Subtractive Hybridization

Common tracer sequences are removed by solution hybridization with the biotinylated driver, followed by removal of all biotin-containing hybrids after addition of streptavidin and organic extraction.

1. To a 0.5-mL tube, add 200 ng tracer to 4 |g driver. Then add 5 |g tRNA to act as a carrier, adjust the volume to 80 ||L with HE buffer, mix well and boil for 2 min. To precipitate, add 8 |L 3 M NaAC and 200 |L 100% ethanol. Vortex briefly, chill on ice for 20 min, and spin at full speed in a refrigerated centrifuge. Wash pellet with 70% ethanol and air-dry.

2. Carefully resuspend pellet in 9 ||L 1X hybridization buffer, cover with 70 ||L mineral oil, put on tube cap locks to prevent tubes from bursting open then boil for 5 min.

3. Place sample PCR machine preheated to 80°C. Run the following thermal profile: 5 min at 80°C, ramp 80-68°C over 15 min, 90 min at 68°C, 10 min at 80°C.

4. To the sample at 80 °C on the PCR machine immediately add 91 |L of extraction buffer (preheated to 80°C). Centrifuge briefly to ensure all the aqueous layers have combined. Remove as much mineral oil from the top as possible.

5. Centrifuge again, and transfer the bottom 75 ||L of hybridization/extraction mixture to a fresh tube containing 21 |L of fresh extraction buffer and 4 |L of 4 |g/|L streptavidin. Mix and incubate at room temperature for 2 min.

6. Add 100 |L TE-saturated phenol/chloroform, vortex for 30 s, and spin at full speed room temperature for 5 min. Remove top three-quarters of the aqueous phase (~75 |L) avoiding the interphase and transfer to a fresh tube.

7 Extract the transferred aqueous phase with 50 mL of chloroform vortex for 30 s, spin at full speed at room temperature for 5 min, and transfer as much of the aqueous layer as possible to a fresh tube. Use ~60 |L of this for the next round of subtraction. Save the remainder ~10 |L for later amplification. Use 5 | L of subtracted material for amplification with the T7Unique oligo.

8. The 60 |L of extracted tracer removed in step 8 are then carried through further sequential rounds of subtraction, and the above steps are directly repeated with the extracted tracer substituting the 200 ng of starting tracer in step 1.

9. Repeat extraction cycles 3-4 times.

10. Amplification of subtracted material: Add 5 |L of subtracted material to a 100 |L PCR reaction using T7Unique as the amplification oligo. Thermal profile, 30 s at 94°C, 30 s at 60°C, 90 s at 72°C x25 cycles.

cDNA LIBRARY REPLICA FILTERS

Fig. 1. Southern hybridization of starting PolyAcDNA with enriched and nonenriched probes. The starting PolyAcDNA samples U and M are pools of PolyAcDNAs derived from 20 unilineage mouse hematopoietic precursors (U) and five multilineage mouse hematopoietic precursors (M) (6). Replica filters of the starting samples were hybridized with unsubtracted (probes U and M) and with PolyAcDNA remaining after four sequential rounds of subtraction (probes U-M and M-U).

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