DOP-PCR OF DNA FOR COMPARATIVE GENOMIC HYBRIDIZATION

 

In designing the PCR reaction, be sure to include a negative PCR control (blank without DNA), a positive control (we use MPE-600 breast tumor cell line DNA) and enough reference DNA for all of the planned CGH reactions.

 

METHOD:

1. Add 1 ul (~50 ng) DNA to each of the sample tubes.

    

2. Add 5 ul of the Pre-Amplification mix to each tube.

    

3. Overlay with 20 ul oil and start PCR Program #91. This step is only necessary if Topo-1 preamplification is used.

    

4. PCR #91: 30 min @ 37 C; link to #92.

    

5. PCR #92: 10 min @ 96 C; link to #93.

    

6. During Program #92 make up a 1:8 dilution of Sequenase in Sequenase dilution buffer (enough for 5 aliquots of 0.3 ul for each sample).

    

7. PCR #93: 5 min @ 30 C, 2 min @ 37 C, 1 min @ 94 C, 10 sec @ 30 C, 5 cycles. Then link to #94.

    

8. For all 5 cycles of #93, Sequenase (0.3 ul) is added through the oil during the initial 5 mins.

    

9. Add 4.0 ul of Taq Polymerase (0.4 ul per sample) to the remaining 450 ul of PCR buffer , mix.

    

10. PCR #94: 10' @ 95 C; link to #95.

     

11. During the last 5 mins of #94, add 45 ul of Taq/PCR buffer, mix (just uncap the tubes, don't take them out; add through the oil)

     

12. Add 45 ul more oil to cover, flick tubes to make sure there are no bubbles before #95 starts.

     

13. PCR #95: 1' @ 94 C, 1' @ 56 C, 3' @ 72 C; 35 cycles; link to #96.

     

14. PCR #96: 5' @ 72 C; link to #97.

     

15. PCR #97: soak at 4 C.

     

16. Remove the oil onto parafilm, and pipette samples into new tubes.

     

17. Load 3 ul of each sample onto 1% agarose gel (using small comb) to define amount and size of product DNA.

     

18. Store remaining DNA PCR product at -20 C until nick translation.

 

Notes

Remember to include sufficient normal DNA reactions for the planned CGH (usually two double PCR reactions: 100 ng in 100 ul each).

We usually include a 50 ng MPE-600 cell line positive PCR/CGH control, and a PCR blank negative control.

For PCR of fresh DNA (from unfixed material), 50 ng input DNA is used per 50 ul PCR reaction. Then use 25 ul of the PCR product for each 50 ul nick translation. Then use 20 ul of FITC or Biotin-labeled probe, or 15 ul of Texas Red-labeled probe, or 10 ul of Dioxigenin-labeled probe for each CGH reaction.

For PCR of paraffin DNA (from formalin fixed / paraffin embedded tissue), it is best to add only 1 ul of DNA per 50 ul PCR reaction. The reaction may be inhibited (less product) if too much of the DNA extraction solution is added (perhaps breakdown products from the Proteinase K reaction are inhibiting). Use 40 ul of this PCR product per 50 ul nick translation reaction, and then use all of the nick translation product for the CGH. If the digoxigenin-labeled probe size is good (>600 bp), only 35-40 ul of the probe still yields good CGH.

We have found that it is unwise to amplify more than 13 tubes per PCR reaction.

Materials

PCR Mix (10 reactions): 100 ul 5X PCR buffer + 400 ul ddH20.

Preamplification Mix (10 reactions): 50 ul of the PCR mix + 1 ul Topo-1 (0.1 ul/sample) + 2 ul MgCl2 (50mM; 0.2 ul/sample); mix well.

5X PCR Buffer (Boehringer): 500 ul 10X Taq buffer; 1 0ul dATP, 10 ul dCTP, 10 ul dGTP, 10 ul dTTP, 125 ul MgCl2 (50mM); 6.7 ul of 1.4 mM DOP Primer, 335 ul dH20. Total 1000 ul (store frozen in 200 ul aliquots).

10x Taq Buffer (Boehringer): 100mM TrisHCl; 2mM MgCl2; 500mM KCl; 1mg/ml gelatin.

DOP Primer : 5'-CCG-ACT-CGA-GNN-NNN-NAT-GTG-G-3'. Use at 1.4 mM.

Topo-1 : Promega.

Sequenase -Vers 2 (USB/Amersham): Use at 1:8 with Sequenase dilution buffer

Taq Polymerase : Boehringer: 5U/ul (use 2U/sample)

dNTP 's (Boehringer): use stock of 100 mM each.

 

PCR Programs

#91: 30 min @ 37 C; Link to #92.

#92: 10' @ 96 C; Link to #93.

#93: 5' @ 30 C, 2 min @ 37 C, 1 min @ 94 C, 10 sec @ 30 C, 5 cycles; Link to #94.

#94: 10' @ 95 C; Link to #95.

#95: 1' @ 94 C, 1' @ 56 C, 3' @ 72 C; 35 cycles; Link to #96.

#96: 5' @ 72 C; Link to #97.

#97: soak at 10 C.