FILTRE PREPARATION

1. Genomic DNA samples

Genomic DNA was extracted from the liver of individual rats by the phenol and chloroform extraction method. You can use genomic DNA extracted from the tail by DNA extraction machine.

Prepare the following PCR solution.
                         Stock        to be       final
                        solution      used     concentration
10x PCR buffer III*      10 x        1.5 µL        1 x
dNTP                   2 mM each      3 µL       0.3 mM each
AP-primer*               50 µM       0.6 µL       1.5 µM
Taq polymerase           5 u/µL      0.25 µL     0.0625 u/µL
dH2O                                   -
Genomic DNA                           50 ng (20-100 ng OK)
Total volume                          20 µL

AP-PCR in a 96 well plate.
PCR conditions: for initial denaturation 3 min at 95°C followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 40°C and extension for 2 min at 72°C.
If you want, confirm good amplification by electrophoresing 5 µl of PCR solution in 2 % NuSieve agarose gel.

Prepare denatured PCR solution by mixing AP-PCR solution with equal volume denature solution contains 0.8 M NaOH and 50 mM EDTA.
Blot the solution onto Hybond-N+ (Amersham) nylon filters by dot-blotter (ATTO Multi-pin-blotter AB-6690 or Kriplanker).
Cross-link the filters by UV. Rinse in 6x SSC, and dry filters.

HYBRIDIZATION

1. Preparation of probes.

Mix 6 µL of marker DNA solution (gel sol.) and 11 µl of dH2O and, denature the solution in boiling water for 5 min. Two µL of marker DNA solution (PCR sol.) and 15 µl of dH2O are also available.
Label the gel using Gene Images labeling kit (Amersham) in the following manner.
                            Vol. to be
                               used
Denatured DNA                  17 µL
solution of nucleotide          5 µL
Random primer                 2.5 µL
Klenow fragment               0.5 µL
Total                          25 µL
Incubate at 37°C for 2 hours.

Prehybridize the filters.  Hybridize the filters with the probes prepared.
Wash the filters.  Detect according to the manufacturer's protocol. We use X-ray film for detectuon.

Analyze with Mapmaker/EXP software (Lander et al. 1987).
With the markers isolated by AP-RDA using ACI as the tester and BUF as the driver, homozygotes for the ACI allele (AA) or heterozygotes (AB) gave positive signals, and homozygotes for the BUF allele (BB) gave negative signals. In this case, AA and AB were together genotyped as D, and BB as B. With the markers obtained by AP-RDA in an opposite combination, AA (negative signal) was genotyped as A, and AB and BB (positive signals) were together genotyped as C.

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Carcinogenesis Division National Cancer Center Research Institute 5-1-1 Tsukiji Chuo-ku, Tokyo 104-0045, Japan Send mail to: tushijim@gan2.ncc.go.jp