—and 10× buffers
  • 5 Weiss U/µl T4 DNA ligase and 1× T4 DNA ligase buffer ( appendix 2D)
  • 100 mM ATP ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D) containing 10 mM MgCl 2
  • 100 µM primary PCR primers: primers A and D (left‐ and right‐end primers; Fig. B)
  • 100 µM universal vectorette primer (primer G, Fig. A)
  • 1 mM 4dNTP mix ( appendix 2D)
  • 1 U/µl Taq DNA polymerase
  • 100 µM secondary PCR primers: primers B and E (left‐ and right‐end nested primers; Fig. B)
  • 100 µM universal nested vectorette primer (primer H, Fig. A)
  • 10 U/µl EcoRI restriction endonuclease and 10× buffer
  • 100 µM sequencing primers: primer C, primer F, and universal sequencing primer I (Fig. A & B)
  • 1.5‐ml microcentrifuge tube, screw‐cap
  • 0.5‐ml microcentrifuge tube
  • 15‐ml centrifuge tube
  • 100°C water bath
  • Thermal cycler
  • Additional reagents and solutions for preparative PAGE and UV shadowing (e.g., CPMB UNIT ) and sequencing PCR products (e.g., CPMB UNIT )

Basic Protocol 4: Generation of ALU‐PCR Products from YAC Clones for Fluorescence in Situ Hybridization

  Materials
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 2 mM 4dNTP mix ( appendix 2D)
  • 100 ng yeast genomic DNA from a YAC‐bearing clone (see protocol 10)
  • 2.5 U/µl Taq DNA polymerase
  • 25 µM Alu primer CL1: 5′‐TCC CAA AGT GCT GGG ATT AC AG‐3′ (see Fig. )
  • 25 µM Alu primer CL2: 5′‐CTG CAC TCC AGC CTG GG‐3′ (see Fig. )
  • 25 µM Alu primer CL1 + Alu primer CL2
  • Mineral oil
  • 7.5 M ammonium acetate
  • 100% and ice‐cold 70% ethanol
  • 1× and 0.25× TE buffer ( appendix 2D)
  • recipe10× polishing buffer (see recipe)
  • 3 U/µl T4 DNA polymerase
  • 10 U/µl T4 polynucleotide kinase
  • 1 Weiss U/µl T4 DNA ligase and 10× T4 DNA ligase buffer ( appendix 2077)
  • 0.5‐ml microcentrifuge tubes
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), phenol extraction and ethanol precipitation of DNA( appendix 3C), and preparation of biotinylated probes by nick translation or random oligonucleotide–primed synthesis (unit 4.3 and CPMB UNIT )

Alternate Protocol 4: Scrambling ALU Elements within YACS to Generate Fluorescence In Situ Hybridization Probes

  • Total yeast DNA from a YAC‐bearing strain prepared in agarose plug (unit 5.1)
  • 1% (w/v) low gelling/melting temperature agarose
  • recipe10× agarase buffer (see recipe)
  • 1 U/µl β‐agarase
  • HinfI restriction endonuclease and 10× buffer
  • 40% (w/v) polyethylene glycol (PEG) 8000
  • 25 µM Alu primer CL2 (Fig. )
  • 1.5‐ and 0.5‐ml microcentrifuge tubes
  • 15°, 42°, and 65°C water baths
  • Additional reagents and equipment for PFGE purification of YACs (unit 5.7), isolation of DNA fragments from agarose gel (e.g., CPMB UNIT ), and phenol extraction and ethanol precipitation of DNA ( appendix 3C)

Support Protocol 2: Isolating Total DNA from YAC‐Bearing Yeast by Spheroplast Method

  Materials
  • Single colony from freshly grown AHC plate or small number of cells from frozen glycerol stock of YAC‐bearing S. cerevisiae (unit 5.2)
  • AHC medium or plates (Ura, Trp; unit 5.5)
  • 50 mM EDTA, pH 8.0
  • SCE buffer (unit 5.1)
  • SCE buffer containing 200 U/ml Zymolyase (ICN Biomedicals) or equivalent
  • 0.28 M 2‐mercaptoethanol (2‐ME)
  • recipeLysis solution (see recipe)
  • TE buffer ( appendix 2D)
  • 10 mg/ml DNase‐free RNase A ( appendix 2D)
  • 50‐ml polypropylene centrifuge tubes
  • 30°C shaking incubator
  • IEC Centra‐4B centrifuge with swinging‐bucket rotor (or equivalent)
  • 50°C water bath
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA( appendix 3C)
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Figures

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Literature Cited

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Key References
   Lengauer et al. 1992., See above.
  Demonstrates the utility of the basic IRS‐PCR approach for generating of high‐quality FISH signal from YACs on both metaphase chromosomes and interphase nuclei.
   Marchuck, D., Drumm, M., Saulino, A., and Collins, F.S. 1990. Construction of T vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucl. Acids Res. 19:1154.
  Original papers describing IRS‐vector PCR, inter‐IRS PCR, IRS‐bubble PCR, and T vector cloning.
   Munroe et al., 1994. See above.
  First description of the concept of inverse PCR.
   Nelson et al., 1989. See above.
  Demonstrates how rapidly the vectorette/bubble PCR methodology can be used for isolating the ends of YACs.
   Nelson et al., 1991. See above.
   Ochman et al., 1988. See above.
   Riley et al., 1990. See above.
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