| Separation and purification of nucleic acid from paraffin-containing samples -> Monitor Keywords |
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Separation and purification of nucleic acid from paraffin-containing samplesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidSeparation and purification of nucleic acid from paraffin-containing samples description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128634, Separation and purification of nucleic acid from paraffin-containing samples. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. Provisional Patent Application No. 60/686,522, filed May 31, 2005 and U.S. Provisional Patent Application No. 60/773,027, filed Feb. 13, 2006. The entire content of both of these priority applications is incorporated herein by reference. TECHNICAL FIELD [0002] The present disclosure relates to separation and purification of nucleic acids from paraffin-containing samples. More specifically, the disclosure relates to the separation and purification of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissue samples. BACKGROUND [0003] Genotyping and gene expression analyses of tissue samples can be of significant importance for the identification of disease biomarkers (e.g., genetic determinants), for the accurate diagnosis of disease, and for the determination of patients' course of treatment. Pharmacogenomic methods can identify patients likely to respond to a particular drug and lead to new therapeutic approaches. For example, tumor tissue excised from a patient can be analyzed for the increased or decreased expression of particular disease biomarkers and thereby help clinicians identify therapeutic agents that could be useful in treating the patient. [0004] Genotyping and gene expression studies (e.g., by reverse transcriptase polymerase chain reaction (RT-PCR) amplification) of tissue samples often is performed using frozen tissue samples. However, many pathological samples are not prepared as frozen tissues, but rather are formalin-fixed and paraffin-embedded (FFPE) to allow histological analysis and archival storage. Thus, rapid and reliable methods for extracting nucleic acids from paraffin-containing tissue samples would greatly aid the study of disease mechanisms and biomarkers. The inventions disclosed herein address this need. SUMMARY [0005] Provided are methods for separating ribonucleic acid from a paraffin-containing sample, that involve: (a) heating the sample at about 50-85.degree. C. for about 1-60 minutes in the presence of an ionic detergent to produce a paraffin phase and an aqueous phase; (b) removing the aqueous phase from the paraffin phase, and (c) adding a protease to the aqueous phase and incubating the aqueous phase at about 25-80.degree. C. for about 5-60 minutes. In some embodiments, the paraffin-containing sample is a formalin fixed paraffin embedded (FFPE) tissue sample. In some embodiments, the heating is between about 64-85.degree. C. In some embodiments, the heating is between about 60-75.degree. C. In some embodiments, the heating is at about 72.degree. C. In some embodiments, the heating is between about 1-30 min. In some embodiments, the heating is for about 10 min. In some embodiments, the ionic detergent is sodium dodecylsulfate (SDS), or Sarcosine. In some embodiments, the protease in Proteinase K. In some embodiments, the incubating is at about 35-70.degree. C. In some embodiments, the incubating is at about 55-65.degree. C. In some embodiments, the incubating is at about 56-58.degree.C. In some embodiments, the incubating is for about 5-30 min. In some embodiments, the incubating is for about 10 min. In some embodiments, the methods further involve purifying the ribonucleic acid from the aqueous phase. In some embodiments, the purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch.RTM. purification, or nucleic acid hybridization. [0006] Also provided are methods for separating ribonucleic acid from a paraffin-containing sample, that involve: (a) heating the sample at about 50-85.degree. C. for about 1-60 minutes in the presence of an ionic detergent to produce a paraffin phase and an aqueous phase; and (b) adding a protease to the aqueous phase and incubating the aqueous phase at about 25-80.degree. C. for about 5-60 minutes. In some embodiments, the methods further involve removing the aqueous phase from the paraffin phase. In some embodiments, the paraffin-containing sample is a formalin fixed paraffin embedded (FFPE) tissues sample. In some embodiments, the heating is between about 64.degree. C. and about 85.degree. C. In some embodiments, the heating is at about 60-75.degree. C. In some embodiments, the heating is at about 72.degree. C. In some embodiments, the heating is for about 1-30 min. In some embodiments, the heating is for about 10 min. In some embodiments, the ionic detergent is SDS or Sarcosine. In some embodiments, the protease is Proteinase K. In some embodiments, the incubating is at about 35-70.degree. C. In some embodiments, the incubating is at about 55-65.degree. C. In some embodiments, the incubating is at about 56-58.degree. C. In some embodiments, the incubating is for about 5-30 min. In some embodiments, the incubating is for about 10 min. In some embodiments, the methods further involve purifying the ribonucleic acid from the aqueous phase. In some embodiments, the purifying involves TRIZOL precipitation, guanidinium isothicyanate, anion exchange chromatography, silica-based purification, ChargeSwitch.RTM. purification, or nucleic acid hybridization. [0007] Also provided are methods for separating ribonucleic acid from a paraffin-containing sample, that involve heating the sample to about 50-85.degree. C. for about 1-60 minutes in the presence of an ionic detergent and a protease to produce a paraffin phase and an aqueous phase. In some embodiments, the methods further involve removing the aqueous phase from the paraffin phase. In some embodiments, the paraffin-containing sample is a formalin fixed paraffin embedded (FFPE) tissue sample. In some embodiments, the heating is at about 64-85.degree. C. In some embodiments, the heating is at about 60-75.degree. C. In some embodiments, the heating is at about 65.degree. C. In some embodiments, the heating is for about 1 min to about 30 min. In some embodiments, the heating is for about 10 min. In some embodiments, the ionic detergent is SDS or Sarcosine. In some embodiments, the protease is Proteinase K. In some embodiments, the methods further involve purifying the ribonucleic acid from the aqueous phase. In some embodiments, the purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch.RTM. purification, or nucleic acid hybridization. [0008] Also provided are methods for separating deoxyribonucleic acid from a paraffin-containing sample, that involve: (a) heating the sample at about 75-100.degree. C. for about 1-60 minutes in the presence of a detergent to produce a paraffin phase and an aqueous phase; (b) removing the aqueous phase from the paraffin phase; and (c) adding a protease to the aqueous phase and incubating the aqueous phase at about 25-80.degree. C. for about 5-60 minutes. In some embodiments, the paraffin-containing sample is a formalin-fixed paraffin embedded (FFPE) sample. In some embodiments, the heating is at about 85-100.degree. C. In some embodiments, the heating is at about 100.degree. C. In some embodiments, the heating is for about 1-30 min. In some embodiments, the heating is for about 10 min. In some embodiments, the detergent is an ionic detergent. In some embodiments, the ionic detergent is sodium dodecyl sulfate (SDS) or Sarcosine. In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the non-ionic detergent is Triton X-114, NP-40 or Tween-20. In some embodiments, the protease is Proteinase K. In some embodiments, the incubating is at about 35-70.degree. C. In some embodiments, the incubating is at about 55-65.degree. C. In some embodiments, the incubating is at about 62.degree. C. In some embodiments, the incubating is for about 5-30 min. In some embodiments, the incubating is for about 10 min. In some embodiments, the methods further involve purifying the deoxyribonucleic acid from the aqueous phase. In some embodiments, the purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwtich.RTM. purification, or nucleic acid hybridization. [0009] Also provided are methods for separating deoxyribonucleic acid from a paraffin-containing sample, that involve: (a) heating the sample to about 75-100.degree. C. for about 1-60 minutes in the presence of a detergent to produce a paraffin phase and an aqueous phase; and (b) adding a protease to the aqueous phase and incubating the aqueous phase at about 75-100.degree. C. for about 5-60 minutes. In some embodiments, the paraffin-containing sample is a formalin-fixed paraffin embedded (FFPE) sample. In some embodiments, the heating is at about 85-100.degree. C. In some embodiments, the heating is about 100.degree. C. In some embodiments, the heating is for about 1-30 min. In some embodiments, the heating is for about 10 min. In some embodiments, the detergent is an ionic detergent. In some embodiments, the ionic detergent is sodium dodecyl sulfate (SDS) or Sarcosine. In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the non-ionic detergent is Triton X-114, NP-40 or Tween-20. In some embodiments, the protease is Proteinase K. In some embodiments, the incubating is at about 35-70.degree. C. In some embodiments, the incubating is at about 55-65.degree. C. In some embodiments, the incubating is at about 62.degree. C. In some embodiments, the incubating is for about 5-30 min. In some embodiments, the incubating is for about 10 min. In some embodiments, the methods further involve the deoxyribonucleic acid from the aqueous phase. In some embodiments, the purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch.RTM. purification, or nucleic acid hybridization. [0010] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The disclosed materials, methods, and examples are illustrative only and are not intended to be limiting. Skilled artisans will appreciate that methods and materials similar or equivalent to those described herein can be used to practice the invention. [0011] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. DETAILED DESCRIPTION OF THE INVENTION [0012] Provided are materials and methods for rapid separation and purification of nucleic acids from paraffin-containing samples. Methods of the invention may be used for separating and purifying nucleic acid from any paraffin-containing sample, including preserved (e.g., with formalin) tissue samples. Paraffin-containing samples may include tissue from any organism: vertebrate (e.g., mammals such as humans, primates, canines, felines, porcines, equines and bovines, as non-mammals such as birds, fish, amphibians and reptiles) or invertebrate (e.g., insects). Normal and diseased (e.g., tumor) tissue may be processed using the disclosed methods. Tumor types include those derived from skin, prostate, ovary, uterus, breast, lung, pancreas, small intestine, colon, liver, kidney, and brain. [0013] Methods of the invention may be used for separating and purifying any nucleic acid, the quality of which is suitable for genetic manipulation (e.g., cloning, amplification, sequencing, RT-PCR and cDNA library construction), genotyping, and gene expression studies. Single stranded (ss) DNA, ssRNA (e.g. micro-RNA), double stranded (ds) DNA, and ds RNA may be separated and purified from paraffin-containing samples using the disclosed methods. The target nucleic acid may be linear or circular, and may be total RNA, mRNA, and chromosomal or genomic DNA (gDNA). [0014] Methods of the invention generally involve a melting step, where paraffin-containing tissue samples are heated in the presence of a detergent, causing the paraffin to melt and tissue sample cells to lyse. From the resulting two-phase mixture (i.e., a paraffin phase and an aqueous phase), the aqueous phase is collected for purification of nucleic acid. Protease(s) can be used at one or more points in the separation process to facilitate tissue cell lysis and/or degrade proteins that could degrade nucleic acid or interfere with subsequent genetic manipulation or analysis. For example, a protease can be included before or during the melting step, and/or can be added to the aqueous phase of the two-phase mixture either before or after it is collected from the paraffin phase. These processes are further described herein. Melting [0015] In the melting step, a paraffin-containing sample is heated in the presence of a detergent, causing the paraffin to melt and tissue sample cells to lyse. For separation of RNA (e.g., mRNA), a paraffin-containing sample is heated at about 50.degree. C. to 85.degree. C. In some RNA-separation embodiments, the sample is heated at about 64.degree. C. to 85.degree. C., or at about 60.degree. C. to 75.degree. C. In one RNA-separation embodiment, the sample is heated at about 72.degree. C. For separation of DNA (e.g., gDNA), a paraffin-containing sample is heated at about 75.degree. C. to 100.degree. C. In some DNA-separation embodiments, the sample is heated at about 85.degree. C. to 100.degree. C. In one DNA-separation embodiment, the sample is heated at about 100.degree. C. [0016] The melting step generally is performed for 1 to 60 minutes. In some embodiments, a paraffin-containing sample is heated for 1 to 30 minutes, or for about 5 to 20 minutes. In one embodiment, the melting step is performed for about 10 min. [0017] The melting step is performed in the presence of a detergent-containing buffer that facilitates cell lysis. The detergent-containing buffer may include one or more ionic and/or one or more non-ionic detergents. Suitable ionic detergents include sodium dodecyl sulfate (SDS), lauroylsarcosine, Na+ salt ("sarcosine" or Sarkosyl), bile salt detergents (e.g., CHAPS, CHAPSO, sodium deoxycholate, sodium taurocholate, sodium glychocholate, sodium glychodeoxycholate) and certyltrimethylammonium bromide (CTAB). Nonionic detergents include sarcosine, Tween-20 NP-40, Triton X-100, NP-10, Triton X-114, Tween-80 and n-octanoyl-.beta.-D-glucosylamine (NOGA). The detergent-containing buffer may also include a buffer salt such as Tris-HCl (pH 7.5-8.5), phosphates, HEPES, PIPES, MOPS, MES, TABS, TRICINE, NaCl, KCl, MgCl.sub.2, a chelating agent (e.g., EDTA or EGTA), and/or a preservative. [0018] Ionic and/or non-ionic detergents can be used for separation of RNA. In some embodiments, ionic detergents are used for separation of RNA. Ionic and/or non-ionic detergents can be used for separation of DNA. In some embodiments, non-ionic detergents are used for separation of DNA. Continue reading about Separation and purification of nucleic acid from paraffin-containing samples... Full patent description for Separation and purification of nucleic acid from paraffin-containing samples Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Separation and purification of nucleic acid from paraffin-containing samples patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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