Column device for isolation and labeling of nucleic acids

Details Column device for isolation and labeling of nucleic acids Column device for isolation and labeling of nucleic acids

2000-12-29

2004-11-16

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100

Reexamination Certificate

active

06818398

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a device for manipulating genetic material, and more specifically, this invention relates to a column device for isolation, fractionating, fragmentation, labeling and purification of total nucleic acid material, DNA and RNA in a minimal number of steps.
2. Background of the Invention
Traditional methods of bacterial identification are usually based on morphological and/or physiological features of a microorganism or on analysis of 16S rRNA gene sequences. These methods require considerable amounts of time.
PCR and other amplification techniques are utilized for bacteria identification. Immunological methods and mass-spectrometry also have been adapted for this purpose, but are expensive and cumbersome.
DNA microchip technology is a rapid, high throughput platform for nucleic acid hybridization reactions. However, nucleic acid fragmentation and labeling are two of the limiting steps in the development of rapid protocols for DNA/RNA microchip technology.
Several enzymatic and chemical protocols are available for fluorescent labeling of nucleic acids. All of these methods are expensive and time consuming. Lastly, most of these protocols demand careful prerequisite nucleic acid isolation, fractionation (generally requiring one or more hours), labeling, separate sample fragmentation procedures and a final purification step.
Typical nucleic acid labeling methods adopt a myriad of approaches. For example, M. D. Schena et al.,
Science
270, 467-470 (1995); J. L. DeRisi et al.,
Science
278, 680-686 (1997); G. P. Yang et al.,
Nucl. Acid Res
. 27, 1517-1523 (1999); K. Wang et al.,
Gene
229, 101-108 (1999), and M. Wilson et al.
Proc. Natl. Acad. Sci USA
96, 12833-12838 all rely on effecting labeling using reverse transcriptase. Typically, this process requires from one to two hours to complete.
D. Guiliano et al.
Bio Techniques
27 146-152 (1999) and G. T. Hermanson,
Bioconjugate Techniques
(Academic Press, Inc. San Diego, Calif., 1996) utilize random priming. However, these protocols require from 3 to 10 hours to complete.
Terminal transferase protocols are featured in K. L. Gunderson et al.
Genome Res
. 8, 1142-1153 (1998) and L. Wodicka et al.
Nat. Biotechnol
. 15, 1359-1367. However, these processes also require between 1 and 2 hours to run.
Polymerase Chain Reaction (PCR) protocols for labeling are widespread. Typical references for PCR processes include R. J. Sapolsky et al.
Genomics
33, 445-456 (1996); M. T.
Cronin et al. Hum. Mutat
. 7, 244-255 (1996); S. Tyagi et al.
Nat. Biotechnol
16, 49-53 (1998); and P. N. Gilles et al.
Nat. Biotechnol
17, 365-370 (1999). However, PCR protocols require between 1 and 2 hours to complete.
A need exists in the art for a high throughput fractionation and labeling protocol for nucleic acid materials. The protocol should require mild conditions of reaction, and decrease the number of solutions and reaction steps compared to typical protocols. The method should yield high amounts of cross-linked complexes in short incubation times. And the method should be applicable to both DNA and RNA sequences.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for labeling nucleotide molecules that overcomes many of the disadvantages of the prior art.
Another object of the present invention is an economical method for labeling DNA and RNA molecules. A feature of the invention is that a column, which very easily may be automated, is utilized to immobilize and subject genetic material to reaction sequences. Another feature is that fluid transport through the column can be facilitated using low speed centrifugation in the laboratory or via syringe-imparted pressure in the field. An advantage of the method is that the process is portable and can be easily juxtaposed to any arrays for subsequent analysis of hybridizations.
Yet another object of the present invention is to provide a method for modifying nucleic acid. A feature of the invention is that the modification occurs on a column in the presence of hydrogen peroxide and ultimately lead to the formation of an aldehyde group for subsequent labeling. An advantage of the present method is that the reaction is simple, requires mild conditions, and produces high yields of cross-linked complexes which are utilized in hybridization experiments.
As a result, a universal method of preparation of labeled nucleic acids samples from studied bacterial or eucaryotic cells is provided for use with any type of hybridization experiments, including microarray assays. The method allows all chemical manipulations to be provided in a minimal number of steps (typically three steps) on a single column, in a span of approximately 30 minutes.
Briefly, the invention provides a method for manipulating genetic material, the method comprising: disrupting cells so as to liberate genetic material contained in the cells; fractionating, if necessary, the genetic material so as to separate DNA from RNA, contacting the genetic material to a silica column in a manner to cause the genetic material to become immobilized to the column; separating cell detritus from the immobilized genetic material; fragmenting and labeling the immobilized genetic material; separating the labeled genetic material from excess label, and eluting the labeled material from the column.
Also provided is a two-buffer process for manipulating genetic material, the process comprising contacting cells containing the genetic material to a silica column; creating a first fraction of cell detritus and a second fraction containing the genetic material; confining the genetic material to the column; removing the cell detritus; subjecting the genetic material to radicals so as to produce reactive aldehyde groups on the genetic material; and attaching chromophore to the genetic material.


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Chakrabarti et al. Fluorescent labelling of closely-spaced aldehydes induced in DNA by bleomycin-Fe(III). Int. J. Radiat. Biol., vol. 75, No. 8, pp. 1055-1065, 1999.*
Proudnikov et al. Chemical methods of DNA and RNA fluorescent labelling. Nucleic Acids Res., vol. 24(22): 4535-4542, 1996.*
Proudnikov D et al. Chemical methods of DNA and RNA fluorescent labelling. Nucleic Acids Res., vol. 24(22): 4535-4542, 1996.*
Proudnikov et al. Chemical methods of DNA and RNA fluorescent labeling. Nucleic Acids Research, vol. 24, No. 22, pp. 4535-4532, 1996.*
Eldadah et al. A new approach for the electrophoretic detection of apoptosis. Nucleic Acids Research, 24 (20): 4092-4093, 1996.

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