CN101657711A - Alkyl amine improves the detection of the biological sample component of formaldehyde fixed - Google Patents
Alkyl amine improves the detection of the biological sample component of formaldehyde fixed Download PDFInfo
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Abstract
The formaldehyde crosslinking that alkyl amine works and takes place in the biological sample to be released in.Therefore, the sample that alkyl amine is contacted formaldehyde fixed is to be used to make the biological components of sample, comprises that nucleic acid or protein are easier to the method that detects and characterize.
Description
Background technology
In in the past 100 years, the virologist is routinely by preserving them as tissue with the formaldehyde fixed biological sample.Though formaldehyde treated has kept the cell characteristic of tissue, formaldehyde treated also can cause chemical crosslinking, and this makes many biological components of sample can not fully be suitable for or be unsuitable for detecting, quantize and characterize.Formaldehyde is by making primary amine group in the protein and near the nitrogen-atoms among protein or the DNA other by-CH
2-key is crosslinked, and preservation or fixing organization or cell.Therefore, although for example PCR (PCR) can be used for detecting and quantize nucleic acid in the biological sample, PCR usually can not be fully or can not be analyzed nucleic acid in the sample of formaldehyde crosslinking effectively, particularly when the expectation quantitative result.
Therefore, nucleic acid is linked to cellular component and has proposed challenge to detecting various cellular components, comprised and detect nucleic acid and protein under the effect of formaldehyde.Be used to improve sample amplification nucleic acid from formaldehyde crosslinking though described some approach, this improvement mostly just comprises the protein in the degraded sample or provides and do not change the washing agent that forms crosslinked covalent bond usually.The present invention solves this and other problem.
Summary of the invention
The invention provides the method for one or more components of the biological sample that is used to analyze formaldehyde crosslinking.In some embodiments, this method comprises makes sample contact with the alkyl amine of sufficient quantity, with the crosslinked component of release at least a portion, thus but the degree of detection (accessibility) of one or more components that improvement is used to analyze.
In some embodiments, biological sample is from the tissue sample of animal.
In some embodiments, the amount of alkyl amine at 0.01% (about 2 mM) between 5% (the about 800mM).
In preferred embodiments, sample and alkyl amine are heated a period of time.
In other the embodiment preferred, this method further comprises detected components at some.
In some embodiments, before detecting step, alkyl amine gone up from sample substantially remove.In some embodiments, before detecting step, the concentration of alkyl amine is reduced to less than about 0.5% (about 80mM) (for example less than about 0.2% or 0.1%).
In some embodiments, detect step and comprise the detection by quantitative component.
In some embodiments, component is a nucleic acid.In some embodiments, nucleic acid is DNA.In some embodiments, component is RNA.
In some embodiments, this method further comprises detection nucleic acid.In some embodiments, detect step and comprise amplification of nucleic acid.In some embodiments, under the condition that allows formation probe and nucleic acid, make the nucleic acid component contact probe, and detect the existence of duplex.In some embodiments, probe is connected on the solid phase carrier.In some embodiments, amplification step comprises the PCR.
In some embodiments, component is a protein.In some embodiments, this method further comprises detection protein.In some embodiments, detect step and comprise mass spectrum or electrophoresis.In some embodiments, mass spectrum comprises substance assistant laser desorpted/ionization (MALDI).
In some embodiments, before contact procedure, sample is embedded in the paraffin.
In some embodiments, alkyl amine is selected from ethylenediamine, monoethanolamine and propylamine.
In some embodiments, if compare with not carrying out the part that contact procedure can be used for analyzing, the part of the component that can be used for analyzing is enhanced at least about twice.In some embodiments, if compare with not carrying out the part that contact procedure can be used for analyzing, the part of the component that can be used for analyzing is enhanced at least about ten times.
In some embodiments, this method further comprises contacts with the protein in the degraded sample sample with proteinase, thereby makes that nucleic acid more can be used for analyzing.
The present invention also provides kit, and it is used to improve the availability of one or more components of the biological sample of formaldehyde crosslinking.In some embodiments, this kit comprises alkyl amine; With the equipment that is used for removing alkyl amine from biological sample, for example proteinase or reagent or equipment.
In some embodiments, this kit comprises reagent or the equipment that is used for removing from biological sample alkyl amine.In some embodiments, this equipment is the post that is used for purification of nucleic acid.
In some embodiments, this kit comprises proteinase.In some embodiments, proteinase is Proteinase K.
In some embodiments, this kit further comprises nucleotide and/or heat-stabilised poly synthase.In some embodiments, the heat-stabilised poly synthase is the Taq polymerase.
The present invention also provides reaction mixture.In some embodiments, reaction mixture comprises the biological sample of formaldehyde crosslinking; With the alkyl amine of sufficient quantity to discharge the crosslinked component of at least a portion.
In some embodiments, the amount of alkyl amine is between 0.01% to 5%.In some embodiments, alkyl amine is selected from ethylenediamine, monoethanolamine and propylamine.In some embodiments, biological sample is from the tissue sample of animal.
Definition
" biological sample of formaldehyde crosslinking " is meant by formaldehyde treated and becomes the crosslinked biological sample of formation between nitrogen and other nitrogenous protein and/or the nucleic acid in protein.Biological sample typically comprises cell.Biological sample for example can be, from the tissue sample of animal.By they being embedded in the sample of storing many formaldehyde treated in the paraffin.
As used in this article, term " alkyl amine " is meant straight or side chain, saturated or undersaturated molecule, and it has 1-10 or more carbon atoms and one or more amino.The moieties of alkyl amine can be methyl, ethyl, propyl group, butyl, amyl group, hexyl, isopropyl, isobutyl, sec-butyl, the tert-butyl group etc.Amino can be primary amino radical or secondary amino group.Alkyl amine can further be not more than 2, and (promptly 0,1 or 2) individual substituting group replaces, and it includes but not limited to one or more oh groups.Can be used for alkyl amine of the present invention including, but not limited to ethamine, propylamine, isopropylamine, ethylenediamine and monoethanolamine.It will be understood by those skilled in the art that other alkyl amine also can be used for the present invention.
Phrase " detected components " is meant the existence of measuring component at least or does not exist, and can comprise further quantitative measurement or other sign of component or part component.
" component " of biological sample is meant that molecule (for example protein, nucleic acid etc.) or people wish the concrete target that detects such as concrete protein or nucleotide sequence.
As used in this article, term " nucleic acid " is meant polymkeric substance (comprising the 2-deoxy-D-ribose) (being DNA), polynucleotide (comprising D-ribose) (being RNA) and the N-glucosides analog of any other purine or pyrimidine bases or the modified purine or the pyrimidine bases of deoxyribonucleotide.
Phrase " to discharge the crosslinked component of at least a portion " is meant that change forms crosslinked covalent bond at two kinds of components (for example nucleic acid and albumen) finger tip of biological sample, so that these two kinds of components no longer connect by covalent bond.This phrase includes but not limited to reverse fully cross-linking process.
As used in this article, phrase " but the degree of detection that is used to analyze " is meant and measures the detection method that specific target molecule exists or do not exist and/or measure.For example many detection methods are stop to detect protein or nucleic acid in the biological sample of formaldehyde crosslinking to small part, so some crosslinked component " not can be used for " detecting.In case crosslinked by handle discharging with alkyl amine, then the raising amount of component (for example at least many about 10% and typically at least about 2 times, or be about at least 10 or 100 times sometimes) can detected and quantitative measurement.
Description of drawings
Fig. 1 has described the example of nucleic acid formaldehyde crosslinking to lysine and crosslinked reverse after adding alkyl amine.
Fig. 2 has described the mass spectrophotometry of undressed oligonucleotide described in the embodiment 1.
Fig. 3 has described the mass spectrophotometry of the oligonucleotide of the processing of formalin described in the embodiment 1.
Fig. 4 has described the oligonucleotide of the processing of formalin described in the embodiment 1 and the mass spectrophotometry of lysine.
Fig. 5 has described to use ethanol diamines (ethanoldiame) as described in example 1 above thereby has handled formalin is handled behind the crosslinked DNA regeneration initiate dna the oligonucleotide and the mass spectrophotometry of lysine mixture.
Fig. 6 has described the mass spectrophotometry of undressed synthetic RNA.
Fig. 7 has described the mass spectrophotometry of synthetic RNA after hatching 1 hour with formalin.
Fig. 8 has described to hatch with formalin the mass spectrophotometry of synthetic RNA after 5 hours.
Fig. 9 has described to hatch with formalin the mass spectrophotometry of synthetic RNA after 24 hours.
Figure 10 has described the mass spectrophotometry of synthetic RNA after hatching 1 hour with formalin and lysine.
Figure 11 has described to be that formalin and lysine hatched 1 hour and utilize ethanol diamines (ethanoldiame) (EDA) to discharge the mass spectrophotometry of removing synthetic RNA behind the cross-linking chemistry subsequently.This figure shows from crosslinked RNA (ribonucleic acid)-lysine adduct initial RNA that regenerates.
Figure 12 has described the effect of ethanol diamines (EDA).The concentration of the EDA that uses shows as percentage.Notably, in this system, find that 0.2% (about 50mM) and higher EDA can suppress the PCR reaction.
It is crosslinked that Figure 13 has described to utilize EDA to reverse in paraffin-embedded tissue (FFPET) sample in formalin fixed.The upper part of figure shows the EDA of low concentration, no matter whether carries out QIAquick
TMPurifying all increases.Yet the amount of amplification is compared low with other swimming lanes.Under the suppression of amplification EDA concentration of (for example being higher than 0.1% (approximately 25mM)), only carrying out QIAquick
TMJust increase during purifying, this had proved before amplification and has removed or the advantage of deactivation EDA otherwise.The part of figure provides the chart of cycle limit contrast signal, and the proof amount that improves EDA is removed the amount that step can significantly improve the DNA that can be used for carrying out pcr amplification in the sample effectively in conjunction with EDA.
Figure 14 has also described to utilize the EDA of raising amount to handle to reach the improvement of the availability that nucleic acid is used to increase in the specimen preparation thing.At the upper part of figure, cycle limit (x-axle) is mapped at amplified signal.In the part, cycle limit is the y-axle below, and the concentration of EDA is the x-axle.
Figure 15 has described the SDS-PAGE gel, and shows the crosslinked bovine serum albumin(BSA) of formalin (BSA) and follow-up by reversing crosslinked result with the EDA processing.
Detailed Description Of The Invention
I. introduce
As shown in Figure 1, formaldehyde cause in the albumen nucleic acid and primary amine, particularly amino acid for example Lysine and arginic crosslinked. As crosslinked result, various lifes in the sample that formaldehyde is fixed The thing component can not be detected by the modern measure method. The invention provides reverse crosslinked, thus so that The method that has more biological components to be detected.
Sample crosslinked that reverses formaldehyde treated be by sample is contacted with the sufficient quantity alkylamine with The release cross-linking reaction realizes. The example that Fig. 1 has described crosslinked reverse (in this example Shown that nucleic acid is crosslinked by formaldehyde and lysine, with alkylamine reaction has been reversed subsequently.
In case crosslinked sample contacts with alkylamine, the then crosslinked minimizing of nucleic acid and protein or disappear Remove, thereby permission improves the detection of these components.
II. so that the more spendable method of crosslinked component
The invention provides for so that the component of the formaldehyde crosslinking of biological sample more can be used for detecting Method, it is by making sample contact with alkylamine. Be used for so that the more spendable alkyl of component The quantity of amine can change, and partly depends on the concrete alkylamine, to be detected that uses Component and employed detection method are because different detection methods has different sensitiveness Therefore may the different spendable component of desirability.
Ideally, to can be used for the amount of the component of specific detection method be component in the sample All measure. Yet the amount that usually is available to the user for the component of detection is lower than component in the sample All measure. In some embodiments of the present invention, processing the sample phase with not using alkylamine Ratio, the amount that can be used for the component used that detects is at least about 2 times and (uses identical detection side Method) under the condition, uses the alkylamine of sufficient quantity. In some embodiments, with do not make Process sample with alkylamine and compare, the amount that can be used for the component used that detects is at least about 5,10, Under the condition of 20,100 times (using identical detection method), use the alkylamine of sufficient quantity. In some embodiments, the concentration that be used for to discharge the crosslinked alkylamine of sample about 0.01% to approximately Between 5% (or more), for example between about 0.01% to about 1%, about 0.05% to about 2% Between, between about 0.05% to about 1%, between about 0.1% to about 1%.
Those skilled in the art can understand state (for example time and the temperature of sample and alkylamine combination Degree) will affect ability and the amount of crosslinked reverse. Alkylamine is processed in environment temperature (for example at 20-40 Or 50 ℃) between under be effectively, therefore do not need heating steps crosslinked to discharge not inevitablely. This When detecting relatively unsettled component, be useful especially, such as RNA. Yet, higher temperature Degree (such as 80～100 ℃, 90-100 ℃, 90-99 ℃ etc.) can further improve nucleic acid or egg But white matter for detection of degree of detection.
In addition, the amount of alkylamine and sample time of hatching is available to the user for detection with impact The amount of component. For example, sample can hatch at least about 5,10,20,30,60 with alkylamine, 120 minutes or more. Although longer incubation time can improve from the component of crosslinked release Amount, but this need to balance each other with the unstable degree of specific components. For example, unstable when detecting When fixed component such as RNA, can expect to use the short incubation time. On the contrary, more stable Component such as protein or DNA can be exposed long event and not damage component.
Need to recognize that can to use different alkylamine to discharge crosslinked. Do not limiting the scope of the invention Prerequisite under, selected alkylamine can discharge component also from the cross-bond of formaldehyde inducement usually And restore component (for example nucleic acid and/or protein) and become before basically existing with formaldehyde crosslinking Identical component. Cross-linking reaction is considered to the reversible process of being undertaken by the reaction of formaldehyde, First amine forms hemiacetal amine (hemiaminal), and subsequent dewatering obtains imines. Imines and second The amine reaction obtains product aminal (aminal). The method replaces second amine by imines and water Reaction be returned to initiation material. Believe that alkylamine of the present invention releases from the cross-bond of formaldehyde inducement Put component, it passes through as the competitive reaction thing in imines and aminal (aminal) formation. When cross-bond discharges as the partial equilibrium process, imine intermediate and formaldehyde and alkylamine, Thereby the cross-bond of component from formaldehyde inducement discharged. Usually, any alkylamine and primary amine (with have The time secondary amine) will be effective. Need to understand and can carry out various replacements to alkylamine, and basically Do not affect amine reverse crosslinked or produce other will with the energy of the reactive part of sample component reaction As long as power is the ability that such replacement does not hinder amine to react basically. Monoethanolamine energy for example Effectively reverse cross-bond. Ethylenediamine also is effectively, although need to admit other diamines will with Sample can be effectively used to method of the present invention. Further, although sometimes can be preferably than short alkane Base chain (for example having 1,2,3,4,5 carbon), but long carbochain also can be used.
The method according to this invention can be used the biological sample of the formaldehyde crosslinking of any type. Usually, tissue sample stems from animal tissue. In some embodiments, sample is embedded in stone In the wax. For example sample can be the fixing paraffin-embedded tissue (FFPET) of formalin. At some In the embodiment, sample is to obtain and be kept at subsequently to comprise the molten of formaldehyde from animal (for example people) In the liquid, before analyzing, stablizing sample, thus the nucleic acid in the crosslinked sample and/or protein. For example cervix or other gynaecology's swab (for example for detection of sexually transmitted disease) can be stored in Comprise in the solution of formaldehyde, thus the nucleic acid in the crosslinked sample and/or protein. Subsequently can root It is crosslinked to use alkylamine to reverse according to method of the present invention.
So that sample component can be used for detecting, comprise in the method for the invention volume for further Outer purifying or other steps. For example, if nucleic acid component that will test sample may have What help is to use Protease Treatment sample (for example before or after alkylamine is processed), perhaps Otherwise degrade protein in the sample. Exemplary protease is Proteinase K, although Need to understand and to replace to various other protease.
Depend on equally the detection method of using subsequently, expectation is removed or is reduced at least and test set The amount of the alkylamine of being combined with sample before dividing. For example, the inventor has been found that and helpfully is From other components and the nucleic acid from the alkylamine purification of samples of sample, it is by using reagent Or equipment for example centrifugal column with other partial purification nucleic acid from sample. Exemplary equipment be with Nucleic acid have affinity silica-based centrifugal column (for example from Qiagen, Valencia, CA's QiaquickTMCentrifugal column), although can certainly use other purification process to remove alkylamine.
Perhaps, amine can be by chemical neutralization no longer can significantly disturb the detection of specific components.
II. the detection of the component of crosslinked biological sample
Abovementioned alkyl amine can be processed with any detection method and be combined with crosslinked sample before detecting The component of product. Just as will be described in further detail below, the component of crosslinked Interference Detection in the sample Comprise nucleic acid and protein. The detection of component can comprise only determines specific components or component one The partly existence of (for example specified protein or nucleotide sequence) or do not exist. Interchangeable, inspection Survey can comprise the quantitative assay component, and/or characterizes component. Sign can comprise for example peptide or nuclear After acid order-checking and/or the definite meal or translation modify and comprise such as glycosylation, phosphorylation etc.
A. nucleic acid
Known many methods for detection of nucleic acid in this area. DNA or RNA (comprise MRNA, rRNA etc.) or the two can be detected. Detection can comprise that quantitative assay is specific Sequence or RNA and/or characterisation of nucleic acids, for example by the hybridization of nucleotide sequencing or sequence-specific Technology (such as such as for detection of SNP (SNP) etc.).
Because the sample of many paraffin-embedded formaldehyde treated is relatively little, therefore, usually wish to make Increase specific nucleic acid with auxiliary detection nucleic acid with amplification method. Can use the expansion of any type Increase method, comprise the index amplification method, linear amplification, thermal cycle, isothermal method etc. Suitably Amplification method include but not limited to PCR (PCR) (Principles and Applications for DNA Amplification(ed.H.A.Erlich，Freeman Press，
NY，N.Y.，1992)；
PCR Protocols：A Guide to Methods and Applications(eds.Innis waits the people, Academic Press, San Diego, Calif., 1990); Current Protocols in Molecular Biology, Ausubel, 1994-1999, including Supplemental updates through April 2004; Sambrook ﹠ Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001)), ligase Chain reaction (LCR) (U.S. Patent No. 5,185,243,5,679,524 and 5,573,907; EP 0 320 308 B1; WO 90/01069; WO 89/12696; With WO 89/09835), circulation is visited The pin technology (U.S. Patent No. 5,011,769,5,403,711,5,660,988 and 4,876,187 and PCT openly apply for WO 95/05480, WO 95/1416, and WO 95/00667), InvaderTMTechnology (U.S. Patent No. 5,846,717; 5,614,402; 5,719,028; 5,541,311; With 5,843,669), Q-β replicase technology (U.S. Patent No. 4,786,600), NASBA (U.S. Patent No. 5,409,818; EP-0 329 822), TMA (U.S. Patent No. 5,399,491,5,888,779,5,705,365,5,710,029), SDA (U.S. Patent No. 5,455,166 and 5,130,238). In amplification, can use multiple different Polymerase. Hot in nature steady from the representative that thermus aquaticus (Thermus aquaticus) (Taq) separates Decide enzyme and be described in U.S. Patent No. 4,889, in 818, in conventional PCR, use its method to retouch Be set forth in the people such as Saiki, among 1988, the Science 239:487-91. Another kind of representative thermally-stabilised Enzyme comprises Thermus (Thermus) species Z05DNA polymerase. Special referring to for example U.S. Sharp No.5,674,738. Randomly, can use PCR in real time or other quantitative techniques with quantitatively Measure specific nucleotide sequence. The method of quantitative amplification is disclosed in for example U.S. Patent No. 6,180,349; 6,033,854; With 5,972,602, and such as people such as Gibson, Genome Research 6:995-1001 (1996); DeGraves waits the people, Biotechniques 34 (1): 106-10,112-5 (2003); Deiman B waits the people, Mol Biotechnol. 20 (2): among the 163-79 (2002). It is useful especially to follow reverse transcription reaction (RT-PCR), so that can To measure the rna level of one or more genes in the sample. The RT-PCR method is this area The technical staff is known (referring to for example Current Protocols in Molecular Biology People such as (, eds., 2002) Ausubel), and transformed easily and be used for the quantitative amplification method.
Also can use additive method for detection of nucleic acid. For example, can be from sample separation nucleic acid And and Probe Hybridization. In some cases, probe can be connected to (for example little gust of solid carrier Row).
B. protein
Also can be after using alkylamine the protein component of test sample. According to side of the present invention Method can adopt various any protein detection and characterizing method.
Exemplary protein detection method is mass spectrum. Exemplary mass spectrometry method comprises but does not limit Comprise that in electrospray ionization and substance assistant laser desorpted/ionization (MALDI) MALDI flies Line time (MALDI-TOF) method. Referring to for example Karas, M.; Hillencamp, F.Anal. Chem.60:2301 1988); Beavis, R.C.Org.Mass Spec.27:653 (1992); Creel, H.S.Trends Poly.Sci.1 (11): 336 (1993).
Utilizing the alternative of Mass Spectrometer Method is to utilize electrophoresis to separate and to detect subsequently interested albumen Matter. Electrophoresis comprises bidirectional electrophoresis method. The method may comprise randomly that utilization subsequently is anti-Body Western trace detects protein.
Other options comprise immune detection protein. Various ELISA reach and are used for immune detection albumen Other forms of matter are known.
III. kit
The present invention also provides kit, and it can be used for using the above-mentioned method of the present invention. Cause And kit can comprise one or more described reagent herein. Randomly, kit Can comprise for the specification of writing (paper) or electronics that uses them.
In some embodiments, kit of the present invention will comprise alkylamine and at least a Extra reagent for detection of or improve to detect nucleic acid or protein. For example, in some enforcement sides In the case, kit comprises that alkylamine and protease (including but not limited to Proteinase K) are used for the degraded egg White matter and so that nucleic acid more can for detection of. For detection of or improve to detect nucleic acid or protein Other reagent comprise and for example can be used for the reagent that increases. Typical PCR for example Can include but not limited to the upstream and downstream primer as reagent, at least a template, deoxidation Ribonucleotide triphosphate comprise (dATP, dCTP, dGTP, TTP, dUTP), polymerase, Buffer solution, metal cation and salt. The kit that is used for the RT-PCR reaction can also comprise contrary Transcriptase and/or primer. For quantitatively (for example " in real time ") amplification, use one or more many Nucleotide probe is to hybridize to the target of expectation. Probe is typically for example glimmering by detectable label Signal institute mark. Exemplary probe is TaqmanTMProbe can make although be appreciated that With the probe of other types with the target in the reaction of monitoring quantitative amplification. Expansion based on nucleotide sequence Increase (NASBA) reaction and can comprise primer, reverse transcriptase, RNase H and archaeal dna polymerase. The amplification of transcriptive intermediate (TMA) reaction can comprise primer, reverse transcriptase and RNA polymerase. Strand displacement amplification (SDA) reaction can comprise nucleotides and the restriction endonuclease of modification. Some amplified reaction can also comprise that BrdU glycosylase (UNG) is as auxiliary amplifing reagent (Amperase for example
, Roche Molecular Sciences, Alameda, CA) and (ginseng See Kleiboeker, Virol J (2005) 11:29).
Be used to detect or improve other reagent that detect nucleic acid or protein and comprise reagent or the equipment that for example is used for protein purification or nucleic acid, for example described herein.
IV. reaction mixture
The present invention also provides reaction mixture.As described here, exemplary reaction mixture will comprise the sample of formaldehyde fixed, randomly comprise paraffin and alkyl amine.Reaction mixture can comprise the alkyl amine of above-mentioned concentration.Further, reaction mixture is randomly with said temperature.Reaction mixture may randomly further comprise proteinase (for example Proteinase K).
Embodiment
This embodiment has described to utilize alkyl amine to reverse the cross-linking chemistry of nucleic acid.
When having lysine (0.3M), use formalin (formalin buffer solution, 10%, Sigma-Aldrich, HT50-1-1) handle synthetic oligonucleotide (dna sequence dna: AAG TCA GAA GGE AAA[E=5-methyl-dC], SEQ ID NO:1; 3 μ M) or the RNA sequence SEQ ID NO:2: FCC CUC GCA GCC GUC CAA CAA CUC A[F=fluorescein]; 3 μ M) and under 4 ℃, hatched 24 hours.Dynamics by LC-MS analysis monitoring cross-linking chemistry.Product in the MS data suggest reaction mixture is by by methylene bridge and the crosslinked oligonucleotide of lysine, and oligonucleotide-formalin adduct constitutes.As if after 24 hours, all detected oligonucleotides all are crosslinked at reaction mixture.Before ethyleneamines is handled, from the formalin and the lysine of reaction mixture excessive separation.In this reaction mixture (400 μ l), (100 μ l 2.0M), and were at room temperature hatched 1.0 hours to add ethyleneamines.The LC-MS analysis confirmation of sample the quantitative reverse of cross-linking chemistry, from all crosslinked adducts initial oligonucleotide of regenerating.Changing the result that LC-MS analyzes in each step of program is depicted in Fig. 2～5.Other embodiment of crosslinked reverse are depicted in Fig. 6～11, at this moment use synthetic RNA molecule as an example.
The exemplary rules that detect DNA are provided below:
Step 1:
Histotomy
Use Microtom RM2255 to cut out 20 μ histotomies, and section is placed 1.5mL microcentrifugal tube (Eppendorf) or screw top pipe .0.0.
Step 2:
Lytic reagent
EDA is joined lytic reagent, and to reach final concentration be 500mM/225 μ L.Contain to each and to add 200 μ L lytic reagent/EDA in the test tube of sample.
Step 3:
Heating steps
In being arranged on 98 ℃ heat block, hatched each sample 30 minutes.After initial 5 minutes, remove each sample and slight vortex from heat block.After vortex, 20, centrifugal under the 817rcf (Eppendorf 5417C for example, 14,000rpm) 5 seconds, so that all paraffin and tissue enter in the solution.Guarantee not remaining paraffin and tissue on the side of pipe.Get back to heat block and continue residue 25 minutes.
After 25 minutes, remove sample and 20 from heat block, centrifugal under the 817rcf (Eppendorf 5417C for example, 14,000rpm) 5 seconds, so that all paraffin and tissue enter in the solution.At room temperature cooled off each sample 5 minutes.
Step 4:
Cracking+Proteinase K step
Contain to each and to add 20 μ L PK in the test tube of sample.Slight vortex, then 20, centrifugal under the 817rcf (Eppendorf 5417C for example, 14,000rpm) 5 seconds, so that all paraffin and tissue enter in the solution.Guarantee not remaining paraffin and tissue on the side of pipe.
In being arranged on 65 ℃ heat block, hatched each sample 1 hour.Slight vortex, then 20, centrifugal under the 817rcf (Eppendorf 5417C for example, 14,000rpm) 5 seconds, so that all paraffin and tissue enter in the solution.
Step 5:
The Proteinase K inactivation step
In being arranged on 98 ℃ heat block, hatched each lysate sample 10 minutes.
After hatching 10 minutes, fast shift out each sample soon and 20 from the heating that is set in 98 ℃, and 817rcf (Eppendorf 5417C for example, 14,000rpm) centrifugal 20 minutes, to remove fragment from lysate.If lysate is cooled off before centrifugal excessively, then will can not form paraffin and solidify top layer, and paraffin will be moved out of together with lysate.Preferably, paraffin forms the top layer that solidifies.
Step 6:
Centrifugation step is to remove fragment
Differentiate with suitable sample, to new 1.5mL screw top pipe of every kind of sample mark.
Lysate is transferred to the 1.5mL test tube of new mark.Avoid paraffin top layer and the cell fragment in the precipitation to appear at the bottom of test tube.
If sample needs further cleaning, with further centrifugal extra 15 minutes of lysate, 20,817rcf (Eppendorf 5417C for example, 14,000rpm).Lysate is transferred to the 1.5mL test tube of new mark.
Lysate cleaning before the PCR
100 μ L lysates are forwarded in the 1.5mL test tube of new mark.According to QIAquick
The method of PCR purification kit (QIAGEN Sciences).Use the volume elution samples of final 100 μ L.
Present embodiment has described to utilize ethylenediamine to reverse the bak protein cross-linking chemistry.
With bovine serum albumin(BSA) (BSA) protein (100 μ g, 10 μ g/mL) join formalin (65 μ l, buffered formaldehyde liquid, 10%, Sigma-Aldrich, HT50-1-1) in and hatch at 4 ℃.After 14 hours and 36 hours, get the sample (each point is got 25 μ L) of five equilibrium.Then, (25 μ l 2.0M) join in these five equilibriums, and at room temperature hatch 1 hour with ethylenediamine.Then by sds gel analytic sample (Figure 15).(hatched protein 14 hours under 4 ℃ in formalin) shown in the swimming lane 4 of Figure 15, protein-protein cross is completely.The swimming lane 5 of Figure 15 (at room temperature the product of protein-protein cross and ethylenediamine were hatched 1 hour) shows that protein-protein cross chemistry is reversible existing under the ethylenediamine.If yet prolonging crosslinked in formalin, crosslinked meeting not exclusively reverses (the swimming lane 6-8 of Figure 15).
Needing described embodiment of understanding and embodiment herein only is for illustrative purposes, under the instruction of this paper, can hint various changes and variation for those skilled in the art, and be also included within the purport and scope of this application, and within the scope of the appended claims.
Sequence table
<110>Roche?Diagnostics?GmbH，F.Hoffmann-La?Roche?AG
＜120〉alkyl amine improves the detection of the biological sample component of formaldehyde fixed
<130>24201WO
<140>US?Not?yet?assigned
<141>Not?yet?assigned
<150>US?60/920,939
<151>2007-03-30
<150>US?60/954,721
<151>2007-08-08
<160>2
<170>PatentIn?version?3.3
<210>1
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide dna sequence dna
<220>
＜221〉modified base
<222>(12)..(12)
<223>n＝m5c
<400>1
aagtcagaag?gnaaa 15
<210>2
<211>25
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide RNA sequence
<220>
＜221〉modified base
<222>(1)..(1)
＜223〉c that is modified by fluorescein (F)
<400>2
cccucgcagc?cguccaacca?acuca 25
Claims (17)
1. be used to analyze the method for one or more components of the biological sample of formaldehyde crosslinking, this method comprises sample contacted with the alkyl amine of sufficient quantity discharging the crosslinked component of at least a portion, thereby but improve one or more components for the degree of detection of analyzing; Detected components randomly.
2. the process of claim 1 wherein that the amount of alkyl amine is between 0.01% to 5%.
3. the process of claim 1 wherein before detecting step, alkyl amine is gone up from sample substantially removed.
4. the method for claim 3 wherein before detecting step, is reduced to the concentration of alkyl amine less than about 0.5%.
5. the process of claim 1 wherein that described component is a nucleic acid.
6. the method for claim 5 wherein detects step and comprises amplification of nucleic acid.
7. the method for claim 5 wherein under the condition that allows formation probe and nucleic acid, makes the nucleic acid component contact probe, and detects the existence of duplex.
8. the process of claim 1 wherein that component is a protein.
9. the method for claim 8 wherein detects step and comprises mass spectrum or electrophoresis.
10. the process of claim 1 wherein that sample is embedded in the paraffin before contact procedure.
11. the process of claim 1 wherein that alkyl amine is selected from ethylenediamine, monoethanolamine and propylamine.
12. if the process of claim 1 wherein and compare that the part of the component that can be used for analyzing is enhanced at least about twice with not carrying out the part that contact procedure can be used for analyzing.
13. according to the method for claim 1 and 5, described method further comprises sample is contacted with proteinase with the protein of degraded in the sample, thereby makes that nucleic acid more can be used for analyzing.
14. kit, it is used to improve the availability of one or more components of the biological sample of formaldehyde crosslinking, and this kit comprises alkyl amine; With the equipment that is used for removing alkyl amine from biological sample.
15. the kit of claim 14, described kit further comprise nucleotide and/or heat-stabilised poly synthase.
16. reaction mixture, described reaction mixture comprises the biological sample of formaldehyde crosslinking; With the alkyl amine of sufficient quantity to discharge the crosslinked component of at least a portion.
17. the reaction mixture of claim 16, wherein the amount of alkyl amine is between 0.01% to 5%.
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| CN201510970816.1A CN105441549A (en) | 2007-03-30 | 2008-03-26 | Alkyl amines improve detection of components of formaldehyde-fixed biological samples |
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| US92093907P | 2007-03-30 | 2007-03-30 | |
| US60/920,939 | 2007-03-30 | ||
| US60/954,721 | 2007-08-08 |
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| CN201510970816.1A Division CN105441549A (en) | 2007-03-30 | 2008-03-26 | Alkyl amines improve detection of components of formaldehyde-fixed biological samples |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655471A (en) * | 2013-11-15 | 2015-05-27 | 莱卡生物系统努斯洛赫有限责任公司 | Fixing tissue samples using nitrogen-containing compounds that release aldehydes |
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2008
- 2008-03-26 CN CN200880010876A patent/CN101657711A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655471A (en) * | 2013-11-15 | 2015-05-27 | 莱卡生物系统努斯洛赫有限责任公司 | Fixing tissue samples using nitrogen-containing compounds that release aldehydes |
| CN104655471B (en) * | 2013-11-15 | 2018-11-30 | 莱卡生物系统努斯洛赫有限责任公司 | Use the nitrogenous compound fixing organization sample of release aldehydes |
| US10345202B2 (en) | 2013-11-15 | 2019-07-09 | Leica Biosystems Nussloch Gmbh | Fixing tissue samples using nitrogen-containing compounds that release aldehydes |
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