Amplification of high-quantity serial analysis of gene expression ditags and improvement of concatemer cloning efficiency.
As a method for accurate quantitative and qualitative analysis of cell transcription, serial analysis of gene expression (SAGE) has been widely used in biological, medical, and pharmaceutical areas of research. The numerous steps required in the SAGE protocol, however, limit the efficiency of SAGE library construction. PCR amplification of ditags and concatemer separation are two such steps. Primer-2, which was used to amplify ditags in the original SAGE protocol, was prone to self-anneal and resulted in a decreased efficiency of the PCR amplification step. We have designed a new primer pair, which substantially enhances the efficiency of PCR amplification. Incorporating this modification reduces the number of reactions required for SAGE library construction. Sixty 50-microL reactions are now sufficient for SAGE library construction (up to an 80% reduction in comparison to the original protocol). Concatemer separation by column filtration and subsequent gel separation has been modified and has proven to be more efficient than simple heat treatment of the ligation mixture. Both of the above modifications may also be suitable for the newly developed LongSAGE protocol.
Du
Zeji
Z
Samuel Roberts Noble Foundation, Ardmore, OK, USA.
Scott
Angela D
AD
May
Gregory D
GD
D003001:Cloning, Molecular / Q000379:methods*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D014158:Transcription, Genetic / Q000235:genetics*