Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-05-16
2003-03-18
Horlick, Kenneth R. (Department: 1631)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S453000, C204S456000, C204S604000, C356S344000, C435S006120, C435S091200
Reexamination Certificate
active
06533912
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to method and apparatus for increasing the throughput of analysis of compounds of a discrete size range.
BACKGROUND OF THE INVENTION
The references mentioned herein are expressly incorporated by reference.
Separation of compounds is key to sample analysis in biology, chemistry, and physics. In numerous linear type (two-dimensional) separation systems, compounds are introduced into one end of a separation length and a flow force is then applied separating the compounds. Once the compounds are separated, the compounds can then be detected, analyzed and possibly isolated. Such separation systems include chromatography, electrophoresis, and centrifugal separation systems.
The very large number of compounds that need to be separated and analyzed has led to an increasing emphasis on high throughput systems. High throughput systems utilize automation, miniaturization, and parallel reactions or separations to increase the speed at which samples may be analyzed. This allows for an assay to maximize the information gained while minimizing the resources required to effect analysis. This achieves the benefit of lowering the amount of material required for each reaction, maximizing the value of costly analytical equipment, and efficiently using experimentalist time.
A number of fields require increased throughput systems. Environmental sampling, combinatorial chemistry, genomic assay and other fields require very large numbers of samples to be analyzed. High throughput analysis is also increasingly important to the analysis of nucleic acid sequences.
The development of the ability to generate very large numbers of discrete length oligonucleotide in samples has increased the demand for high throughput of oligonucleotide analytical systems. The polymerase chain reaction (PCR) (described in U.S. Pat. No. 4,683,202 to Mullis, K. et al.) discloses a method for amplification of nucleic acid sequences. In this method, high copy numbers of single strands of template nucleic acid sequences are generated. A short nucleic acid sequence primer is annealed to a specific sequence of the single stranded template nucleic acid. An initiator (DNA polymerase) adds nucleotide bases from solution to the 3′-end of the primer. By using matched sets of two primers, oligonucleotide products of specific known length may be amplified. In addition to PCR, use of restriction enzymes to cut nucleic acid strands into fragments of discrete lengths is used to detect specific sequence differences in DNA.
An additional need for high throughput nucleic acid analytical systems results from the very large size and variability of the genomes of various organisms. For example, human genome contains over three billion nucleotide base pairs. These three billion bases represent many thousands of genes. When analyzed on the base pair level, millions of single base variations exist in the human population. High throughput is required for systems used to analyze genomic variation of an organism or to track segregation within a genome.
Various methods can be used to detect genetic variability. These include restriction fragment length polymorphism (RFLP), analysis of short tandem repeat (STR) loci, and analysis of single nucleotide polymorphism.
RFLP is an assay for variation in DNA sequences at a site at which a particular restriction enzyme cuts. DNA variants will give different sizes of DNA fragments after digestion with a specific restriction enzyme. The use of probes to detect known sequences allows determination of restriction site variability proximate to a specific locus. RFLP can be combined with PCR to produce oligonucleotides of a discrete size range. In “The use of capillary electrophoresis for point mutation screening” in Trends Biotechnol., November 1997; 15(11):448-51 by Mitchelson, K.; Cheng, J.; Kricka, L., the authors described the widely used PCR-RFLP technique wherein the PCR reaction is run to produce amplified DNA fragments from a target template DNA. The amplified product is cleaved by a restriction endonuclease at specific cleavage sites to which the endonuclease binds and the size of resulting fragments determined. Presence or absence of a cleavage site is indicative of detection of the mutation of interest.
Short tandem repeat loci (variation in the number of short, tandem repeat units at a locus causing DNA length variability between alleles at that locus) may be used to assay DNA variability. The highly variable copy number of the short repeats serves as a genetic marker. Distinct polymorphic short tandem repeat (STR) loci are amplified and analyzed. U.S. Pat. No. 5,843,660 to Schumm et al. describes a procedure for using PCR to amplify STR loci. The amplification procedure produces a set of nucleic acid fragments within a discrete size range. The nucleic acid sequences of the discrete size range may then be separated and analyzed. In addition to RFLP and STR, single nucleotide polymorphisms may be used as markers of the genetic or phenotypic variability and as genetic markers. The use of single nucleotide polymorphism may present various advantages over either RFLP or STR analysis.
Numerous advantages are presented by single nucleotide polymorphism (SNP) analysis. Generally SNP analysis protocols produce small nucleic acid sequences of a discrete size range. SNP analysis may be rapid and adaptable to automation. This results in a reduction of the amount of reagents and other materials that is needed to effect this analysis. SNP genotyping is highly adaptable to high throughput systems. In the human genome, millions of individual single nucleotide polymorphisms exist. In contrast, much lower frequency of STR or RFLP variants occur.
The advantages of SNP analysis have made this analysis a preferred genomic marker assay. SNP analysis is useful for genotyping or enabling mutation scoring, genetic mapping, pharmacogenetic typing, phylogeny typing, and is adaptable to forensic and identity analysis. Single nucleotide polymorphisms may correlate to phenotypic expression and some SNP variants could serve as markers for disease states enabling simplified diagnostics. A relatively large number of SNP variants exist in the human genome, numbering in the millions. At specific loci, the variation may be between two nucleotides: a common-type variant nucleotide (common variant allele) and a rare-type variant nucleotide (rare variant allele). This binary variation (diallelic) enables allele analysis in which a variant may be categorized as either primary type or variant type. Thus unlike STR variation, SNP variation more rapidly may yield binary allele calls. RFLP variants are binary but cumbersome and expensive.
A number of techniques have been described to use single nucleotide polymorphism in genetic analysis. One such procedure is single nucleotide primer extension (SNuPE). An example is seen in U.S. Pat. No. 5,888,819 to Goelet et al. which describes the use of a PCR-like analysis system to gain information on genetic variation. In this procedure a single stranded template nucleic acid sequence is obtained and a primer is hybridized onto a conforming template sequence to form a template-primer duplex. A DNA polymerase extends the nucleic acid-primer duplex by one nucleotide. This can be enabled by using a terminator, such as a radio-labeled or fluorescence-labeled dideoxynucleotide, to ensure that only a single base is attached to the 3′-end of the primer. This protocol extends the primer by one base. Determination of which type of nucleotide was incorporated onto the 3′-end of the primer is enabled by the presence of the type of the detectable label.
U.S. Pat. No. 5,846,710 to Bajaj describes similar technology for single nucleotide polymorphism analysis. A single stranded sample oligonucleotide is mixed with a labeled nucleotide, a primer having a sequence complementary to a known sequence on the template oligonucleotide and an agent to induce DNA extension (such as a DNA polymerase). Other than the labeled nucleotide, no other nucleotides are include
Bashkin John S.
Kautzer Curtis R.
Mansfield Elaine S.
Peponnet Christine
Molecular Dynamics, Inc.
Schneck David M.
Schneck Thomas
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