Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.
We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.
Shibata
Y
Y
RIKEN Tsukuba Institute, Ibaraki, Japan.
Carninci
P
P
Watahiki
A
A
Shiraki
T
T
Konno
H
H
Muramatsu
M
M
Hayashizaki
Y
Y
D020121:5' Untranslated Regions; D000818:Animals; D001923:Brain Chemistry; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D015723:Gene Library; D016678:Genome; D008099:Liver / Q000737:chemistry; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010865:Pilot Projects; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011088:DNA Ligases