PCR:

We generally use 1-2 ul of starting paraffin microdissected DNA for each 50 ul DOP-PCR reaction.

We assume that about 1 ug of product is produced in each DOP-PCR reaction. The entire product is then nick translated as described below.

Nick Translation of the PCR Product:

For the first CGH, our approach is to start with predefined test and reference labels, and then to do a replicate ("inverse") reaction based on the initial results.

For the first CGH, the sample DNA is nick translated with digoxigenin-dUTP and stained with anti-digoxigenin-TRITC. 45 ul of the nick translation product is used. The amount of the digoxigenin sample should be reduced to 35 ul if the PCR product size is larger than ~600 bp.

The reference DNA is directly labeled with FITC-dUTP (20 ul is used for the CGH).

We use PCR-amplified DNA from the MPE600 cell line for a positive control CGH. Since this is good quality fresh DNA, only 10 ul of the dig-labeled DNA is necessary.

CGH Hybridization (see Direct CGH for details of the hybridization)

1) Reprecipitate DNA's

• Add the following DNA's to a 1.5 ml centrifuge tube, mixing with pipet:20 ug Cot-1 DNA (~20 ul)~900 ng FITC labeled DNA (~45 ul)~200 ng Texas Red labeled DNA (~22 ul)
• Add 1/10th volume of 3M Na Acetate, mixing with pipet.
• Add 2.5 X (original) vol 100% EtOH to ppt DNA, vortex gently.
• Spin 30 mins at 14K rpm, 4 C.
• Decant supernatant; blot dry, being careful to avoid DNA pellet.
• Add 10 ul of MM1/H20 mix (70% MM1/30% H20).
• Carefully dissolve with pipet, and gently vortex.
• Quickly spin (1 sec) to bring volume to bottom of tube.

(see directcgh or indirectcgh for further instructions)

The Inverse Hybridization

Based on the initial CGH hybridization using digoxigenin labeling, and the PCR product size, the following algorithm is used to decide on the second hybridization:

If the initial CGH using the digoxigenin labeled DNA was bad, the PCR is repeated using more DNA, and if the CGH still fails, a second microdissection is considered.

If the probe size was good (>600bp) and the CGH using digoxigenin labeled probe was bright and uniform, then the second inverse reaction uses sample DNA directly labeled with FITC-dUTP (40 ul) and reference DNA directly labeled with Texas Red dUTP (22 ul).

If the CGH using the Digoxigenin labeled DNA looked good, but the probe size wasn't ideal (or the probe size was good but the dig CGH wasn't good quality) then the test sample is labeled with Digoxigenin and stained with anti-digoxigenin-TRITC (45ul), and the reference DNA is labeled with biotin (22ul) and stained with FITC-Avidin (1 layer on day 2).