Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-04-26
2002-10-22
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C435S091200, C435S810000, C435S040500
Reexamination Certificate
active
06469159
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and kits for the extraction of nucleic acids. In particular, the present invention relates to methods for extracting nucleic acids from tissue samples and paraffin-embedded tissue samples.
2. Background Information
Within the field of biological diagnostics, molecular-based techniques involving the amplification of nucleic acids are being used increasingly for the detection of inherited diseases, cancer, and infectious diseases. However, in applying such amplification techniques with tissues or other important clinical samples, impurities in nucleic acid preparations can inhibit or reduce the sensitivity and efficiency of amplification (Wilson, I. G., Applied and Environmental Microbiology 63(10):3741-3751, 1997).
DNA extraction from archival paraffin-embedded pathology tissue samples is particularly useful in retrospective studies in which the determination of a molecular diagnosis can be correlated with patient outcome. As pointed out by Crisan and Mattson (DNA and Cell Biology 12:455-464, 1993), the advantages of retrospective DNA analysis are multiple and can be applied to: (i) the study of numerous disease processes, where viral, bacterial, or parasitic agents are suspected to play an etiologic role becomes possible and epidemiological or prognostic correlation may be derived; (ii) the study of endogenous DNA abnormalities associated with various types of malignancies; (iii) the study of inherited DNA in genetic diseases; (iv) retrospective studies of rare diseases for which use of archival specimens would allow larger patient study groups than would be possible in prospective studies requiring fresh tissues; and (v),the possibility of correlating the presence or absence of a particular disease, morphological diagnosis or type, disease stage, prognosis, and response to treatment, where the clinical outcome is already known.
Methods reported for the amplification of DNA are based either on the use of a DNA polymerase (e.g., polymerase chain reaction, PCR), a ligase (ligase chain reaction, LCR), or both (GAP-LCR). Of these methods, PCR has been the most widely used to date. PCR involves the hybridization of primers to the strands of a target nucleic acid in the presence of a DNA polymerization agent and deoxyribonucleoside triphosphates under appropriate conditions. The result is the formation of primer extension products throughout several cycles of amplification, and exponential multiplication of the number of original target sequences. Further details about PCR can be obtained by consulting U.S. Pat. No. 4,683,195 (Mullis, et al), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al).
Because of its inherent sensitivity, product carryover and contamination between samples is a problem with PCR and nucleic acid based amplification systems in general. Product carryover during sample preparation is a serious problem. It is a function of the amount of time that a sample is exposed to the external environment, and related to the number of times the sample containment device must be opened, thereby exposing the sample to the external environment. Thus, it is advantageous to have a sample preparation method that is rapid and allows reduced or minimal exposure of the sample to the external environment; particularly, it is an advantage to provide a method of nucleic acid extraction where the number of times the sample containment device has to be opened is minimal.
Point mutations in the ras proto-oncogenes occur with great frequency in many human cancers, and are a potentially important diagnostic target (Bos, J. L., Cancer Res. 49:4682-4689, 1989). For example, as much as 90% of pancreatic cancer involves a mutation in the K-ras gene; the majority occurring in codon 12 (Almoguera et al., Cell 53:549-554, 1998). There are a number of methods for detecting the presence of a ras mutation. One method, restriction endonuclease mediated selective-PCR (REMS-PCR) has been described recently (WO 9632500). REMS-PCR is based upon the use of a thermostable restriction enzyme during PCR thermocycling. REMS-PCR greatly simplifies and decreases the time required for analysis and detection.
As pointed out by Volenandt et al., the amount of extracted DNA can dramatically affect the yield in a PCR reaction (Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. Methods in
Molecular Bioloqy Vol
. 15
: Current Methods and Applications
, 1993, edited by: B. A. White, Humana Press Inc., N.J.) When analyzing DNA from fixed tissue, an inverse relationship between the volume of extracted sample added and PCR amplification yield often is observed. This is due to the effects of certain fixatives and other inhibitors on Taq DNA polymerase activity. Furthermore, nucleic acid fragmentation occurring during fixation or DNA extraction also can be a problem in amplification of DNA (Greer et al., Am. J. Clin. Pathol. 95:117-124, 1991; and Crisan et al., Clin. Biochem. 25:99-103, 1992).
In the case of DNA extraction from paraffin or fresh tissue sections, complex methods for DNA preparation are typically used. Such methods require long incubations with protease enzymes in the presence of surfactants to release DNA and to degrade proteins that can interfere in nucleic acid amplification. Other subsequent steps in purification of extracted DNA may include treatment with an RNAase to remove contaminating RNA, followed by DNA precipitation with a solvent such as ethanol or a mixture of solvents such as phenol and isoamyl alcohol to remove protein and other cellular material, followed by DNA hydration (Volenandt et al., Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue. Methods in
Molecular Biology Vol
. 15
: Current Methods and Applications
, 1993, edited by: B. A. White, Humana Press Inc., N.J.). In the case of paraffin-embedded tissues, paraffin is usually removed by extraction with solvents such as xylene in a multiple step procedure prior to the proteinase step. A recent report by Banerjee et al. provides a protocol for DNA release from paraffin-embedded tissues (BioTechniques 18:768-773, 1995). The method involves the following steps: (1) microwave treatment, (2) removal of the paraffin by a centrifugation step, (3) Proteinase K digestion, and (4) a heat step to destroy Protease K activity.
Slebos and his associates have reported a method for releasing DNA from paraffin-embedded tissue which includes the use of three 10 micron sections, an incubation with a non-ionic detergent, and an 18-24 hr incubation with Proteinase K, followed by centrifugation (Diagnostic Molecular Pathology 1(2):136-141, 1992). The resultant supernatant is used directly in PCR amplification.
To overcome the need for long incubation with Proteinase K and the need for a heat inactivation step, the provisional specification of NZ 233270 describes the use of a thermostable proteinase instead of Proteinase K for digestion of cell protein and release of nucleic acid. This method provides an improvement in speed and ease-of-use. However, it is limited by the amount of amplifiable DNA that is released from paraffin-embedded tissue. Thus, there is still a need in the art for a rapid and highly effective means of extracting nucleic acids from tissue samples in a manner that is compatible with subsequent amplification procedures.
SUMMARY OF THE INVENTION
The present invention overcomes the above-noted problems and provides a needed means of extracting nucleic acids from tissue samples in a manner compatible with subsequent amplification methods. Thus, it is an object of the present invention to provide methods and kits for extracting nucleic acids from tissue samples and paraffin-embedded tissue samples.
Various other objects and advantages of the present invention will be apparent from the detailed description of the invention.
In one embodiment, the present invention relates to a method of extracting nucleic acids from tissue samples. The method comprises contacting the tissue sample with
Belly Robert T.
Chilson Gary J.
LandOfFree
Methods for extracting nucleic acids from tissue samples and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for extracting nucleic acids from tissue samples and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for extracting nucleic acids from tissue samples and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2970550