Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis

The serial analysis of gene expression (SAGE) technique is an important tool for genome-wide gene expression analysis. However, the requirement of a large amount of mRNA for the analysis and the difficulties in generating high-quality tag and ditag fragments for the construction of a SAGE library often interfere with the successful performance of the SAGE technique. We developed two procedures to solve these issues: (i) introducing low-cycle PCR amplification of the 3' cDNA before the BsmFI digestion of the 3' cDNAs and (ii) gel purifying the BsmFI-released tag fragments before ditag formation. These modifications provide a large quantity of initial 3' cDNAs and high-quality tags and ditags for the construction of SAGE libraries.     

Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) by adding a heating step to the original protocol.

The efficiency of the original SAGE (Serial Analysis of Gene Expression) protocol was limited by a small average size of cloned concatemers. We describe a modification of the technique that overcomes this problem. Ligation of ditags yields concatemers of various sizes. Small concatemers may aggregate and migrate with large ones during gel electrophoresis. A heating step introduced before gel electrophoresis breaks such contaminating aggregates. This modification yields cloned concatemers with an average size of 67 tags as compared to 22 tags by the original protocol. It enhances the length of cloned concatemers substantially and reduces the costs of SAGE.     

Discarding duplicate ditags in LongSAGE analysis may introduce significant error

Background  

During gene expression analysis by Serial Analysis of Gene Expression (SAGE), duplicate ditags are routinely removed from the data analysis, because they are suspected to stem from artifacts during SAGE library construction. As a consequence, naturally occurring duplicate ditags are also removed from the analysis leading to an error of measurement.     

3' end cDNA amplification using classic RACE

Having knowledge of the entire 3' sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3' RACE, the poly(A) tail functions as a non-specific tag at the 3' end of the mRNA. cDNA ends can be obtained in 1-3 days using this protocol.     

5' -and 3'-terminal nucleotide sequences of Tetrahymena pyriformis 17S rRNA.

Ribonuclease T1 oligonucleotides arising from the 5' and 3' termini of the 17S rRNA of Tetrahymena pyriformis were isolated by the diagonal method of Dahlberg (Dahlberg, J. E. (1968), Nature (London) 220, 548), and their nucleotide sequences were determined. The base sequence of the 3'-terminal fragment is (G)AUCAUUAoh, which is identical to that found in other 17S-18S eucaryotic rRNA species. The nucleotide sequence of the 5'-terminal oligonucleotide is pAACCUGp, which is identical in length to that found in other eucaryotes and shows a partial but significant sequence homology to the 5' RNase TI oligonucleotides isolated from other eucaryotic species.