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CONCLUSIONS AND PERSPECTIVES

Stringent quality control on sequence databases is required

These findings may contribute to a more stringent implementation of a quality control within nucleotide databases, as well as the professional analysts of molecular artifacts and related experiments, not to mention the rational human engineering of pharmaceutical drugs and proteins. We must first tame, through its controlled use, those palindromic linkers. Then, the use of sequence quality control (to detect those molecular artifacts) can successfully be applied. Improvements can be made in the design of Affymetrix microarrays, i.e., by removing the palindromic linker from human sequences such as AA557228, AA113291, AI798671, AA864645, AA780435, W90032, AA810599, T52176, AA976510, AI380906, AA535275, AI792166, T67559, L04270, U83598, D59474, T03148, T54342, D59674, D59787, D80164, H90908, N80129, C14426, D59619, D80240, T56800, C14298, W72424, IR1056496, T69555, D80337, C14227, C14407, C14344, D80210, etcetera.

Possible use of palindromes and its heterotranscripts to reverse hereditary diseases

Another possibility is the use of palindromes for the attachment of two different genes (genetic modules) for the engineering of therapeutic proteins, i.e., for the engineering of antiobesity treatments that may be customized and prescribed according to the particular metabolism of each person (21).

Artificial vectors for gene therapy may produce heterotranscription

It is possible that palindromic linkers like the ones reported in this article, or using other linkers, may be produced by artificial vectors for gene therapy also. If this is the case, an unsuspected side effect for humans may result. To date, those linkers are still present in those thousands of artificial sequences submitted to the Genbank, and are an allegory of things that can be prevented in human gene therapy. This article has been written to raise the awareness to the scientific community on the presence of these thousands of artificial hetero-transcripts by current methodologies producing the sequences reported to the Genbank as well as the vectors used in gene therapy.

Published and submitted heterotranscripts are still awaiting its correction

In doing an analysis of the sequences presented as chimerical mRNAs in Table 4 of reference (22), I also found in that article the EcoR1-related palindromic linker present inside the sequence AY029161 (16), already seen. In that sequence, there is a fragment for a putative human tumor suppressor LPTL, AF418553, originated in the human chromosome 8, and linked with a fragment for phosphohistidine phosphatase PHP14, NM_014172 originated in the human chromosome 9.

Until the submission of this article, none of the sequences presented here had been corrected in the Genbank. Could this be because they have not been determined as artifact products to date?

Another sequence in reference (22) that contained the EcoR1 related palindromic linker was BC000519, identified as an artifact and has already been removed from the Genbank.

A possible transposon-like removal of palindromic linkers seem to have happened on other chimeras

Awaiting correction are thousands of sequences like the ones presented in Table 2 (7). Sequences like AF176705 for the human F-box protein FBX10 (23), and Z28355 from the atrium heart (24), are both containing fragments of the vector and of the linker. Another sequence, HTCBYB08, contains fragments of the vector only, lacking the linker. Experiments were also done with such sequences lacking any evidence of a palindromic linker, also without obtaining amplified products by RT-PCRs (unpublished results). Both of the sequences Z28355 and HTCBYB08 exhibit traces of the same cloning vector, vector from the sequence X52324 (ARBLSKM), the pBluescript SK(-) vector (25-27). A transposon-like self-removal of the palindromic linker may be one way in which many heterotranscripts appear to be lacking the linker, as in the sequence HTCBYB08. This hypothesis also needs to be experimentally evaluated. If this is the case, palindromic linkers may be used for a possible artificial reversal of mutations in vivo (28).

On the other hand, heterotranscripts lacking the linker, and present in Affymetrix' microarrays, may show two different expression profiles, for example, a zero expression on one side while a contrasting higher expression on the other side (data not shown). A similar pattern is present when non-expressed introns are included within Affymetrix' probes side by side with sequences expressed by the exons, as in Figure 1 from reference (2).

Final comment

The key palindrome described in this article has already been introduced in reference (2) as CTCGTGCCGAATTCGGCACGAG and it has been reported elsewhere (2, 3, 29), also.

ACKNOWLEDGMENTS

Tracy Lynn Duncan helped in preparing this review and supported me during the last two years.

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