Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

Nucleic acid extraction solution and use thereof

Nucleic acid extraction solution and use thereof description/claims

This application claims the benefit of U.S. Provisional Application Ser. No. 60/261,845, filed Jan. 15, 2001, the disclosure of which is incorporated herein by reference.

The present invention relates to a nucleic acid extraction solution and to uses thereof, and more particularly, the invention relates to a nucleic acid extraction solution containing an ethylene glycol-type reagent and to uses thereof.

Nucleic acid-based assays have a wide variety of applications in the biological sciences. One important application is the detection of certain nucleic acid sequences in a biological sample. This type of assay has become useful in determining whether certain organisms, for example, microbial or viral pathogens (for example, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), or human papillomavirus (HPV)), are or have been present in a particular sample of interest. This can be helpful in determining whether an individual of interest, for example, a human, has or has not been infected with a particular organism. This type of information can be important for treating or managing the health of an individual.

A variety of nucleic acid extraction solutions have been developed over the years for extracting nucleic acid sequences from a sample of interest. See, for example, Sambrook et al. (Eds.) Molecular Cloning, (1989) Cold Spring Harbor Press. Many such methods typically require one or more steps of, for example, a detergent-mediated cell lysis step, a proteinase treatment step, a phenol and/or chloroform extraction step, and an alcohol precipitation step. Such methods typically require multiple steps, and can be time consuming. Inadvertent omission or improper sequence of one or more steps may result in less efficient nucleic acid extraction. Furthermore, when extracting nucleic acids from multiple samples, the more steps a particular method has, the more likely it is that samples become cross-contaminated during processing. When the resulting nucleic acid sample is analyzed, for example, by using an amplification-based protocol, for example, via polymerase chain reaction, cross-contamination may lead to false-positive results, which, in a clinical setting, may be significant.

Accordingly, it is desirable to produce a nucleic extraction solution that can be used in a simple, quick, and reliable nucleic acid extraction protocol that minimizes the risk of cross-contamination between samples, and that the resulting nucleic acids may be analyzed using conventional methodologies. These and other objects and features of the invention will be more clearly understood from the following description and claims.

The present invention provides a nucleic acid extraction solution containing an ethylene glycol-type reagent and methods using such an extraction solution. Once extracted by the solution of the invention, the isolated nucleic acid samples can be analyzed to determine whether particular nucleic acid sequences, for example, microbial or viral nucleic acid sequences, are present in a biological sample of interest. As a result, the methods and compositions can be used to determine quickly and reliably whether microbial or viral nucleic acids are present in the sample, which can thus act as an indicator of microbial or viral infection or contamination. The nucleic acid extraction solution can be used to extract nucleic acids of interest, for example, microbial or viral nucleic acids from biological samples containing cells and/or cellular debris, for example, biological samples containing mammalian cells and/or mammalian cell debris.

In one aspect, the nucleic acid extraction solution comprises an ethylene glycol-type reagent or an ethylene glycol derivative having the formula R1O—CH2—CH2—OR2. R1 and R2 are selected independently from the group consisting of hydrogen and an alkyl group. Preferably, the alkyl group of R1 or R2 has 1-12 carbon atoms, and more preferably, has 1-6 carbon atoms. More specifically, the alkyl group is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, and iso-hexyl. In a preferred embodiment, the alkyl group is selected from the group consisting of methyl, ethyl, n-butyl, iso-butyl, sec-butyl and tert-butyl.

In a preferred embodiment, R1 and R2 simultaneously are not both hydrogen atoms. More preferably, when R1 is a methyl group, an ethyl group or a butyl group, R2 is hydrogen. The ethylene glycol-type reagent preferably is 2-methoxyethanol, 2-ethoxyethanol or 2-n-butyloxyethanol. Most preferably, the reagent is 2-methoxyethanol.

In another preferred embodiment, the solution comprises from about 0.5% (v/v) to about 5% (v/v) of the reagent. More preferably, however, the solution comprises about 1% (v/v) of the reagent.

In another preferred embodiment, the solution optionally comprises a buffering agent. It is contemplated that a variety of buffering agents may be useful in the practice of the invention, however, the buffering agent preferably is a Tris buffer, a MOPS buffer or a borate buffer. In another embodiment, the solution preferably has a pH greater than about 7. The solution preferably has a pH in the range from about 7 to about 13, and under certain circumstances the solution preferably has a pH of from about 9 to about 11.

In another embodiment, the solution optionally further comprises a detergent. Although a variety of detergents can be useful in the practice of the invention, preferred detergents include, for example, Tween®, Brij®, and Triton-X100®. In addition, the solution may further comprise a chaotropic salt, for example, a guanidium salt, for example, guanidium thiocyanate.

In another aspect, the invention provides a method of extracting nucleic acids from a biological sample. The sample may be derived from a mammal, more specifically, a human, and may be, for example, a tissue sample, body fluid sample, or another cell containing sample, for example, a cervical smear. The method comprises mixing the biological sample with a nucleic acid extraction solution comprising a reagent having the formula R1O—CH2—CH2—OR2. R1 and R2 independently are selected from the group consisting of hydrogen and an alkyl group. In a preferred embodiment, the alkyl group of R1 or R2 has 1-12 carbon atoms, more preferably, has 1-6 carbons. The alkyl group preferably is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, and iso-hexyl. More preferably, the alkyl group is selected from the group consisting of methyl, ethyl, n-butyl, iso-butyl, sec-butyl and tert-butyl.

In a preferred embodiment, R1 and R2 simultaneously are not both hydrogen atoms. For example, when R1 is methyl, ethyl, n-butyl, iso-butyl, sec-butyl or tert-butyl, R2 is hydrogen. The solution preferably comprises from about 0.5% (v/v) to about 5% (v/v) of the reagent. More preferably, however, the solution comprises less than about 5% (v/v) of the reagent. Most preferably, the solution comprises about 1% (v/v) of the reagent.

In another embodiment, the solution optionally further comprises a buffering agent for maintaining the pH of the solution. Preferred buffering agents include, for example, a Tris or Tris-like buffer, a MOPS buffer or a borate buffer. Most preferably, the buffer is a borate buffer. The solution preferably has a pH greater than about 7. More preferably the solution has a pH in the range from greater than about 7 to less than about 13, and in certain circumstances, the solution has a pH in the range from about 9 to about 1. In a preferred embodiment, the solution comprises about 1% (v/v) 2-methoxyethanol and borate buffer, pH 9.5. A buffering agent, therefore, preferably has a pKa of greater than about 7.

In another embodiment, the solutions optionally further comprises a detergent. A variety of detergents may be used in the practice of the inventions, however, preferred detergents include, for example, Tween®, Brij®, and Triton®-X100. In addition, the solution optionally may further comprise a chaotropic salt, for example, a guanidium salt.

In another embodiment, the extraction method comprises an additional step of heating the mixture to a temperature within the range from about 50° C. to about 100° C., more preferably from about 75° C. to about 100° C., and most preferably from about 90° C. to about 100° C. The method, however, preferably lacks one or more of a phenol extraction step, a chloroform extraction step or an alcohol, for example, ethanol, precipitation step.

In another embodiment, the method comprises the optional, additional step of detecting, for example, via hybrid capture or other hybridization protocol, a particular nucleic acid sequence, for example, a microbial or viral nucleic acid sequence, in the sample. This method can be used to determine simultaneously the presence or absence of one or more contaminating agents, for example, microbial or viral pathogens, in the sample. Prior to detecting the nucleic acid sequence, the method of the invention may also include an optional step of amplifying the nucleic acid sequence of interest by, for example, polymerase chain reaction, ligase chain reaction, or the like.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws