Modified microporous membrane for non-specific and sequence-specific nucleic acid capture and methods of use

Abstract

Disclosed herein is a novel multi-layered, composite microporous membrane comprising in at least one layer a highly electropositive hydrophilic material distributed throughout wherein the material is capable of irreversibly binding nucleic acid and, optionally, at least one layer where the material is associated with sequence-specific peptide nucleic acids, permitting the simultaneous or sequential capture, amplification and / or identification of specific nucleic acid sequences of interest. Also disclosed herein are methods of use of the composite membranes of the invention in applications based on the sequence-specific capture and / or amplification and identification of nucleic acid from complex biological samples.

Description

REFERENCE TO RELATED APPLICATIONS[0001] This application claims benefit under Title 35, U.S.C. .sctn.119(e), of U.S. application Ser. No. 09 / 873,675, filed Jun. 4, 2001.BACKGROUND OF THE DISCLOSURE[0002] The present disclosure relates to articles of manufacture comprising a multi-layer composite microporous membrane, wherein at least one layer of the membrane has associated therewith highly electropositive solid phase hydrophilic materials useful for highly efficient and irreversible binding of nucleic acids, optionally modified with sequence specific peptide nucleic acids (PNA's); methods of fabricating such articles of manufacture; and methods of using such articles of manufacture to identify, separate and / or amplify target nucleic acid and to optionally store the membrane and bound nucleic acid for archival purposes.[0003] Detection of Nucleic Acid Through Use of Probe Complementarity[0004] The molecular structure of nucleic acids provides for specific detection by means of compl...

AI Technical Summary

Benefits of technology

This approach enhances the sensitivity and specificity of nucleic acid detection by enabling the capture and amplification of low copy number targets from complex samples, reducing labor and the need for multiple steps, and improving the detection of specific sequences in the presence of high background nucleic acid.

Problems solved by technology

In currently employed conventional protocols each of these three steps is performed separately, making nucleic acid analysis labor intensive.

For analysis purposes, nucleic acid must frequently be extracted from extremely small specimens in which it is difficult, if not impossible, to obtain a second confirmatory specimen.

In such examples, the extent of the genetic testing and confirmation through replica testing is, thus, limited by the nucleic acid specimen size.

Using conventional extraction protocols for these small specimens, the nucleic acid is often lost or yields are such that only a single or few amplification analyses are possible.

Specimens that contain high levels of endogenous or background nucleic acid such as blood are extremely difficult to analyze for the presence of low level specific targets.

Since conventional purification methods require elution of the bound nucleic acid, these solid phase materials are widely considered to be of little use for DNA purification.

Generally, nylon membranes must be treated, however, to immobilize the DNA after it has been transferred, as by way of thorough drying, or exposure to low amounts of ultraviolet irradiation (at 254 nm), and such immobilization is not irreversible.

Moreover, these techniques are labor intensive and require technicians with considerable expertise in molecular biology techniques.

Using the current nucleic acid hybridization formats and stringency control methods, it remains difficult to detect low copy number (i.e., 1-100,000) nucleic acid targets even with the most sensitive reporter groups (enzymes, fluorophores, radioisotopes, etc.) and associated detection systems (fluorometers, luminometers, photon counters, scintillation counters, etc.)

This difficulty is caused by several underlying problems associated with direct probe hybridization.

One problem relates to the stringency control of hybridization reactions.

Unfortunately, the application of these stringency conditions results in a concomitant decrease in the number of hybridized probe / target complexes remaining for detection.

Another problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples.

A third problem relates to the unfavorable hybridization dynamics between a probe and its specific target.

A fourth problem for most present hybridization formats is the high level of non-specific background signal.

These problems, either individually or in combination, lead to a loss of sensitivity and / or specificity for nucleic acid hybridization in the above described formats.

This is unfortunate because the detection of low copy number nucleic acid targets is necessary for most nucleic acid-based clinical diagnostic assays.

As is known to those skilled in the art, such detection is not easily performed using currently available technologies, such as those described above.

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