Notes

1. Hybridization conditions were optimized after a series of test subtractions in which tracer cDNAs were radioactively labeled and the removal of shared sequences monitored by measuring the amount of radioactivity remaining in the extracted aqueous phase. Over 80% of tracer sequences could be reproducibly removed in one round using the above hybridization conditions leading to 99% removal of common sequences after four sequential rounds of subtraction.

2. With some preparations of the oligonucleotide T7dT, we have noticed PCR artifacts which were generated in the initial conversion of the primary NotI dT-

PROBE

Fig. 2. Differential screening of a human X cDNA library with subtracted PolyAcDNA probes. Two replica filters were produced from a single Petri dish containing approx 2000 X plaques. One filter was hybridized with a probe enriched for sequences expressed in unilineage cells (U-M), and the other hybridized with a probe enriched for sequences expressed in multilineage cells. The arrows indicate clones that hybridized strongly with one probe and not the other.

Fig. 2. Differential screening of a human X cDNA library with subtracted PolyAcDNA probes. Two replica filters were produced from a single Petri dish containing approx 2000 X plaques. One filter was hybridized with a probe enriched for sequences expressed in unilineage cells (U-M), and the other hybridized with a probe enriched for sequences expressed in multilineage cells. The arrows indicate clones that hybridized strongly with one probe and not the other.

amplified PolyAcDNA to T7 amplifiable tracer cDNA. Since these artifacts are not present in the NotI dT driver cDNA, they are enriched during subtraction and can form a significant part of the subtracted material if there are only minor differences between the subtraction partners. If this is a problem, to avoid this possibility, the tracer is maintained as with the may be avoided as follows.

Subheading 3.1., step 1: Substitute T7dT for NotI 24 in the PCR reaction to generate the driver.

Subheading 3.1., step 2: Substitute NotI dT for T7dT in the PCR reaction to generate the tracer.

Subheading 3.3., step 6: Substitute NotI Unique for T7Unique to amplify enriched tracer sequences after subtractive hybridization.

3. When washing Southern filters containing PolyAcDNAs hybridized with a PolyAcDNA probe, the probe will anneal to the polyA/T ends present in all PolyAcDNA molecules making it necessary to wash at high stringency (0.2X SSC) at temperatures up to 73°C.

4. Hybridization and washing conditions for library screening should be carried out at a lower stringency, particularly when probe and library are derived from dif ferent species. In the example shown in Fig. 2, a mouse was used to screen a human library, and washing stringency was kept low (45°C and 2X SSC).

5. The ultimate success of a screening procedure will often be judged by a functional test of the identified gene. For this reason, it is important to consider parameters, such as the average size of inserts in the library (reflecting the probability of having full-length cDNAs) and the presence of suitable selection markers and promoters in the cDNA vector.

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