Efficient cloning of full-length cDNAs based on cDNA size fractionation.
The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries.
Draper
Michael P
MP
Paratek Pharmaceuticals, 75 Kneeland Street, Boston, Massachusetts 02111, USA. mdraper@paratekpharm.com
August
Paul R
PR
Connolly
Timothy
T
Packard
Brian
B
Call
Katherine M
KM
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification*; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data
D018076:DNA, Complementary