cDNA Library Construction for the Lambda ZAP-Based Vectors

Because the vast majority of plant viruses have a positive-sense RNA genome, which acts as the viral mRNA, the RNA must first be converted into cDNA before cloning, amplification, and subsequent manipulation. Successful cDNA synthesis should yield full-length copies of the original population of mRNA molecules. Hence, the quality of the cDNA library can be only as good as the quality of the mRNA. Pure, undegraded mRNA is essential for the construction of large, representative cDNA libraries (1 ). Secondary structure of mRNA molecules can cause the synthesis of truncated cDNA fragments. In this case, treatment of the mRNA with a denaturant, such as methylmercuric hydroxide, prior to synthesis may be necessary (2 ). Other potential difficulties include DNA molecules contaminating the mRNA sample. DNA can clone efficiently and their introns can confuse results. RNase-free DNase treatment of the sample is recommended.