Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-12-12
2004-10-12
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S239000, C536S023100, C210S634000, C210S656000, C210S658000, C210S198200
Reexamination Certificate
active
06803200
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions and methods for releasing nucleic acid molecules from solid matrices. The invention further relates to compositions and methods for purifying and isolating nucleic acid molecules from biological materials such as animal tissues and plant matter.
BACKGROUND OF THE INVENTION
It is desirable in many instances to generate and analyze nucleic acid molecules samples obtained from numerous individual entities and/or organisms of populations. Often, such samples are used to identify the genotypes of individuals which reside either in the same or different geographic locations. Thus, the collection and analysis of samples are often employed to determine the genotypes of individual members of populations. Such analyses generally result in the generation of data relating to both the individuals from which the samples are obtained and the populations as a whole.
The collection and analysis of samples which contain nucleic acid molecules from populations of organisms are often performed to obtain genotype data from viral, plant and animal populations. One example of a situation where genotype analysis of large numbers of individuals of members vof populations is commonly performed is where data regarding the genotypes of plants in a geographic location is sought. These data may be generated to determine the spread rate of particular plant strains or to identify genetically modified plants which either have been grown from seeds sold to farmers or are the progeny of plants grown from such seeds.
A number of companies currently sell genetically modified plants and seeds derived from these plants. In some cases, these seeds are sold under the condition that the purchasers, generally farmers, repurchase seeds from their suppliers instead of growing plants from seeds which are obtained from plants grown from purchased seeds. Further, a number of consumer groups, as well as governmental organizations, have objected to the sale of agricultural products prepared from genetically modified plants.
In each instance described immediately above, genotype analyses can be performed to identify genetically modified plants. Such analyses often begin with the collection of large numbers of plant samples obtained in rural settings. Thus, there is a need for methods which allow for the collection and convenient storage of large numbers of samples containing nucleic acid molecules derived from plants which can then be used for genotype analyses.
In other situations, genotype analyses are performed on samples derived from animals (e.g., humans) to generate data related, again, to either individuals or populations of which these individuals are members. Further, genotype analyses performed on samples derived from either animals or plants may be used to obtain data relating to entities associated with these organisms. Examples of such associated entities include viruses such as Human Immunodeficiency Viruses (HIVs). In particular, genotype analyses of HIV populations can be performed using nucleic acid molecules obtained from human blood samples. Due to the rapid rate with which HIVs alter their genomes, genotype analyses have been employed to track the spread and regional predominance of various viral strains.
The use of filter paper (e.g., Whatman 3MM filter paper) provides an inexpensive method for the collection, shipment, and storage of samples which contain nucleic acid molecules (e.g., RNA, plasmids, viral vectors and chromosomal DNA). This is especially the case when samples are collected in remote areas where there is no access to refrigeration.
One example, of a filter paper based medium used for the collection, shipment, and storage of blood samples is FTA® paper, which is composed of cellulose material impregnated with (i) a monovalent weak base; (ii) a chelating agent; (iii) an anionic detergent; and, optionally, (iv) uric acid or a urate salt. FTA® paper can be used to store human genomic DNA, for example, in the form of dried spots of whole blood, the cells of which lyse after making contact with the paper. Stored at room temperature, genomic DNA on FTA® paper is reported to be stable for at least 7.5 years. (Burgoyne et al., CONVENTIONAL DNA COLLECTION AND PROCESSING: DISPOSABLE TOOTHBRUSHES AND FTA® PAPER AS A NON-THREATING BUCCAL-CELL COLLECTION KIT COMPATIBLE WITH AUTOMATABLE DNA PROCESSING, 8
th
International Symposium on Human Identification, Sep. 17-20, 1997.) Thus, the placement of nucleic acid samples on filter paper (e.g., FTA® paper) offers a compact archival system compared to glass vials or plastic tubes located in precious freezer space.
DNA from blood spots has been used in newborn screening programs to identify genetic mutations implicated in several diseases and to provide a means for identifying military personnel. (See, e.g., Seltzer et al.,
Biochem. Med. Metab. Biol.
46:105-109 (1991); Jinks et al.,
Hum. Genet.
81:363-366 (1989); Skogerboe et al.,
Clin. Chem.
37:454-458 (1991); McEwen et al.,
Am. J. Hum. Genet.
55:196-200 (1994).)
The storage of blood samples on dried filter paper has the additional advantage of pathogen inactivation. More specifically, HIV, as well as a number of other infectious agents, are believed to lose viability upon drying. Further, nucleic acid molecules obtained from these dried blood spots, as well as other dried samples containing nucleic acid molecules, can also be used to isolate and reverse transcribe messenger-RNA (mRNA).
The spotting of bacterial nucleic acids on filter paper can also be used as part of a sample storage and retrieval system. Recently, Rogers and Burgoyne characterized samples of several bacterial strains of Staphylococcus and
Escherichia coli
stored on FTA® paper by PCR-ribotyping. (Rogers et al.,
Anal. Biochem.
247:223 (1997).)
Before analysis of nucleic acids captured by filter papers, washing steps generally need to be performed to remove stabilizing chemicals, if present, and cellular inhibitors of enzymatic reactions. Since DNA, for the most part, remains with the paper through these washing steps, manipulations to purify such nucleic acids are simplified and amenable to automation.
Several methods have been developed for releasing nucleic acids from materials such as FTA® paper. For example, Burgoyne demonstrated that purified plasmid DNA, stored on paper encased in polystyrene, can be recovered using a uric acid solution. (Burgoyne, U.S. Pat. No. 5,496,562, the entire disclosure of which is incorporated herein by reference.) Another method for nucleic acid release employs a buffer containing a chelating agent in an aqueous solution. (See, e.g., PCT Publications WO 99/39010, WO 99/38962, and WO 99/39009, each of which is incorporated herein by reference.)
The invention provides methods for releasing DNA from solid matrices which are relatively simple in comparison to methods currently in use in the art. Further, the DNA released by methods of the invention can be used directly in a number of processes (e.g., genotyping analyses).
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for the removal of nucleic acid molecules (e.g., DNA) from solid matrices. In particular, the methods of the invention employ releasing reagents to facilitate the release of nucleic acid molecules. The invention further provides compositions relating to these methods.
The present invention also relates to methods for purifying and/or isolating nucleic acid molecules.
In one general aspect, the invention provides methods for removing nucleic acid molecules from solid matrices comprising contacting the solid matrices with releasing reagents which comprise one or more alkanol amines.
In another general aspect, the invention provides methods of purifying and/or isolating nucleic acid molecules comprising:
(a) contacting the nucleic acid molecules with solid matrices under conditions which favor adherence, attachment, association, and/or binding (covalently or non-covalently) of the nucleic acid molecules to the solid matrices: and
(b) con
Connolly Michael A.
Gebeyehu Gulilat
Goldsborough Mindy D.
Xia Jiu-Lin
Benzion Gary
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
Strzelecka Teresa
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