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Buffers for electrophoresis and use thereof

Inactive Publication Date: 2007-06-21
APPL BIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The present teachings provide, among other things, buffer compositions and / or sieving (gel) formulations that improve the performance of electrophoresis instruments, such as capillary electrophoresis (CE) devices.

Problems solved by technology

Unfortunately, it is not uncommon to encounter sequencing errors involving specific sequences which are difficult to resolve.
One such problem, called a “compression,” occurs when the single-stranded fragments anneal to themselves to form a “hairpin” structure at a particular position.
This may cause the fragment to migrate more quickly through the gel than one would expect from its length, which can result in bands that run very close together, sometimes overlapping one another.

Method used

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  • Buffers for electrophoresis and use thereof
  • Buffers for electrophoresis and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0054] A buffer solution in accordance with the present teachings was formulated as: 100 mM Bis-Tris (bis[2-Hydroethyl]imino-tris[hydroxymethylmethane), 100 mM TAPS (N-tris[hydroxylmethyl]methyl-3-aminopropane-sulfonic acid), and 1 mM EDTA (Ethylenediaminetetraacetic acid, pH 8).

[0055] 1 mM EDTA was used to eliminate or alleviate damage to the capillary coating due to metals.

example 2

[0056] Less temperature induced DNA band broadening was observed with TrisTAPS than with NaTAPS (Tris-(hydroxymethyl)-methyl-amino-propanesulfonic acid, sodium salt) running buffer. To understand how the cations, Tris and Na, influence DNA band broadening during electrophoresis, DNA separation and mobility were examined during capillary electrophoresis with running buffers containing the different metal cations: Li, Rb, Ca, Mg, K, and Cs; and organic buffers, Bis-Tris, TrisBispropane, imidizole, arginine, ammonium, and tetrapentylammonium. It was noticed that the sizing of a fragment, referred to herein as GS700 250 nucleotide (nt) fragment, or simply the 250 nt fragment, which has putative DNA secondary structure that causes it to run anomalously under low DNA denaturant conditions, was running near its predicated size (250 nt) with Bis-TrisTAPS as the running buffer during capillary electrophoresis. From an examination of the structure of the Bis-Tris molecule, it is believed that...

example 3

[0057] The ability of the buffer of the present teachings to improve nucleic acid denaturation during electrophoresis was observed in the following assays:

EXAMPLE 3(A)

[0058] One assay measured the ability of the present buffer to reduce the anomalous mobility of the GS700 250 nucleotide (nt) fragment—the anomalous mobility is thought to be due to DNA secondary structure. The results from this assay are reported in Table I. During electrophoresis at 70° C. on the ABI Prism 310 instrument using POP37 polymer (Applied Biosystems; Foster City, Calif.), with TrisTAPS as the running buffer, the 250 nt fragment migrated as 244.5 nt, and with an additional 1M urea in the polymer formulation, the 250 nt fragment migrated as 245.5 nt. Using Bis-Tris buffer (without additional urea) the 250 nt fragment migrated as a 247.5 nt fragment—closer to the correct 250 nt than obtained by adding an additional 1 M urea to the polymer.

TABLE IMigration of the250 nt fragmentbuffer cation50° C.60° C.70° ...

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Abstract

Various embodiments provide, for example, buffer compositions and / or sieving formulations useful in connection with electrophoresis instruments, such as capillary electrophoresis (CE) devices. In various embodiments, a buffer composition can include Bis-Tris, TAPS and / or TAPSO, and, optionally, a chelating agent, such as EDTA. Methods of separating samples containing bio-molecules, such as DNA or RNA, are also described.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application No. 10 / 198,832, filed Jul. 19, 2002, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 909,649, filed Jul. 19, 2001, the entireties of both of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] Electrophoresis is commonly used to separate biological molecules, such as deoxyribonucleic acid (“DNA”), ribonucleic acid (“RNA”), proteins, etc., according to size or length. [0003] DNA base sequencing and fragment analysis are among the most useful embodiments of electrophoresis separations. In DNA base sequencing, for example, a DNA sequencing product is denatured and the resulting single stranded DNA sample is applied to an electrophoresis gel for separation. [0004] Unfortunately, it is not uncommon to encounter sequencing errors involving specific sequences which are difficult to resolve. One such problem, called a “compression,” oc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68B01D57/02B03C5/00C08F2/58C12M1/00C12N15/09G01N27/447
CPCG01N27/44747
Inventor HACKER, KEVIN J.VOSS, KARL O.
Owner APPL BIOSYSTEMS INC