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The Genetics of Pancreatic Cancer

-- Technical Section

 

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Representational Difference Analysis
(RDA) Protocol

MS 6/95

  1. GENERATION OF AMPLICONS
    1. Digestion genomic DNA

        1 g genomic DNA (driver and tester, respectively)
        10 l buffer B (BM)
        x l loTE
        1 l BamHI (10U/l; BM)
        100 l

        > 1 hr/37C
        PC8, EPpt, 3W, resuspend in 6 l loTE

        Repeat digestion and purification

       

    2. Ligation to RBamHI primers

        6 l digest (1 g driver and tester, respectively)
        10 l RB12 primer (0.05 mM)
        10 l RB24 primer (0.05 mM)
        3 l 10x ligase buffer (NEB)
        29 l

        Annealing: 50C -- 1 hr, then decrease to 10C
        Ligation: add 1 l ligase (400 NEB-U/l); O/N 15C

       

    3. PCR-1 amplicons

        PCR-mix: ~100 ng template (driver and tester, resp.)
        20 l 10x RDA buffer
        16 l MgCl2 (50 mM)
        4 l dNTPs (10 mM)
        x l H20
        200 l final volume, including enzyme and primers

        3 min/72C
        Add 20 l Taq polymerase (20U, pre-diluted 1/5 in 1x RDA buffer; Gibco-BRL)
        3 min/72C
        Add 4 l RB24 primer (0.05 mM)

        Perform an amplification curve with 14, 16, 18, and 20 cycles of 1 min/94C and 3 min/72C, with a final extension of 5 min/72C

        To ensure the production of double-stranded products, perform an additional cycle of amplification and final extension.

        Therefore add:
        6 l primers
        1 l a10x RDA buffer
        2 l H2O
        1 l Taq polymerase (5U)
        10 l

        1 cycle of 1 min/94C and 8 min/72C

        PC8, EPpt, 2W, resuspend in 10 l loTE

        Analyze 10% of the products on a 1% agarose gel. Run the gel exactly 3 minutes at 100 Volts, and quantify by comparison to a DNA standard. 5-10 g of amplicons should be generated per 200 l PCR reaction. Run the gel for another 30 minutes, and choose the number of cycles that gives maximal recovery, but does not show smearing of the products toward the well (indicative of single-stranded DNA, and thus exhaustion of one of the PCR reagents).

       

    4. Size selection tester amplicons

        Load 5-10 g of tester amplicons, from the chosen optimal number of PCR cycles, on a 1.5% LMP agarose gel, separate and excise the amplicons from the gel between 150-1500 bp.

        Purify the amplicons by Quiagen chromatography:

          1 vol agarose block
          0.8 vol 0.5 M MOPS buffer
          1 vol 5 M NaCl
          5.2 vol H2O

        Vortex to pieces
        10 min/70C
        Load on a Q20 tip that is equilibrated with 1 ml QBT; keep the gel mix at 70C; load in ~500 l aliquots
        Wash with 6 ml QC
        Elute with 800 l QF
        Precipitate by adding 2 l glycogen and 650 l isopropanol
        Microfuge 10 min/RT, 2W, resuspend in loTE at ~100 ng/l (assume 30% recovery)
        Gel-quantify and bring the DNA to a final concentration of 100 ng/l

        Perform PCR on ~1.5 ng of gel-purified tester amplicons as template, in 200 l PCR reactions, as above.
        Generate an amplification curve with 14, 16, 18, and 20 cycles, plus additional cycle.
        Analyze 10% on gel; quantify and choose the optimal number of cycles.

       

    5. PCR-2 amplicons

        Generate ~5 g of tester amplicons and ~120 g of driver amplicons, according the optimal number of PCR cycles (including the additional cycle).

        PC8, IPpt, 2W, and resuspend in loTE.
        Gel-quantify and bring the DNA at a final concentration of ~1 g/l.

       

    6. Digestion amplicons

        1 g amplicons
        1 l buffer B
        0.4 l BamH1 (10 U/l)
        x l loTE
        10 l

        Scale the reaction up according to the amount of amplicons

        > 1 hr/37C

        PC8, IPpt, 3W, resuspend in loTE (no glycogen)
        Gel-quantify

       

    7. Ligation tester amplicons to JBamHI primers

        4 l tester amplicons (2 g)
        20 l JB12 primer (0.05 mM)
        20 l JB24 primer (0.05 mM)
        6 l 10x ligase buffer
        8 l loTE
        58 l

        Annealing: 50C -- 1 hr, then decrease to 10C
        Ligation: add 2 l ligase (400 NEB-U/l); O/N 15C

        Test the ligation in PCR with previous primers (RB24) and new primers (JB24), with ~35 ng template in a 50 l PCR reaction, for 25 cycles. The amplification with the previous primers generally gives some products, but the amount of products should not be more than half the amount generated with the new primers.

     

  2. SUBTRACTIVE DNA HYBRIDIZATION
    1. Hybridization-1

        500 ng tester-JB amplicons
        40 g driver amplicons
        x l loTE
        200 l

        PC8, IPpt, 2W (no glycogen)
        Carefully resuspend in ~3.5 l 3xEE buffer to a final volume of 4 la
        Overlay with 1 drop of mineral oil
        5 min/98C
        Add 1 l prewarmed 5 M NaCl, carefully mix by pipetting
        20 hr/67Cb
        Dilute to 500 l final volume with loTE

        Notes:

        1. The amount of tester and driver amplicons should be quantified accurately. The hybridization reaction is driven by the DNA concentration (C0t: C0 times t, where C0 is the DNA concentration at the beginning of the hybridization reaction, and t is the time of reannealing). 10 g/l is around the limit of solubility of DNA; after resuspension in 4 l 3xEE buffer, the solution should be viscous and look cloudy.
        2. For hybridizations with xenograft DNA, where the genomic complexity is theoretically doubled, increase the hybridization time to 40 hours.

       

    2. PCR-1 of Hyb-1

        Perform 2 PCR reactions with 50 l of Hyb-1 dilution as template in each, in 200 l PCR reactions, for 10 cycles. Perform PCR under above described conditions, except that the Taq polymerase is added after 3 minutes at 85C instead of 72C. Bring down to 72C before adding the Taq polymerase.
        The "super-hot-start" is intended to reduce priming mediated by duplexes of near-identical repetitive elements.

       

    3. . Mung bean nuclease treatment Hyb-1

        Both PCR samples are purified by PC8, EPpt, 2W, and resuspended in 5 l loTE

        Add:
        29 l loTE
        4 l 10x MBN buffer (NEB)
        2 l MBN enzyme (10 U/ul; NEB)
        40 l

        30 min/30C
        Add 160 l 50 mM Tris, pH 8.9
        5 min/98C
        Pool both MBN-treated Hyb-1 samples

       

    4. . PCR-2 of Hyb-1

        Perform 3 PCR reactions with 30 l of MBN-treated Hyb-1 sample as template in each, in 200 l PCR reactions. Perform amplification curves with 15, 17 and 19 cycles, plus additional cycle.
        PC8, EPpt, 2W, resuspend in 10 l loTE, and pool the samples.
        Analyze 10% on a 10% polyacrylamide gel.

       

    5. . Next round of RDA For the second round of hybridization and selection, go to steps # 6 and 7 for changing primerset. JB and NB primers alternate with each round.

      Proceed with hybridization-2 as for hybridization-1 (steps # 8-11). Adjust the quantities of tester samples according the table below. These amounts serve as a guideline; the amounts depend on the fraction of target sequences in the tester sample, and on the enrichment factor of the previous round(s).

      # Hyb Amount of DNA used in hybridization reaction Primer set*
      1 500 ng tester-JB / 40 g driver JBamH1
      2 50 ng Hyb1-NB / 40 g driver NBamH1
      3 100 pg Hyb2-JB / 40 g driver JBamH1
      4 5 pg Hyb3-NB / 40 g driver NBamH1

      * For primer sequences, and background of the RDA procedure, see Lisitsyn et al., Science 259, 946-951 (1993)

    6. . Abbreviations, solutions and procedures
      BM Boehringer Mannheim
      BSA Bovine serum albumine, acetylated, NEB
      EE 3xEE buffer: 30 mM EPPS, 3 mM EDTA pH 8.0; EPPS: Sigma E9502
      EPpt DNA precipitation with ethanol. Use 1/3 volume of 10 M NH4OAc and glycogen carrier, mix, and add 2-3 volumes (aqueous + salt) 100% ethanol, mix, and microfuge for 5 minutes at RT.
      IPpt DNA precipitation with isopropanol; for removing adaptor primers. Use 1 volume of 2 M NaClO4 and glycogen carrier, mix, and add 1.5 volume (aqueous) isopropanol, mix, and microfuge for 5 minutes at RT.
      loTE 3 mM Tris, 0.2 mM EDTA, pH 7.5
      MBN Mung bean nuclease
      NEB New England Biolabs
      dNTPs 10 mM of each 4 dNTPs, Pharmacia
      O/N Overnight
      PC8 Extraction with phenol:chloroform:isoamylalcohol (25:24:1), pH 8.0; includes 1 minute micro-centrifugation.
      PCR Done in tubes, with 2 drops of mineral oil, in an Omnigene thermocycler (Hybaid) using the tube control mode.
      RDA 10x RDA buffer: 670 mM Tris pH 8.8, 160 mM (NH4)2SO4, 100 mM -Mercaptoethanol, 1 g/l BSA
      RT Room temperature
      W Wash with 70% ethanol; includes 1 minute micro-centrifugation. Routinely, a DNA precipitation is followed by two washes. When the next procedure is a ligation, the DNA is washed three times.
      QBT, QC, & QF Qiagen buffers