Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-19
2003-11-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C210S650000
Reexamination Certificate
active
06645722
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of ultrafiltration. More specifically, the invention relates to a method for purifying sequencing reactions.
BACKGROUND OF THE INVENTION
Ultrafiltration with small devices is becoming a standard procedure used in DNA and protein research which is steadily requiring smaller and smaller quantities of materials. Methods of ultrafiltration have, in the past, relied on centrifugal forces to filter a liquid component through an ultrafiltration membrane. However, as the quantities of materials needed have become increasingly smaller, centrifugal methods no longer suit current needs, especially because these centrifugal methods are not conducive to automation.
Specifically, for example, DNA sequencing reactions must be purified prior to analysis by automated fluorescent sequencing (AFS) conducted on sequencing instruments such as the models ABI 377 and ABI 3700 from PE Biosystems, MegaBACE 1000 from Amersham Pharmacia Biotech and CEQ 2000 from Beckman. Introduction of the latter three high throughput capillary electrophoresis instruments has put new demands on sample purity and handling requirements.
Purification is required because contaminants that interfere with resolution of the sequencing products during electrophoretic separation will prevent determination of all or some of the DNA sequence. The identity of these interfering contaminants is determined, in part, by the sequencing chemistry used for labeling the sequencing products for fluorescent detection (e.g., dye primer or dye terminator chemistry), and the type of DNA sequencing instrument used for electrophoretic resolution of the labeled sequencing products (e.g., slab polyacrylamide gel or capillary electrophoresis).
Dye terminators are fluorescently labeled dideoxynucleotide triphosphates (ddNTP's) which, once incorporated in a base-specific manner, prevent further polymerization of the sequencing product. Primers, template, DNA polymerase, dNTP's, buffer, salts and dye terminators are added to a sequencing reaction at concentrations sufficient to allow production of sequencing products ranging from approximately 10 to 1200 nucleotides in length. Because dye terminators are not natural substrates of DNA polymerase, high concentrations must be provided relative to the natural dNTP substrates to ensure their incorporation into the polymerizing sequencing products. The consequence of this inefficient incorporation is that a large amount of unincorporated dye terminator is still present after the reaction is completed. Unincorporated dye terminators co-migrate with short sequencing products during electrophoresis, and produce a variety of artifacts that interfere with sequence analysis.
The most common methods for removing unincorporated dye terminators from sequencing reactions prior to electrophoresis are alcohol precipitation, typically using ethanol, and gel filtration. However, salts compete with sequencing products for electrokinetic injection onto capillary sequencing instruments and must also be removed. Ethanol precipitation has poor salt removal capabilities which detracts from its utility as a method for preparing samples prior to capillary electrophoresis because the efficiency of electrokinetic injection of sequencing products is inversely proportional to the salt concentration. As such, for purposes of removing these salts, alcohol precipitation is a poor and variable method for preparing samples. Although gel filtration is better suited for removing salt than alcohol precipitation, both gel filtration and alcohol precipitation are centrifuge-based methods which, as noted, are difficult to automate. Centrifugal methods are often sufficient for low throughput DNA sequencing carried out on older slab gel sequencing instruments, but the Genomics industry is scaling up DNA sequencing to a point where centrifugal sample preparation methods are no longer practical or sufficiently robust. Fueled by fierce competition and by new high throughput capillary DNA sequencing instruments, the Genomics industry is demanding unprecedented sample purity, automation capability and high throughput. However, such automatable methods, which are capable of removing salts and dye terminators, are not currently available.
Similarly, purified primer extension products may also be analyzed by gel analysis, such as capillary electrophoresis, or by mass spectrometry. For example, single nucleotide polymorphisms (SNPs) are single-base differences that genetically distinguish individuals within a population and as such can act as markers for diseases caused by the interaction of multiple genes. Differential termination of primer extension reactions is a commonly used method for detecting SNPs, whereby a dideoxy nucleotide analog is incorporated at the site of the sequence variation. Primer extension reactions contain many of the same components as sequencing reactions used to generate dideoxy-terminated sequencing ladders of indeterminant length. As such, the same contaminants must likewise be removed prior to analysis. The contaminants may comprise salts and dideoxy terminators, both of which pass through an ultrafiltration membrane, while the primer extension products do not.
SUMMARY OF THE INVENTION
It is therefore a primary object of this invention to provide a method for purifying DNA sequencing reactions or SNP assays that is capable of automation.
It is a further object of this invention to provide an automated method of ultrafiltration capable of removing salts and dye terminators.
It is a further object of this invention to provide a method of ultrafiltration capable of producing greater sample purity and recovery.
It is a further object of this invention to provide a method of ultrafiltration capable of high throughput.
It is a further object of this invention to provide an automated method for removing salts and dye terminators from DNA sequencing reactions capable of high throughput.
It is a further object of this invention to provide a method for removing salts from DNA sequencing reactions which is more efficient and consistent than salt removal by alcohol precipitation.
The method of the invention is the result of efforts to develop an ultrafiltration-based sequencing reaction cleanup method that is capable of automation and high throughput. Separation of impurities, such as salts and dye terminators, can be driven by vacuum using common laboratory equipment and, as such, is amenable to automation. Historically, ultrafiltration membranes in centrifugal devices were used to remove low molecular weight contaminants from higher molecular weight solutes, but fractionation can be time-consuming and inefficient. As noted, DNA sequencing reaction cleanup is a demanding separation wherein unincorporated fluorescent labels and salts must be removed from polymerase extension products in order to produce usable data. Unlike centrifugal ultrafiltration, which cannot produce sufficient purity, constant pressure differential ultrafiltration is used in the method of the invention to accomplish this critical separation. Constant pressure differential ultrafiltration provides highly pure results when the membrane and operating conditions are carefully controlled. An ultrafiltration membrane is employed to retain sequencing products while unincorporated fluorescent labels and salts pass through the membrane during constant pressure differential ultrafiltration.
As noted, constant pressure differential ultrafiltration offers excellent salt removal, fluorescent label removal and sequencing product recovery in a readily automated format. Vacuum-based or positive pressure-based manifolds used in the method are easier to automate than centrifugal ultrafiltration. Also, complete top access (no filtrate collection capabilities necessary) to the purified sequencing reaction allows direct electrokinetic injection from the surface of the ultrafiltration membrane, thereby simplifying sample processing and reducing the need for other consumables. Development of this novel protocol for pro
Gabriels Joseph
Leonard Jack T.
MacDonald Constance
Ketter James
Millipore Corporation
Nields & Lemack
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