Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles

Abstract

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.

Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis

“…Smaller fragments hybridize more efficiently and are amplified more efficiently by PCR. The amplification efficiencies of PCR products are also affected by the nucleotide sequence of the DNA fragment (49). Thus, the probability of identifying differentially expressed genes depends also on the chosen restriction enzyme.…”

Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

“…The approach can be fine-tuned by the user to provide different degrees of coverage and discriminatory power, depending on the choice of fragmenting enzyme. The method is similar to the TALEST modification (Spinella et al 1999) of the original SAGE protocol in that it uses a 16-fold degenerate linker cassette to attach an oligonucleotide adapter to the unknown 3Ј overhangs of MmeI-digested DNAs, thereby taking advantage of being able to use cohesive termini for highefficiency linker addition. Addition of this linker not only provides an appended sequence for PCR amplification but also attempts to reduce bias during amplification by flanking the monomeric GSTs on both sides with distinct long linkers.…”

“…The major difference between the two procedures occurs during the formation and subsequent amplification of the resulting tags. In the long SAGE protocol, selfligation of MmeI overhangs is used to form ditags (Saha et al 2002), whereas in the GST method, an excess of a 16-fold degenerate cassette (Spinella et al 1999) is used to add the oligonucleotide adapter needed for PCR amplification. Although ligation of the degenerate cassette and subsequent PCR of the monotags might be more efficient under some conditions, the orientation of the cloned monotags can only be independently determined by the position of the extra nonpalindromic bases that are added during ligation with the degenerate cassette.…”

“…Since the SAGE technique was first reported, several groups have modified the original procedure in order to increase tag length (Ryo et al 1998(Ryo et al , 2000Spinella 1999). These longer tags are particularly useful in characterizing expression patterns in the absence of complete genome sequence data, that is, from "uncharted transcriptomes," and in designing primers to obtain full-cDNAs from transcripts with tags that are not currently present in RefSeq or similar expression databases.…”

“…Because the last C residue in the MmeI recognition site of the adaptor partially overlaps the NlaIII site of the bound DNA, the released fragments contain 21 bases of sequence information from the starting DNA. These products are recovered and ligated with an adaptor having a 16-fold degenerate 3Ј overhang (Spinella et al 1999) which renders it compatible with all possible two-base 3Ј overhangs released by MmeI. This adaptor was designed to add two consecutive T residues and a second NlaIII site on the ends of the original MmeI-generated fragments (TTCATG…).…”