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

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

      Repeat digestion and purification

       

   2. Ligation to RBamHI primers

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

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

       

   3. PCR-1 amplicons

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

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

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

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

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

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

      PC8, EPpt, 2W, resuspend in 10 µl 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 µg of amplicons should be generated per 200 µl 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 µg 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/70°C
      Load on a Q20 tip that is equilibrated with 1 ml QBT; keep the gel mix at 70°C; load in ~500 µl aliquots
      Wash with 6 ml QC
      Elute with 800 µl QF
      Precipitate by adding 2 µl glycogen and 650 µl isopropanol
      Microfuge 10 min/RT, 2W, resuspend in loTE at ~100 ng/µl (assume 30% recovery)
      Gel-quantify and bring the DNA to a final concentration of 100 ng/µl

      Perform PCR on ~1.5 ng of gel-purified tester amplicons as template, in 200 µl 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 µg of tester amplicons and ~120 µg 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 µg/µl.

       

   6. Digestion amplicons

      1 µg amplicons
      1 µl buffer B
      0.4 µl BamH1 (10 U/µl)
      x µl loTE
      10 µl

      Scale the reaction up according to the amount of amplicons

      > 1 hr/37°C

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

       

   7. Ligation tester amplicons to JBamHI primers

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

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

      Test the ligation in PCR with previous primers (RB24) and new primers (JB24), with ~35 ng template in a 50 µl 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
   
   8. Hybridization-1

      500 ng tester-JB amplicons
      40 µg driver amplicons
      x µl loTE
      200 µl

      PC8, IPpt, 2W (no glycogen)
      Carefully resuspend in ~3.5 µl 3xEE buffer to a final volume of 4 µl^a
      Overlay with 1 drop of mineral oil
      5 min/98°C
      Add 1 µl prewarmed 5 M NaCl, carefully mix by pipetting
      20 hr/67°C^b
      Dilute to 500 µl 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/µl is around the limit of solubility of DNA; after resuspension in 4 µl 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.

       

   9. PCR-1 of Hyb-1

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

       

   10. . Mung bean nuclease treatment Hyb-1

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

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

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

        

   11. . PCR-2 of Hyb-1

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

        

   12. . 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 µg driver	JBamH1
       2	50 ng Hyb1-NB / 40 µg driver	NBamH1
       3	100 pg Hyb2-JB / 40 µg driver	JBamH1
       4	5 pg Hyb3-NB / 40 µg driver	NBamH1

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

   13. . 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 ß-Mercaptoethanol, 1 µg/µl 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