Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing gas sample
Reexamination Certificate
2000-04-28
2004-11-30
Nguyen, Nam (Department: 1753)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing gas sample
C422S105000, C204S518000, C204S542000, C204S543000, C204S544000, C204S627000, C435S287200, C435S288500
Reexamination Certificate
active
06824740
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to devices and methods for performing active, multi-step molecular and biological sample preparation and diagnostic analyses. More particularly, the invention relates to sample preparation, cell selection, biological sample purification, complexity reduction, biological diagnostics and general sample preparation and handling.
BACKGROUND OF THE INVENTION
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein. Many of these techniques and procedures form the basis of clinical diagnostic assays and tests. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and the separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis,
Molecular Cloning: A Laboratory Manual
, 2 Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited in its application by a lack of sensitivity, specificity or reproducibility. For example, these problems have limited many diagnostic applications of nucleic acid hybridization analysis.
The complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved. Broadly speaking, the complete process may be divided into a number of steps and substeps. In the case of genetic disease diagnosis, the first step involves obtaining the sample (e.g., blood or tissue). Depending on the type of sample, various pre-treatments would be carried out. The second step involves disrupting or lysing the cells, which then releases the crude DNA and RNA (for simplicity, a reference to DNA in the following text also refers to RNA, where appropriate) material along with other cellular constituents. Generally, several sub-steps are necessary to remove cell debris and to purify further the crude lysate. At this point several options exist for further processing and analysis. One option involves denaturing the purified sample DNA and carrying out a direct hybridization analysis in one of many formats (dot blot, microbead, microtiter plate, etc.). A second option, called Southern blot hybridization, involves cleaving DNA with restriction enzymes, separating the DNA fragments on an electrophoretic gel, blotting to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the genomic DNA sample, and thereby helps to improve the hybridization specificity and sensitivity. Unfortunately, this procedure is long and arduous. A third option is to carry out the polymerase chain reaction (PCR) or other amplification procedure. The PCR procedure amplifies (increases) the number of target DNA sequences. Amplification of target DNA helps to overcome problems related to complexity and sensitivity in analysis of genomic DNA or RNA. All these procedures are time consuming, relatively complicated, and add significantly to the cost of a diagnostic test. After these sample preparation and DNA processing steps, the actual hybridization reaction is performed. Finally, detection and data analysis convert the hybridization event into an analytical result.
The steps of sample preparation and processing have typically been performed separate and apart from the other main steps of hybridization and detection and analysis. Indeed, the various substeps comprising sample preparation and DNA processing have often been performed as a discrete operation separate and apart from the other substeps. Considering these substeps in more detail, samples have been obtained through any number of means, such as obtaining of whole blood, tissue, or other biological fluid samples. In the case of blood, the sample is often processed to remove red blood cells and retain the desired nucleated (white) cells. This process is usually carried out by density gradient centrifugation. Cell disruption or lysis is then carried out, preferably by the technique of sonication, freeze/thawing, or by addition of lysing reagents.
In certain cases, the blood is extensively processed to remove contaminants. One such system known to the prior art is the Qiagen system. This system involves prior lysis followed by digestion with proteinase K, after which the sample is loaded onto a column and then eluted with a high salt buffer (e.g., 1.25 M NaCl). The sample is concentrated by precipitation with isopropanol and then centrifuged to form a pellet. The pellet is then washed with ethanol and centrifuged, after which it is placed in a desired buffer. The total purification time is greater than approximately two hours and the manufacturer claims an optical density ratio (260 nm/280 nm) of 1.7 to 1.9 (OD 260-280). The high salt concentration can preclude performance of certain enzymatic reactions on the prepared materials. Further, DNA prepared by the Qiagen method has relatively poor transport on an electrophoretic diagnostic system using free field electrophoresis.
Returning now to the general discussion of sample preparation, crude DNA is often separated from the cellular debris by a centrifugation step. Prior to hybridization, double-stranded DNA is denatured into single-stranded form. Denaturation of the double-stranded DNA has generally been performed by the techniques involving heating (>Tm), changing salt concentration, addition of base (e.g., NaOH), or denaturing reagents (e.g., urea, formamide). Workers have suggested denaturing DNA into its single-stranded form in an electrochemical cell. The theory is stated to be that there is electron transfer to the DNA at the interface of an electrode, which effectively weakens the double-stranded structure and results in separation of the strands. See, e.g., Stanley, “DNA Denaturation by an Electric Potential”, U.K. patent application 2,247,889 published Mar. 18, 1992.
Nucleic acid hybridization analysis generally involves the detection of a very small number of specific target nucleic acids (DNA or RNA) with an excess of probe DNA, among a relatively large amount of complex non-target nucleic acids. DNA complexity is sometimes overcome to some degree by amplification of target nucleic acid sequences using polymerase chain reaction (PCR). (See, M. A. Innis et al,
PCR Protocols: A Guide to Methods and Applications
, Academic Press, 1990). While amplification results in an enormous number of target nucleic acid sequences that improves the subsequent direct probe hybridization step, amplification involves lengthy and cumbersome procedures that typically must be performed on a stand alone basis relative to the other substeps. Complicated and relatively large equipment is required to perform the amplification step.
The actual hybridization reaction represents an important step and occurs near the end of the process. The hybridization step involves exposing the prepared DNA sample to a specific reporter probe, at a set of optimal conditions for hybridization to occur to the target DNA sequence. Hybridization may be performed in any one of a number of formats. For example, multiple sample nucleic acid hybridization analysis can be conducted on a variety of filter and solid support formats (See, G. A. Beltz et al., in
Methods in Enzymology
, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dot blot” hybridization, involves the non-covalent attachment of target DNAs to a filter, which are subsequently hybridized with a radioisotope labelled probe(s). “Dot blot” hybridization has gained wide-spread use, and many versions have been developed (See, M. L. M. Anderson and B. D. Young, in
Nucleic Acid Hybridization—A Practical Approach
, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, p
Heller Michael J.
Jackson Thomas R.
Scott Bradley S.
Sheldon, III Edward L.
Swanson Paul D.
Nanogen Inc.
Nguyen Nam
O'Melveny & Myers LLP
Starsiak Jr. John S.
LandOfFree
Apparatus for active biological sample preparation does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Apparatus for active biological sample preparation, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Apparatus for active biological sample preparation will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3277641