A general strategy for cloning viroids and other small circular RNAs that uses minimal amounts of template and does not require prior knowledge of its sequence.
Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.
Navarro
B
B
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Spain.
Daròs
J A
JA
Flores
R
R
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics; D012313:RNA / Q000235:genetics*; D000079962:RNA, Circular; D012367:RNA, Viral / Q000235:genetics; D013698:Templates, Genetic; D014772:Viroids / Q000235:genetics*
D017931:DNA Primers; D000079962:RNA, Circular; D012367:RNA, Viral; D012313:RNA