Reagents for preparing a sample containing biomolecular...

Details Reagents for preparing a sample containing biomolecular... Reagents for preparing a sample containing biomolecular...

1999-12-22

2002-10-15

Kumar, Shailendra (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Amino nitrogen containing

C564S216000, C514S629000, C204S451000

Reexamination Certificate

active

06465692

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Area of the Art
The invention relates generally to electrophoretic separation of biomolecular analytes and, specifically, to reagents for preparing biomolecular analytes for such a separation, and methods for making the reagents.
2. Description of the Prior Art
Capillary electrophoresis (CE) is a technique that has been used to separate proteins or nucleic acids, such as DNAs, from each other. See, for example, Chen, Fu-Tai A. et al., “Capillary Electrophoresis—A New Clinical Tool,”
Clin. Chem.
77/1:14-19 (1991); see, also, U.S. Pat. Nos. 5,120,413 and 5,228,960; see, further, U.S. Pat. No. 5,891,313. These documents are incorporated herein by reference.
In general, CE involves introduction of a sample into a capillary tube, i.e., a tube having an internal diameter of about 2 to about 2000 microns, and the application of an electric field across the tube. Eletrokinetic loading of a DNA sequencing sample mixture into a capillary electrophoresis tube is a preferred method of introducing a sample of analytes into the capillary electrophoresis tube. After the injection of the sample into the tube, an electric field is applied to the tube for separation. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. Each of the sample constituents has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
The results of CE analysis are typically presented as “electropherograms”, i.e., peaks of various widths and heights which correspond to the constituent parts of the sample. For example, a constituent which is present in a sample in a high concentration may evidence a peak having a large height and wide width, compared to a constituent present in a (relatively) low concentration. Typically, the electropherogram is derived by plotting detection units (typically, ultraviolet light absorbance) on the vertical axis and time of constituent traversal through the column to a detection region on the horizontal axis. Results can also be derived in terms of a unit value, typically derived from the areas bounded by the individual peaks.
In capillary electrophoretic separation, formamide is widely used to denature DNA analytes. Typically, analytes are reconstituted in formamide and heated at 90° C. for 2-3 min. to achieve denaturation prior to injection. Efficient sample injection and high-signal intensity are critical for accurate analysis. These two parameters are mainly governed by the pH and conductivity of a sample-formamide mixture.
The pH of DNA sample-formamide mixture plays an important role for DNA separation. Neutral and slightly basic pH is optimum because fluorescent dyes, such as cyanine dyes, are more stable at neutral pH. Furthermore, DNA is negatively charged above pH 6. When the sample/formamide mixture is slightly acidic, DNA is not completely ionized. This low-charge density results in inefficient sample injection and, therefore, insufficient signal intensity.
The conductivity of the DNA-formamide mixture also plays an important role for DNA separation. When the ionic strength of a DNA-formamide mixture is high due to the presence of salts, these highly mobile, smaller, negatively charged ions compete with less mobile, larger DNA fragments under applied voltage during sample injection. Therefore, small amounts of DNA fragments get injected into the capillary. As a result, a low signal will be observed, causing a low accuracy in analysis.
The most common approach to address the issue of high ionic strength is to deionize commercially available formamide by stirring it with a mixed bed resin prior to use. Commercially available formamide contains formate ions (HCOO
−
) and ammonium ions (NH4
+
) as the main impurities. These are the products of hydrolysis of formamide in the presence of water. These ions contribute to the high ionic strength and varying pH of formamide. By mixing with a resin carrying both H
+
and OH
−
charges, most of the ions in formamide can be removed until the capacity of the resin is exhausted.
The deionization process can be described as given in equation (1):
Resin-H
+
+Resin-OH
−
+HCOO
−
+NH
4
+→
Resin-HCOO
−
+Resin-NH
4
+
+H
2
O  (1)
However, the deionization process does not produce formamide with consistently low conductivity and accurate pH. Therefore, a need exists to develop a new and reliable method to prepare formamide with low conductivity and optimum pH for genetic analysis applications.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide formamide that may be used for preparing samples containing biomolecular analytes for electrophoretic separation with improved efficient sample injection and high-signal intensity. It is also an object of the present invention to provide methods for making such formamide.
These and other. objects and advantages are achieved by the reagents and methods of the present invention. One aspect of the present invention provides formamide having a conductivity in the range of about 5 to 7 &mgr;mho and a pH in the range of about 6.5 to 7.5. Another aspect of the present invention provides a method of preparing formamide to be used for preparing samples containing biomolecular analytes for electrophoretic separation. The method comprises (a) purifying the formamide until the conductivity of the formamide is below about 7 &mgr;mho, and (b) adjusting the pH of the purified formamide from step (a) to a range of about 6.5 to 7.5. In one embodiment of the present invention, the formamide is purified by removing water contained in the formamide and distilling the dry formamide until the conductivity of the formamide is below about 7. The pH of the purified formamide may be adjusted by a base that is non-nucleophilic. A reagent comprising the formamide of the present invention is also provided.
The reagents and methods of the present invention provide a number of advantages. The formamide of the present invention, when used in a sample loading solution for preparing samples for electrophoretic separation, improves the sample loading and hence enhances the signal intensity of the separation. Particularly, compared to the regular deionized formamide, the formamide of the present invention provides significantly better performance in DNA applications. In addition, the method for preparing formamide of the present invention is reliable and reproducible.
The reagents and methods of the present invention are well-suited for use in any capillary electrophoretic separation, particularly for DNA-related applications like sequencing and fragment analysis. For example, the formamide of the present invention is well-suited for use in a sample loading solution for reconstituted nucleic acid fragments contained in a sample for capillary electrophoretic separation. Particularly, the formamide of the present invention may be used to prepare sample loading solutions as described in the co-pending U.S. patent application Ser. No. 09/447,386, filed Nov. 23, 1999, the content of which is incorporated herein by reference.
The reagents and methods of the present invention may be used in connection with a capillary electrophoresis system such as, but not limited to, CEQ™ 2000 DNA Analysis System, P/ACE™ MDQ Capillary Electrophoresis System and Paragon CZE® 2000 Capillary Electrophoresis System, which are commercially available from Beckman Coulter, Inc., Fullerton, Calif., USA.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.


REFERENCES:
patent: 4687732 (1987-08-01), Ward et al.
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