Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-02
2001-09-25
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06294329
ABSTRACT:
The invention relates to the use of primers or primer pairs for DNA fingerprint analysis, wherein the use of the primers or primer pairs allows to obtain fingerprints from humans as well as from animals as well as from plants as well as from microorganisms. The invention further relates to primers or primer pairs for the above-mentioned use.
It is generally known that the presence of polymorphic and heterogenously dispersed repetitive sequences such as microsatellites is used for genetic analysis.
It is also well-known that retrotransposons such as copia elements of Drosophila and copia-like elements in other species of the animal and plant kingdom usually are present as multiple copies in the genomes. Repetitive genomic sequences of this type were used in the example of copia-like elements in pisum (pea) for the genetic analysis of this plant species (Lee et al., Plant Mol. Biol. 15 (1990), 707-722). This method designated OFLP is based on a copia-specific primer and a second primer of a sequence of the retrotransposon flanking pea genome for PCR amplification. This made it possible to amplify pea varieties by PCR amplification of specific elements of the pea copia family and to test for polymorphisms by separation of the non-radioactively labeled PCR products on an agarose gel and determine genetic relatedness. Also other retrotransposons, e.g. Tos1-1, Tos2-1 and Tos3-1 from rice have been used as molecular genetic markers for differentiation and identification of rice cultivars by RFLP-analysis (Fukuchi et al., Jap. J. Genetics 68 (1993), 195-204), while, however, also here it has been postulated that for other plant species their endogenous retrotransposons are isolated for use as a molecular marker. Another work (Purugganan and Wessler, Mol. Ecology 4 (1995), 265-269) uses a PCR-based method, which utilizes the variation of restriction sites for restriction enzymes in transposable elements for a fingerprint analysis. All these methods described in the prior art have, however, in common that the described genetic markers or primers cannot be universally used in humans, plants, animals or microorganisms. It is obvious that the provision of such genetic markers or primers would offer essential advantages in many areas of modern biology or medicine.
Thus, the problem underlying the present invention was to overcome the above-mentioned drawbacks of the prior art and to provide methods and means which allow for a maximum degree of universal applicability of a minimum amount of primers or genetic markers for a fingerprint analysis of species from the plant and animal kingdom as well as of humans.
The solution to this problem is provided by the embodiments characterized in the claims.
It has surprisingly been found that the primers which hybridize with copia-like elements in coconut (
Cocos nucifera L
.) and which in this system permit a fingerprint analysis can also be successfully used in many other species of the animal and plant kingdom as well as in humans and even in microorganisms including yeast. This finding permits to universally use said primers for fingerprint analysis in the whole animal and plant kingdom as well as in humans and in microorganisms.
Thus, the present invention relates to the use of a primer or primer pair for DNA fingerprint analysis, characterized in that the primer or primer pair permits a fingerprint from humans as well as from animals and plants as well as from microorganisms, wherein the primer or primer pair hybridizes with a DNA which is comprised in the copia-like element of coconut (
Cocos nucifera L
.) as represented in FIG.
2
B.
In this connection, the surprising results of the present invention are achieved with arbitrary combinations of different primers of opposite polarity with the only requirement that they hybridize with the copia-like element represented in
FIG. 2B
, as well as with the use of a single primer which, due to the repetition of the copia-like elements, albeit in 5′→3′/3′→5′ orientation of two adjacent elements and not, as represented in
FIG. 2B
, in 5′→3′/5′→3′ orientation, likewise provides the highly polymorphic fingerprints. It is self-evident that the aforementioned definition for the primers includes that they also hybridize to DNAs from other organisms as long as they contain DNA sequences which correspond to DNA sequences from the above-mentioned copia-like element.
The requirements for hybridization of the primer and subsequent amplification is derivable to the person skilled in the art without inventive effort from the prior art and the following examples.
The primers of the invention are preferably 15 to 25 nucleotides in length. The invention, however, can also be carried out with primers which are shorter or longer.
The present finding is even more surprising since as a rule the prior art started from the assumption that primers can only be used for reliable fingerprints in taxonomically narrow limits.
In the prior art Rohde et al. (J. Genet. & Breed. 46 (1992), 391-394) it is described that highly repetitive sequences having homology to copia elements described in other species exist in the genome of coconut (
Cocos nucifera L
.), which sequences are visible as two DNA bands, 1.3 and 1.4 kilobases in length, respectively, after restriction of isolated genomic DNA with the restriction enzyme EcoRI and separation on an agarose gel. Three of these “Ecorep”-designated DNA fragments were sequenced after subcloning and sequence deviations could be determined. Attempts to use these differences for the genetic analysis of different coconut types by use of Ecorep sequences as a molecular probe in RFLP analysis or by sequence specific PCR primers were not successful (Rohde et al., J. Genet. & Breed. 46 (1992), 391-394; Rohde in: “La Recherche Europeene au Service du Cocotier—Actes du Seminaire—8-10 septembre 1993, Montpellier”. CIRAD (Collection: Colloques du CIRAD), Montpellier, pages 41-52).
Recently it was found for three coconut types that subfamilies of these 1.3 and 1.4 kilobase Ecorep sequences exist, in which these elements are clustered in the coconut genome, i.e. they represent tandem repeats, and in which usually at least one of the two expected EcoRI restriction sites at the ends of the sequence previously defined as “spacer region” is absent (Rohde et al., J. Genet. & Breed. 49 (1995), 179-186) from the previously identified elements (Rohde et al., J. Genet. & Breed. 46 (1992), 391-394). This spacer region shows high homology to the copia-like BARE-1-element from barley (
FIG. 1A
; Manninen and Schulman, Plant. Mol. Biol. 22 (1993), 829-846). Thus, the subfamily of copia-like sequences in the coconut genome represent tandem repeats, which display homology to the endonuclease and reverse transcriptase RNAse H region of a copia or copia-like element (see FIG.
1
B). The observed sequence deviations in the elements of the subfamily could now—in contrast to the above-described attempts for the Ecorep sequences—be used for a genetic analysis in coconut by appropriate PCR primers. This method for a genome analysis in coconut was designated as ISTR (inverse sequence tagged repeat) analysis.
It was now surprisingly found that this subfamily with its highly conserved sequence appears to be ubiquitous in the plant and animal kingdom since the use of identical ISTR-primers (see also Table 1), which were developed on the basis of coconut sequences determined by us, obtained high polymorphic DNA fingerprints for other plant species as well as for animals and humans as well as for microorganisms. In this context, not only a multitude of polymorphic markers can be discovered which segregate in the progeny (“single locus/multiple allele”-markers) but also new polymorphic markers arise (individual specific markers), which, for example, are present neither in the father nor in the mother in control cross-breedings (for example cattle, sheep) and which can be possibly ascribed to recombination events or to the amplification of specific genomic regions.
Birch, Stewart, Koi & Birch, LLP
Johannsen Diana
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
Myers Carla J.
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