Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes.
In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.
Carninci
P
P
Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, Tsukuba, Japan. rgscerg@rtc.riken.go.jp
Shibata
Y
Y
Hayatsu
N
N
Sugahara
Y
Y
Shibata
K
K
Itoh
M
M
Konno
H
H
Okazaki
Y
Y
Muramatsu
M
M
Hayashizaki
Y
Y
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification*; D005260:Female; D015723:Gene Library; D008297:Male; D051379:Mice; D011247:Pregnancy; D012315:RNA Caps; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D012315:RNA Caps