Target Discovery and Validation Reviews and Protocols

Gene Target Discovery 117
In the first protocols describing subtraction cloning, double-stranded (ds)
driver–tester was removed by hydroxyapatite (HAP) column chromatography,
and single-stranded driver mRNA was removed by RNase or alkali degradation
(5–7). HAP binds double-stranded molecules more tightly than single-stranded
molecules, enabling separation of driver–tester and driver–driver duplexes from
unhybridized tester. Because this method is cumbersome, alternative techniques
for removing driver and tester duplexes have been developed, and they involve
both methods for physical removal, enzymatic degradation, immobilization,
and chemical cross-linking (reviewed in ref. 8).
1.1.3. PCR-Based Subtraction Cloning
Using polyA+ mRNA as the driver is possible only when one is able to
obtain large amounts of starting material and when the biological sources to be
compared are closely related. When material is limited, or the tissue is complex,
amplification must be performed before subtraction. Multiple rounds of subtraction
also may require amplification of the resulting population of tester. A
drawback is that the representation of individual transcripts may be altered during
multiple rounds of amplification. The transcript from both driver and tester
may be amplified from cDNA libraries, by in vitro transcription, or by PCR.
Single-stranded driver is more efficient than double-stranded driver because of
competition between driver–driver duplex formation and the desired driver–tester
duplex formation. This competition necessitates more rounds of subtraction to
ensure optimal subtraction. Amplified single-stranded driver may be obtained
from libraries as single-stranded phagemids (9), by in vitro transcription with T3
or T7 RNA polymerase (10), or by asymmetric PCR on single-stranded template
(11). However, subtraction performed using double-stranded driver also can be
effective (12).
1.1.4. Positive Selection
In positive selection, nucleic acids representing genes uniquely or predominantly
expressed in one source of biological material compared with another
are selected and isolated. Undesired nucleic acids representing genes expressed
in both sources are left behind.
For subtraction cloning, nucleic acids from the interesting material (tester)
are hybridized with nucleic acids from the compared material (driver). In
contrast to subtraction cloning, double-stranded nucleic acids are used both
for driver and tester. The principle is to isolate tester–tester duplexes, representing
genes uniquely expressed in the material of interest. Tester–driver and
driver–driver duplexes as well as unhybridized driver and tester are left behind.
No physical separation of single-stranded and double-stranded nucleic acids
is included.