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

500 Ravassard et al.
Fig. 1. Constructing cDNA libraries using the SLIC strategy. Reverse transcription is
performed with a random primer RA3′ NV. RNAs are digested and primer is removed to avoid
its ligation to the A5′ NV oligonucleotide. A5’ NV bears a phosphate group at its 5′ extremity
to allow ligation to the 3′ end of the ss-cDNA. To avoid formation of concatemers A5′ NV
bears an amino group at its 3′ end. Two rounds of nested PCR are performed to generate a
ready-to-use cDNA library.
short and long molecules with the same primers always leads to a selective amplification
of the shorter ones, even though the longer ones were originally more abundant (1).
Attempts to enlarge the average size of the PCR products have been made with the
help of long-range PCR procedures, such as Pfu dilutions (2), Taq extender (Stratagene,
La Jolla, CA), and Expand PCR system (Boehringer, Indianapolis, IN). The results
were not significantly different if compared with the classic PCR techniques (data
not shown).
In conclusion, the size bias represents the major limitation in constructing PCR
cDNA libraries. Thus, to avoid bias during the amplification step the average size of
the cDNA library must be between 0.8 and 1 kbp, which is far below the usual average
length of a ss-cDNA.