Universal Inheritable Barcodes for Identifying Organisms

Abstract

The needs for recognition of novel conventional or transgenic organisms include protection of patented or Identity Preserved lines, detecting transgenics and tracing dispersal. We propose simple ‘Biobarcodes’ using universal PCR primers to recognize the universal ‘nonsense’ recognition site of all biobarcodes, followed by a variable nonsense sequence. The proposed sequences are long enough to allow recognition in spite of mutations, have stop codons to prevent coding, and will not self anneal. Sequences of PCR-amplified biobarcodes can be compared to a universal database.

These are a variety of needs for devising simpler recognition methods for organisms marketed in commerce or released in the environment; whether they are conventionally selected, mutant or transgenic bacteria, fungi, plants or animals. The needs include: (1) the need for protection for patented or other IP lines, where IP takes on the designation of either ‘Intellectual Property’ or ‘Identity Preserved’. It is often hard to prove that a line has been ‘miss-appropriated’ by a competitor or illegally grown. (2) Labeling regulatory authorities and various consumer groups are demanding labeling of transgenic commodities. This is accomplished by segregating plants or their products and externally following them throughout their commercial life. Internal markers are an adjunct to such external markings. (3) The need to trace organisms in the environment. The use of biocontrol agents to control weed, bacterial, fungal or insect pests and the use of other live organisms as inoculants is increasing. Knowledge about their dispersal in plants and other organisms as well as in the environment is necessary, irrespective of whether the organisms are indigenous or transgenic. Many of the agents are closely related to known pathogens or pests and there are claims that an organism can change its host range and attack valuable species (with consequences of liability). There are also fears that organisms will introgress into other organisms, and there are needs to ascertain whether the new organism changed host range or whether an epidemic was the result of wild strains [1]. These issues will become more acute with transgenically enhanced biocontrol agents [2]. This can still be a problem even if failsafe mechanisms are transformed into biocontrol agents along with hypervirulence genes [3]. Labeling biocontrol agents with selectable markers (such as gus, gfp or antibiotic resistance) has been used for following the movement of biocontrol agents in the environment [4,5] , but will not differentiate between them if the same few markers are continually used. RFLP has been used to follow and differentiate between one organism and closely related strains of the same pathogen [6].

There has been a considerable expenditure on identifying valuable organisms – transgenic and non-transgenic. Seed companies use AFLP and other molecular techniques to ascertain whether competitors have incorporated their genetic material, and the seed industry uses it to ascertain whether farmers have been storing and reusing transgenic seed off license. Consumer advocacy groups and regulators have been using similar techniques to ascertain whether transgenic products have been mixed with non-transgenic organisms. PCR amplification of the DNA in question, using a series of primers for typically used promoters, terminators, marker genes and the genes of importance, is often used to find trace amounts of transgenes in crops and commodities [7–11] . Some processed products might need tens of PCR reactions because of the mixing of crops and the numbers of possible transgenes that they might contain. This problem will be exacerbated as more transgenic crops and organisms reach the market. The multiple sampling and PCR reactions are time consuming and fraught with problems. For example, the commonly used 35S promoter can be found in almost any non-transgenic commodity containing DNA from a cole (Brassica) crop because a proportion of such crops is invariably infected by the ubiquitous cauliflower mosaic virus, the source of that promoter. The PCR results must be further verified by using either restriction endonucleases, Southern blotting or direct sequencing, or using nested PCR or quantitative competitive PCR reactions. Highly processed foods have their DNA fragmented to <400 bp. Detection can be hampered by a lack of availability of DNA reference material and of sequence information [7–11] .

Immunodiagnostic methods have been developed for many of the gene products. Again, each sample would have to be reacted with a series of antibodies when there is a possibility of many different gene products. Even one product can be hard to detect when a single mutation is introduced into a commonly occurring plant gene, such as those encoding many herbicide resistances. It was easier to develop an immunoassay to detect bacterial 5-enolpyruvylshikimate-phosphate synthase encoding resistance to the herbicide glyphosate that could distinguish it from the susceptible plant enzyme [12]. Even then, a triple sandwich technique was required.

Even when transgenics are discovered by such procedures or ‘kits’, there is no information as to source. Thus, regulatory authorities might wish to consider simple, common recognition sequences for detecting transgenic or other organisms in the market place. It is depressing to contemplate how much is being invested in detecting transgenic DNA in commodities (and no one has died from eating it) against how little is being expended towards detecting commonly occurring ‘natural’ bacterial contamination and mycotoxins in foodstuffs (which have negatively affected many lives). We propose a new recognition system, which, if imposed, would require far less expenditure in detecting DNA, allowing resources to be used for detecting truly dangerous contaminants in foodstuffs.

Barcodes and biobarcodes

The barcode system was developed to identify individuals rapidly, whether articles in a store, automobiles or test tubes. A simple barcode reader first identifies that there is a barcode to be read, and in what orientation, by seeing a pattern of bars that is otherwise too rare to be expected randomly. It does so by recognizing a universal sequence of spaced bars in an orientation-dependent manner that appear as universal recognition sites on either end of the barcode ( Fig. 1a). In between the universal recognition sites is a variable series of bars of differing width and spacing. This assigned variable region is read by the reader into a computer and the database then identifies the object as being someone’s car, candy bar or blood sample.

Fig. 1.
(a) A standard barcode with universal recognition sites at either end defining orientation, and a variable sequence of bars of various widths and spacing that can be accessed from a database. (b) A schematic representation of a Biobarcode with fixed universal nonsense DNA sequences defining universal recognition sites at either end defining orientation, and a unique defined variable nonsense DNA sequence that can be accessed from a database containing a stop codon (purple) to prevent frameshifts from defining peptide coding. A special assigned sequence can be added for those groups that are interested in having further definition of typed products. Abbreviations: L, left; R, right.

An analogous DNA-encoded system with universal recognition sites binding a variable region is proposed akin to the barcode system. This biobarcode system would enable material to be assayed in one PCR-sequencing run using universal primers that identify all ‘biobarcoded’ biological materials. In this system, open codes are to be designed and assigned by a single repository. They begin and end with the common universal ‘barcode analogous’ orientation-dependent nucleotide sequences that are recognized by the pair of universal recognition PCR primers, which, under PCR conditions, duplicate the whole intervening sequence ( Fig. 1b). The assigned code is transformed into the target organism, as part of a transgenic construct, or it can be transformed directly into an otherwise non-transgenic organism.

The universal ‘bar code recognition’ nonsense (non-coding) nucleotide sequence is designed to be long enough that a few mutational changes will still allow it to be recognized by a PCR primer set. The initial universal recognition code is followed by a designed nonsense (non-coding) nucleotide sequence that is long enough to allow tens of millions of different such sequences to be generated, and again allow for some mutational changes. Neither the initial common recognition sequence, nor the particular individual strain sequence, should even vaguely resemble nonsense sequences reported in any sequence database. Frame-shift mutations should not render any part of the barcode sequence as an open reading frame long enough to allow significant polypeptides to be made. Stop codons are thus inserted into the assigned sequences in all reading frames to prevent a frame-shift mutation from becoming a genetic coding sequence.

Some people might not care whether there is any biobarcode in an organism, just whether their propriety DNA is found in it. Such people could have a special sequence pair assigned to them that would follow the universal recognition sequence in a biobarcode. Thus, they could use a primer that recognizes this sequence, and start the PCR reaction from there. Others wishing to know whose and what DNA is in the same organism or sample can probe with the universal primer pair.

A considerable amount of computational power with appropriate algorithms is needed to generate the common universal code sequence and the following variable individual strain sequence. The algorithms used to generate the sequences are being designed to exclude sequences that could self anneal, preventing the taq polymerase from amplifying the biobarcoded DNA. The biobarcode can be inserted in tandem with the genes of choice for transformation, or it can be co-transformed with the gene of choice in cases where the organism is transformed with a ‘sense’ transformation. In other cases, an excisable selectable marker will be needed.

If a product contains more than one biobarcode (i.e. a foodstuff concocted from different transgenic crops bearing different transgenes), there will be more than one band on the PCR gel to be sequenced. The sequenced bands are then compared to the public database. This is a positive method, with results from all biobarcode-labeled species, versus the guessing at what transgenes might be present, as occurs using the present technologies.

Regulation

It is envisaged that a single body would assign the biobarcodes and maintain a public database listing the biobarcode sequences of organisms that have been released. It is clearly advantageous to industry, regulators and taxpayers that such a system be instituted because of the amount of resources saved and the protection provided. Because of the savings, it is in the public interest that such a system be universally instituted.

In Canada, for example, the insertion of a biobarcode would probably not come under regulatory scrutiny if biobarcodes are introduced into plants because they are not considered to be ‘plants with novel traits’ if they are non-protein producing. In Europe, any introduced sequence (except antisense) seems to come under regulatory scrutiny, but after due risk assessment it is hoped that a blanket approval could be obtained for biobarcodes that meet the specific criteria listed above (and possibly others).

Acknowledgements

Useful discussions with Eithan Rubin of the Weizmann Bioinformatics Group are gratefully acknowledged. This research is supported in part by the Levin Fund. J.G. is the Gilbert de Botton Professor of Plant Sciences.

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