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AssigneeKeygene N.V.
InventorsMichael Josephus Theresia Van Van Eijk, Johan Dominicus Peleman, Maria Johanna De Ruiter-Bleeker
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The present invention discloses methods for identifying and analysing microsatellite-associated polymorphisms between different DNA samples. Different DNA samples, e.g. from different individuals, are analysed using a PCR based on the combination of a RAMP-primer and an AFLP-primer and polymorphisms between the different DNA samples are identified. The polymorphisms thus identified may be isolated and further analysed by e.g. DNA sequence analysis both upstream and downstream from the microsatellite-associated polymorphism. These sequences may subsequently be used to devise and synthesise new means for analysis of the polymorphic locus, such as e.g. PCR-primer pairs or oligonucleotide probes.
Use of a RAMP-primer and an AFLP-primer in analysing a nucleic acid sequence, in particular in analysing a nucleic acid sequence for the presence polymorphisms associated with microsatellites.
Use of a RAMP-primer and an AFLP-primer in amplifying a nucleic acid sequence.
Use according to any of the preceding claims, in which the restriction fragment is derived from genomic DNA, cDNA;
Use according to any of the preceding claims, in which the nucleic acid/restriction fragment is derived from agronomically important crops such as wheat, barley, maize, tomato, pepper, lettuce, rice or soybean;
Method for analysing a nucleic acid sequence, the method at least comprising the steps of:
Method for analysing a nucleic acid sequence, the method comprising the steps of:
Method for providing a PCR primer or a pair of PCR primers for use in the amplification of a PCR fragment spanning a microsatellite-associated genomic polymorphism, the method comprising the steps of:
Method for providing a PCR-primer, comprising the steps of:
Use of (the combination of) a RAMP primer and an AFLP® primer in the development of PCR-primers.
Dependencies are inferred from the claim text.
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Nucleic acid amplification reactions: allele-specific amplification
For detection of mutation or polymorphism: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
Term end not estimated
The present invention relates to a method for identifying and analysing nucleic acid sequences that contain or are associated with highly repetitive nucleotide sequences known as microsatellites. In particular, the invention provides a method for identifying and analysing nucleic acid sequences based upon polymorphisms associated with such microsatellites.
Various methods for analysing nucleic acid sequences are known in the art. Often, such techniques involve amplification—such as by PCR—of one or more parts of the nucleic acid(s) of a mixture of restriction fragments generated from the nucleic acid(s). The amplified mixture thus obtained is then analysed, e.g. by detection of one or more of the amplified fragments. For example, the amplified fragments may be separated on the basis of differences in length or molecular weight, such as by gel-electrophoresis, after which the amplified fragments are visualised, e.g. by autoradiography of the labelled amplified fragments or blotting followed by hybridisation. The resulting pattern of bands is referred to as a “(DNA) fingerprint”.
Usually in DNA fingerprinting, fingerprints of closely related species, subspecies, varieties, cultivars, races or individuals are compared. Such related fingerprints can be identical or very similar, i.e. contain a large number of corresponding—and therefore less informative—bands. Differences between two related DNA-fingerprints are referred to as “DNA-polymorphisms”. These are amplified fragments—i.e. bands—, which are unique in or for a fingerprint and/or for a subset of fingerprints. The presence or absence of such polymorphic fragments in a fingerprint—or the pattern thereof—can be used as a genetic marker, for instance to identify a specific species, subspecies, variety, cultivar, race or individual; to establish the presence or absence of a specific inheritable trait and/or of a specific gene; and/or to determine the state of a disease. For a further discussion of DNA-fingerprinting, DNA-polymorphisms, genotyping, PCR and similar amplification techniques, as well as the techniques and materials used therein, reference is inter alia made to the prior art mentioned hereinbelow, as well as to the standard handbooks.
FIGS. 1 and 2 are schematic representations of the method of the invention;
FIG. 3 shows a section in the 200-280 basepairs fragment mobility size range of the fingerprint patterns obtained from lettuce lines Rachel (lane 1), Vicky (lane 2), Lianne (lane 3), Quincy (lane 4), Thirza (lane 5), Veraly (lane 6), Clarion (lane 7) and Ballora (lane 8) using the method of the invention, as further described in Example 1;
FIG. 4 shows a section in the 270-400 basepairs fragment mobility size range of the fingerprint patterns obtained from the parental lines and six F2 progeny of a pickle mapping population using the method of the invention, as further described in Example 2;
1. Description of Biological Materials
The biological materials used are lettuce lines Rachel, Vicky, Lianne, Quincy, Thirza, Veraly, Clarion and Ballora.
2. TaqI/MseI AFLP Template Preparation, Pre-Amplification and Amplification Using a Selective TaqI AFLP Primer in Combination with a RAMP Primer