Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization.
Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.
Rebrikov
D V
DV
Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia.
Britanova
O V
OV
Gurskaya
N G
NG
Lukyanov
K A
KA
Tarabykin
V S
VS
Lukyanov
S A
SA
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005189:False Positive Reactions; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D051379:Mice; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D013687:Telencephalon / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger