Differential mRNA fingerprinting by preferential amplification of coding sequences.
The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.
Fuchs
B
B
Mayo Clinic, Medical Sciences 3-69, 200 First Street SW, Rochester, MN 55905, USA.
Zhang
K
K
Bolander
M E
ME
Sarkar
G
G
D000251:Adenosine Triphosphatases / Q000235:genetics; D002460:Cell Line; D016172:DNA Fingerprinting; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D025261:Mitochondrial Proton-Translocating ATPases; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; C501005:MT-ATP6 protein, human; D012333:RNA, Messenger; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases