John M. S. Bartlett.pdf - Bio-Nica.info

502 Ravassard et al.
size dispersion around the mean is extremely low when compared with a conventional
library. Finally, 5000 primary clones were screened with a TH oligonucleotide, yielding
two positive TH clones (0.04%). Thus, no major distortion had been introduced in the
abundance of TH clones, since TH represents 0.05% of CSG mRNA.
1.5. Direct Screening of the PCR Library
with Biotinylated Oligonucleotides
One of the major difficulties in making a cDNA library is the cloning of the
double-stranded cDNA (ds-cDNA). We tested a direct screening protocol of uncloned
ds-cDNA. After the second nested PCR, the amount of ds-cDNA was about 2 to 5 µg.
We directly hybridized the denatured ds-cDNA with a biotinylated TH specific primer.
After the hybridization reaction, probe-cDNA hybrids were separated from unhybridized
DNA using streptavidin-coated magnetic beads (Dynabeads M-280). After various
washing steps, the captured cDNA was amplified using the third nested PCR primers
(A5′3 and A3′3) directly onto the beads. The PCR product was cloned, and more than
85% of them were TH clones as analyzed by partial sequencing. Biotinylated cDNA
probes, instead of oligonucleotides, can also be used to screen a PCR library (4).
1.6. Application of the SLIC Method to Subtractive Libraries
Subtraction cloning strategies could be modified and certainly improved taking
advantage of the generation of cDNA molecules exhibiting two defined extremities.
Basically, tracer cDNA synthesized on mRNA from source A is hybridized to sequences
of driver mRNA, which is isolated from a different but usually related source B. The
tracer cDNAs that do not become hybridized with driver mRNA represent an enriched
population of sequences expressed only in A cells. These are used for constructing an
A-cell specific cDNA library.
The SLIC strategy provides DNA molecules with two defined ends. This offers the
opportunity to work on cDNA from populations A and B with two different sets of
SLIC primers, A (A5′ NV and RA3′ NV) and B (B5′ NV and RB3′ NV; Fig. 3). During
the PCR amplification of the tracer population B, a pair of biotinylated primers is used.
Thus, this population can be captured and pure single-stranded molecules immobilized
on magnetic beads. Then, after hybridization with the amplified A population, the
unhybridized population corresponds to the A-cell specific sequences and can be used
easily to generate substracted libraries or probes. This strategy gives for the first
time the opportunity to realize such subtractive libraries with a very small amount
of input material.
1.7. Conclusion
The SLIC method is a powerful and unique tool to synthesize cDNA and substractive
libraries from a limited number of cells. It is unfortunately extremely difficult to
generate full-length libraries. Nevertheless, cloning 5′ or 3′ ends of a cDNA is no
longer a limiting step because anchored PCR can be easily performed. In this case, the
same ss-cDNA that was used to generate the library can also be used as a matrix to
isolate both ends of the incomplete clone.