Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-12-28
1998-06-02
Jones, W. Gary
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
536 243, C12P 1934, C07H 2104
Patent
active
057598215
DESCRIPTION:
BRIEF SUMMARY
This invention relates to improvements to methods of random amplification of polymorphic DNA.
This invention has particular but not exclusive application to methods of random amplification of polymorphic DNA for production of molecular markers for genetic studies such as production of genetic maps and for development of correlations between map markers and valuable traits in genetic improvement of plants and animals, and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in other applications apparent to those skilled in the art.
The method of Random Amplification of Polymorphic DNA (RAPD), an application of the Polymerase Chain Reaction (PCR) method (Mullis et al. 1987), to produce molecular markers for genetic studies is known. These procedures dramatically extend and improve the productivity and reliability of the RAPD method, providing much more information than that obtainable with preceding methods. The RAPD method involves the use of an oligonucleotide of arbitrary short sequence, generally 10 bases long, as a primer in the Polymerase Chain Reaction. This oligonucleotide primer will bind to numerous complementary sites which occur randomly in the DNA of the genome under study. Where two such sites are suitably juxtapositioned on opposite chains, the intervening sequences will be copied in each direction, concomitantly also producing new binding sites, so that in each cycle of the PCR process doubling of these sequences occurs.
In principle, the quantity of DNA increases exponentially from as little as a single molecule up to several .mu.g over repeated cycles in the PCR process. These products from various sites will generally be of different length and are usually separated by electrophoresis through an agarose gel, then detected as discreet bands by fluorescence emission following ethidium bromide staining and excitation with ultra violet light. Alternative separation and detection methods may also be used, such as polyacrylamide gel electrophoresis and silver staining. Typically 2 to 10 bands (products) can be detected which are from independent sites randomly dispersed across the genome.
The total product yield in a PCR reaction is limited, presumably by primer limitation and/or product (pyrophosphate) inhibition, so that high efficiency in production from one (or a few) more effective sites will lead to reaction termination before some potentially valuable products reach detectable levels, so that these potential products are competitively displaced from detection. Use of a number of oligonucleotides of specific sequence will each yield a characteristic set of bands from a particular set of sites randomly dispersed over the genome. Genetic changes at particular sites will lead to loss of some DNA products, changes in size, or generation of new products, which can be scored as genetic polymorphisms and can be used as genetic markers (molecular markers). The number of different oligonucleotide primers 10 bases in length is 4.sup.10 =1048576, of which ca 30% are of generally preferred GC content (50 to 80%) so that the method provides approximately 300,000 possible primers, which is an effectively unlimited number for testing.
Relevant disclosures indicating the state of the art are as follows: Tingey (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18(22): 6531-6535. Tingey & P. A. Scolnik (1992) Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs. Proceedings National Academy of Science U.S.A. 89: 1477-1481. identification of markers linked to a Pseudomonas resistance gene in tomato using random markers and near-isogenic lines. Proceedings National Academy of Science U.S.A. 88: 2236-2340. detecting polymorphisms on the basis of nucleotide differences. U.S. Pat. No. 5,126,239. 30 Jun. 1992. Assignee: E. I. Du Pont de Nemours and Company. Wilmington, Del., U.S.A. Process for ampl
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Atzel Amy
F B Investments Pty Ltd
Jones W. Gary
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