WO2012071985A1 - 从ffpe样本中提取dna的方法及其用途 - Google Patents

从ffpe样本中提取dna的方法及其用途 Download PDF

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WO2012071985A1
WO2012071985A1 PCT/CN2011/082374 CN2011082374W WO2012071985A1 WO 2012071985 A1 WO2012071985 A1 WO 2012071985A1 CN 2011082374 W CN2011082374 W CN 2011082374W WO 2012071985 A1 WO2012071985 A1 WO 2012071985A1
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dna
nucleic acid
ffpe
sample
fragment
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French (fr)
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洪雪玉
刘晓
王冠
张敏红
张秀清
杨焕明
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • This invention relates to the field of genomics, and more particularly to the field of genomics research for FFPE samples, and in particular, to methods for extracting DNA from FFPE samples and uses thereof. More specifically, the present invention provides methods for extracting DNA from FFPE samples, methods for constructing nucleic acid libraries of FFPE samples, nucleic acid libraries for FFPE samples, and methods for determining nucleic acid sequences of FFPE samples. Background technique
  • Tissue samples were stored in the form of FFPE (formalin-fixed paraffin-embedded) samples.
  • FFPE formalin-fixed paraffin-embedded
  • the FFPE method has been used in clinical and scientific fields for a century.
  • a large number of archived FFPE samples provide a valuable resource for retrospective studies, clarifying disease mechanisms, finding therapeutic targets, and indicating prognosis, but paraffin-embedded medical samples are very valuable, each sample is limited and irreplaceable, and organized
  • the sample begins to degrade after being isolated.
  • the fixation of formalin causes different degrees of degradation and intermolecular cross-linking of nucleic acids in the tissue.
  • the high-temperature infiltration process of paraffin further accelerates the degradation of nucleic acids, the time and environment of preservation. It also has a huge impact on the nucleic acid in the sample. Therefore, it is very difficult to study the FFPE sample at this stage.
  • the present invention has been completed based on the following findings of the inventors:
  • FFPE sample DNA extraction kits such as Ambion's Recover All Total Nucleic Acid Isolation Kit, QIAGEN.
  • QIAamp® DNA FFPE Tissue kits etc.
  • the DNA extracted from FFPE samples using these kits or according to the methods used in the kits cannot be effectively used for studies at some molecular levels.
  • due to the particularity of FFPE samples, whether it can be used for exome capture sequencing has not yet been demonstrated, and there is no corresponding method reported.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the inventors have completed the present invention through extensive experiments and studies.
  • Implementation in accordance with the present invention For example, the present invention provides a method of extracting DN ⁇ from a FFPE sample and its use.
  • the invention provides a method of extracting DNA from a FFPE sample.
  • the method comprises the steps of: dewaxing the FFPE sample with a dewaxing agent to obtain a dewaxed sample, wherein the dewaxing agent is selected from the group consisting of xylene and d-limonene At least one; digesting the dewaxed sample with a lysate and a protease to obtain a digested product containing the released DNA; incubating the digested product at 75-95 degrees Celsius 30-60 Minutes; and recovery and purification of the DNA.
  • the method for extracting DNA from FFPE samples according to an embodiment of the present invention can efficiently extract DNA from FFPE samples, and the obtained DNA can be effectively applied to subsequent molecular level studies such as exon capture sequencing studies.
  • the present invention provides a method of constructing a nucleic acid library of a FFPE sample.
  • the method comprises the steps of: extracting DNA from the FFPE sample by using a method of extracting DNA from an FFPE sample according to an embodiment of the present invention; fragmenting the DNA to obtain a DNA fragment; The DNA fragment is subjected to terminal repair and the base A is added at the 3' end to obtain a DNA fragment having a sticky terminal A; the DNA fragment having the sticky terminal A is linked to a linker to obtain a ligation product; The product is subjected to fragment selection to obtain a fragment of interest; and the target fragment is subjected to PCR amplification to obtain an amplification product, which constitutes a nucleic acid library of the FFPE sample.
  • the nucleic acid library of the FFPE sample can be efficiently constructed using the method of constructing the nucleic acid library of the FFPE sample according to an embodiment of the present invention, and the nucleic acid library can be effectively applied to subsequent processing, for example, for high-throughput sequencing platforms or exons Capture sequencing technology.
  • the inventors have found that the above method is simple in process, extremely easy to operate, and the operation flow is easy to standardize and easy to promote.
  • the inventors have surprisingly found that when constructing multiple nucleic acid libraries based on the above methods for the same FFPE sample, the stability of the sequencing data obtained by high-throughput sequencing of each nucleic acid library is stable and repeatable. The method is very good, indicating that the method of constructing the nucleic acid library of the FFPE sample of the embodiment of the present invention is effective and reliable.
  • the invention provides a nucleic acid library of a FFPE sample.
  • the nucleic acid library of the FFPE sample is constructed by the method of constructing a nucleic acid library of the FFPE sample according to an embodiment of the present invention.
  • the nucleic acid library of the FFPE sample according to the embodiment of the present invention can be effectively applied to high-throughput sequencing platforms and exon capture sequencing studies.
  • the invention provides a method of determining a nucleic acid sequence of a FFPE sample.
  • the method comprises the steps of: constructing a nucleic acid library of the FFPE sample according to a method of constructing a nucleic acid library of an FFPE sample according to an embodiment of the present invention; sequencing the nucleic acid library of the FFPE sample And obtaining a sequencing result; and determining a nucleic acid sequence of the FFPE sample based on the sequencing result.
  • the method of determining the nucleic acid sequence of the FFPE sample according to an embodiment of the present invention enables accurate and efficient determination of the nucleic acid sequence of the FFPE sample.
  • FFPE-like samples are determined using a method for determining the nucleic acid sequence of a FFPE sample according to an embodiment of the present invention.
  • the nucleic acid sequence of the present invention can effectively reduce the bias of data output, and the repeatability is very good.
  • FIG. 1 is a schematic flow chart showing a method of extracting DNA from a FFPE sample according to an embodiment of the present invention
  • FIG. 2 is a flow chart showing a method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention
  • the electrophoretic detection result of the DNA extracted from the frozen sample of the human gastric cancer tissue by the QIAGEN tissue DNA extraction kit and the DNA extracted from the FFPE sample of the human gastric cancer tissue according to the embodiment of the present invention
  • Figure 4 shows the results of electrophoretic detection of nucleic acid libraries of frozen samples and FFPE samples constructed by the method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention
  • Figure 5 shows the results of electrophoretic detection of DNA fragments obtained by two kinds of disruption treatments according to an embodiment of the present invention
  • Figure 6 shows the results of electrophoretic detection of two nucleic acid libraries constructed by the method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention and omitting a fragment selection step of constructing a FFPE sample nucleic acid library.
  • the invention provides a method of extracting DNA from a FFPE sample.
  • the method comprises the following steps:
  • the FFPE sample is dewaxed using a dewaxing agent to obtain a dewaxed sample, wherein the dewaxing agent is at least one selected from the group consisting of xylene and d-limonene.
  • the thickness of the FFPE sample is not particularly limited, and according to a specific example, it is preferred that the FFPE sample has a thickness of 2 to 10 ⁇ m.
  • the inventors have found that when the FFPE sample is sliced such that the thickness of the FFPE sample is 2-10 microns, the dewaxing process is easy to perform, the dewaxing effect is very good, and the sample is not damaged, but when the thickness of the FFPE sample is greater than 10 Micron is not conducive to dewaxing, and when it is less than 2 microns, it is easy to make samples. Damaged.
  • the FFPE sample is dewaxed by a dewaxing agent, and may further include a step of removing the dewaxing agent by ethanol washing, wherein the ethanol is preferably anhydrous ethanol, thereby being capable of effectively removing the detachment Wax, easy to follow up.
  • FFPE sample as used herein, sometimes referred to herein as "FFPE sample” means a sample that has been treated with formalin-fixed paraffin embedding, formalin-fixed paraffin-embedded
  • the method can be carried out using methods and means conventional in the art.
  • the procedure for making a FFPE sample can include the following steps: The tissue sample is fixed with 4-10% formaldehyde for 14-20 hours, and after thorough dehydration, it is embedded in paraffin.
  • the dewaxed sample is digested with a lysate and a protease to obtain a digested product, wherein the digested product contains released DNA.
  • the lysate may contain: 10-50 mmol/L Tris-HCl, H 7.4; 100-500 mmo VL NaCl; 5-20 mmol/L EDTA, pH 8.0; and 1% by weight to 2 weight %SDS.
  • the lysate contains: 10 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 10 mmol/L EDTA, pH 8.0; and 1.5% by weight SDS.
  • the dewaxed sample when the dewaxed sample is subjected to cleavage digestion with the above preferred lysate, the dewaxed sample can be sufficiently cleaved without damaging the DNA in the sample.
  • the amount of the lysate used for the lysing and digesting treatment of the dewaxed sample is not particularly limited. According to some specific examples, it is preferred to use 200-500 ⁇ l per 10 mg of the FFPE sample.
  • the lysate more preferably, 300 ⁇ l of the lysate is used per 10 mg of the FFPE sample, whereby the dewaxed sample can be sufficiently cleaved without damaging the DNA in the sample.
  • the protease used for the digestion of the dewaxed sample preferably proteinase K
  • the amount of the protease to be used for the digestion treatment of the dewaxed sample is not particularly limited. According to a specific example, it is preferred to use l-3 mg of protease per 10 mg of the FFPE sample, more preferably every 10 mg of FFPE. The sample was incubated with 1.5 mg of protease. Thereby, the protein in the dewaxed sample can be sufficiently digested without damaging the DNA in the sample, facilitating the release of DNA.
  • the temperature and duration of the digestion treatment of the dewaxed sample are not particularly limited, according to a specific example of the present invention, at 50-60 degrees Celsius (also sometimes referred to herein as "°C")
  • the digestion treatment is carried out for 15-20 hours, preferably 16 hours, and more preferably, the digestion treatment is carried out at 56 degrees Celsius for 16 hours.
  • the inventors have found that when the dewaxed sample is digested for 16 hours at 56 degrees Celsius, the dewaxed sample can be fully cleaved and digested, thereby significantly reducing protein contamination, effectively increasing DNA purity and subsequent involvement of DNA. The efficiency of the reaction.
  • digesting the dewaxed sample with the lysate and the protease may further comprise adding an additional protease during the digestion process, and according to some specific examples, preferably adding an additional 2-4 proteases, the inventor Surprisingly, it has been found that when the above operation is carried out, that is, during the process of protease digestion, the addition of 2-4 proteases can completely digest the protein in the dewaxed sample, thereby effectively increasing the yield of DNA in the FFPE sample. with Purity, and can effectively eliminate the cross-linking of protein and DNA, which is conducive to the subsequent reaction. In addition, the inventors found that the number of proteases added should not be too much, otherwise the obtained DNA would not be suitable for subsequent operations. Thus, in accordance with the present invention, it is most preferred to additionally add 2-4 proteases.
  • the digestion product is incubated at 75-95 ° C for 30-60 minutes.
  • the temperature and time at which the digestion product is incubated are not particularly limited, and it is preferably incubated at 75 to 95 ° C for 30 to 60 minutes, more preferably at 90 ° C for 45 minutes. Thereby, it is possible to help the DNA recovery molecule crosslink, and to allow the digestion product to sufficiently release DNA, and the DNA structure is not easily damaged.
  • the DNA is recovered and purified.
  • the method of recovering purified DNA is not particularly limited, and according to some specific examples of the present invention, a purified PCR kit can be used to recover purified DNA.
  • DNA can be efficiently extracted from FFPE samples, and the obtained DNA can be effectively applied to subsequent molecular level studies on FFPE samples, such as exon capture, Nucleic acid sequencing library construction, high-throughput sequencing, and analysis of exon sequence information analysis.
  • the present invention provides a method of constructing a nucleic acid library of a FFPE sample. According to an embodiment of the invention, referring to Figure 2, the method comprises the following steps:
  • DNA is extracted from FFPE samples by a method of extracting DNA from FFPE samples according to an embodiment of the present invention.
  • the resulting DNA is fragmented to obtain a DNA fragment.
  • the method of fragmenting the obtained DNA is not particularly limited, and according to some specific examples, fragmentation may be carried out by at least one selected from the group consisting of atomization, ultrasonication, HydroShear, and enzymatic treatment.
  • the FFPE sample DNA is fragmented using an ovaris ultrasonic interrupter.
  • the mode of the Ovaris ultrasonic interrupter can be selected as Frequency Sweeping mode, and the interruption parameter is Duty Cycle 10%, Intensity 5, Cycles per Burst 200 A total of 2-3 interruptions, each interrupting time is 60-110 seconds, preferably 75 seconds.
  • the loss of DNA can be reduced, and the desired DNA fragment can be efficiently obtained.
  • the length of the DNA fragment obtained after the fragmentation treatment is 200-300 bp, preferably 250-300 bp, whereby the obtained DNA fragment can be effectively used for the construction and subsequent processing of the nucleic acid library.
  • the DNA fragment was subjected to terminal repair and base A was added at the 3' end to obtain a DNA fragment having a sticky terminal A.
  • the DNA fragment can be end-repaired using Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragment has 5 ' ⁇ 3 'polymerase activity and 3 ' ⁇ 5 'polymerization Enzyme activity, but lacks 5' ⁇ 3' exonuclease activity, whereby the DNA fragment can be efficiently repaired at the end.
  • Klenow (3'-5' exo-), ie Klenow with 3' ⁇ 5' exonuclease activity, may be utilized,
  • the base A is added to the 3' end of the terminal-repaired DNA fragment, whereby a DNA fragment having a sticky terminal A can be efficiently obtained.
  • a DNA fragment having a sticky terminal A is ligated to a linker to obtain a ligation product.
  • a DNA fragment having a sticky terminal A can be ligated to a linker using T4 DNA ligase, whereby the ligation product can be efficiently obtained.
  • the linker may further comprise a label, thereby conveniently constructing a nucleic acid library of a plurality of FFPE samples at the same time, and can be effectively applied to an exon capture technology and a high-throughput sequencing platform, thereby By sequencing, the sequence information of the nucleic acid library, the exon and the tag can be accurately obtained, and based on the sequence information of the tag, the nucleic acid sequence information and the exon sequence information of the plurality of FFPE samples can be accurately distinguished, thereby being able to sufficiently Utilize high-throughput sequencing platforms to save time and reduce sequencing costs.
  • the ligation product is subjected to fragment selection to obtain a fragment of interest.
  • the ligation product can be subjected to fragment selection by 2% agarose electrophoresis, whereby the target fragment can be obtained conveniently and efficiently.
  • the target fragment has a length of 350-400 bp, whereby the obtained target fragment can be effectively applied to subsequent PCR amplification, significantly enhancing amplification efficiency, and improving the nucleic acid library of the constructed FFPE sample.
  • the concentration of the nucleic acid library can be effectively applied to subsequent studies.
  • the resulting fragment of interest is subjected to PCR amplification to obtain an amplification product which constitutes a nucleic acid library of the FFPE sample.
  • the step of purifying the fragment of interest may be further included prior to PCR amplification of the fragment of interest.
  • the method of purifying the fragment of interest is not particularly limited.
  • the fraction of interest is preferably purified using a purification kit, whereby the obtained fragment of interest is very high in purity and easy to be subjected to subsequent treatment.
  • the annealing and extension time and the number of cycles of PCR amplification are not particularly limited.
  • the annealing and extension time of the PCR amplification is 40-60 s, and the number of cycles is 6 -10, more preferably, the annealing and extension time is 45 s, and the number of cycles is 8, whereby the efficiency of PCR amplification can be remarkably improved, and the concentration of the nucleic acid library of the FFPE sample can be effectively increased.
  • the nucleic acid library of the FFPE sample can be efficiently constructed by the method of constructing the nucleic acid library of the FFPE sample according to an embodiment of the present invention, and the nucleic acid library can be effectively applied to subsequent genomics research on the FFPE sample, for example, can be applied to the external display
  • Sub-capture sequencing studies can also be directly applied to high-throughput sequencing platforms to determine the nucleic acid sequence information of FFPE samples, which can be effectively applied to subsequent studies.
  • the method for constructing a nucleic acid library of a FFPE sample according to an embodiment of the present invention is simple in process, easy to standardize in the operation flow, simple in implementation, and easy to generalize.
  • the inventors have surprisingly found that when the nucleic acid library is repeatedly constructed based on the above method for the same FFPE sample, the library construction stability and reproducibility are very good.
  • a method of constructing a nucleic acid library of an FFPE sample may further comprise performing a nucleic acid library of the FFPE sample using at least one selected from the group consisting of solid phase hybridization and liquid phase hybridization techniques.
  • the target sequence is captured to obtain a sequencing library of the target sequence.
  • the above-mentioned solid phase hybridization is performed using a 2.1M exon capture chip of NimbleGen, and the nucleic acid library of the FFPE sample is subjected to target sequence capture, thereby enabling the FFPE sample to be conveniently and efficiently obtained.
  • the exon sequencing library enables high-throughput sequencing of exon sequencing libraries to accurately and efficiently determine exon sequence information for FFPE samples.
  • the invention provides a nucleic acid library of a FFPE sample.
  • the nucleic acid library of the FFPE sample is constructed by a method of constructing a nucleic acid library of a FFPE sample according to an embodiment of the present invention.
  • the nucleic acid library of the FFPE sample according to the embodiment of the present invention can be effectively applied to a high-throughput sequencing platform, and based on the sequencing result, the DNA sequence information of the FFPE sample can be accurately obtained, thereby being effectively applied to subsequent molecular level research. .
  • nucleic acid libraries of FFPE samples can also be effectively applied to capture sequencing studies of target sequences such as exons, in particular, by using solid phase hybridization and liquid phase hybridization.
  • the target sequence of the nucleic acid library of the FFPE sample such as exon capture
  • the sequencing target can accurately determine the target sequence of the FFPE sample, such as an explicit display.
  • the present invention provides a FFPE sample nucleic acid library and a method of constructing the same.
  • One aspect of the present invention provides a method for constructing a FFPE sample nucleic acid library, which comprises the following steps: Step 1 Extraction of FFPE sample nucleic acid;
  • Fragmentation methods include atomization, ultrasonic fragmentation, HydroShear or restriction enzyme digestion to break the nucleic acid into fragments of 200-300 bp in size;
  • Step 3 DNA fragment end repair and 3' end connection base A;
  • a DNA random fragment having a terminally linked base A is ligated to a linker having a known sequence
  • the ligation product is subjected to agarose electrophoresis, and the ligation product having a length of 350-400 bp is purified by gelation as a target fragment; Step 6 PCR amplification
  • Primers were designed according to known linker sequences and subjected to PCR amplification to obtain a FFPE sample nucleic acid library.
  • the extracting of the nucleic acid in step 1 comprises the following steps:
  • the FFPE sample was sliced, and then the slice was immersed in diterpene or d-limonene to remove paraffin. After shaking and mixing, the supernatant was removed by centrifugation; washed with absolute ethanol to remove diphenyl or dextran, and the supernatant was removed by centrifugation.
  • the lysis buffer component 10-50 mmol/L Tris-HCl, H 7.4; 100-500 mmol/LnaCl; 5-20 mmol/LEDTA, H 8.0; 1-2 % weight SDS.
  • the extracting of the nucleic acid in step 1 comprises the following steps:
  • the FFPE sample was sliced to 2-10 microns, and then the slice was immersed in xylene or d-limonene to remove paraffin. After shaking and mixing, the supernatant was removed by centrifugation; washed with absolute ethanol to remove xylene or d-limonene, and removed by centrifugation.
  • lysis buffer composition 10 mmol/LTris-HCl, pH 7.4; 150 mmol/LnaCl; 10 mmol/L EDTA, pH 8.0; 1.5% by weight SDS.
  • the existing FFPE nucleic acid extraction kit provides a method for extracting nucleic acid from a FFPE sample, and the digestion time is 1 hour.
  • the inventors found that due to cross-linking of nucleic acid and protein in the FFPE sample, one-hour digestion treatment cannot be performed. Protein digestion of nucleic acid cross-linking results in protein contamination in the extracted nucleic acid, and the nucleic acid is low in purity, thereby affecting subsequent reactions. Since the purity of the nucleic acid directly affects the efficiency of the downstream molecular reaction, the inventors extended the digestion time to 15-20 hours, thereby effectively increasing the purity of the nucleic acid and the efficiency of subsequent reactions involved.
  • the method provided in the existing FFPE nucleic acid extraction kit adds only proteinase K once during digestion, and the inventors have surprisingly found that, in most cases, only one addition is not sufficient to completely digest the protein, and an excess is added in one time. Proteinase K also failed to achieve the effect of complete digestion, whereas the method of multiple addition of proteinase K according to an embodiment of the present invention enabled complete digestion of the protein.
  • the inventors have found that the fractional addition of proteinase K in the present invention and the use of a longer digestion time can effectively increase the yield and purity of DNA in FFPE samples, and can eliminate the cross-linking of proteins and DNA, thereby facilitating the cross-linking of proteins and DNA.
  • the downstream reaction proceeded smoothly.
  • the fragmentation described in step 2 uses an ultrasonic method, and the ultrasound is used with low intensity and multiple interruptions.
  • ultrasonic interrupters have the choice of low-intensity ultrasound and high-intensity ultrasound. For example, if you use the Covaris ultrasonic interrupter to interrupt, you can select the mode Frequency Sweeping mode, the interrupt parameter is set to Duty Cycle 10%, Intensity 5, Cycles per Burst 200, interrupted 2-3 times, each interrupt time is 60 -110 seconds, preferably 75 seconds.
  • the present invention controls the intensity of the ultrasound to minimize the length of the fragment to be 250-300 bp, thereby reducing DNA loss, thereby facilitating library construction; and adding a step to a length of 350-400 bp of the ligation product before the PCR amplification, that is, the purpose
  • the fragment is subjected to a step of purifying the gel, because the tangent of the ligation product is acceptable for many common samples.
  • the fragmented DNA after the disruption described in step 2 includes enzymes including, but not limited to, T4 DNA polymerase, Klenow fragment, and T4 polynucleotide kinase. End-repair, a blunt-ended random DNA fragment, and then ligated at the 3' end of a blunt-ended DNA random fragment, including but not limited to Klenow (3 '-5' exo-) enzyme Base A.
  • the PCR amplification according to step 6 has an annealing and extension time of 40-60 seconds and a cycle number of 6-10.
  • the annealing time of the PCR is generally 10-30 seconds
  • the extension time is 30 seconds per 500 bp, and these times are used during the database construction process of the FFPE sample nucleic acid library.
  • the amplification efficiency of the library is low, and the inventors speculate that the efficiency of the enzyme reaction may be lowered due to the sample.
  • the annealing and extension time are extended to 40-60 seconds, preferably 45 seconds, thereby enabling Significantly improve the success rate of building a library, and obtain a library of suitable size and concentration.
  • the PCR amplification is carried out in step 6, and the annealing and extension time is 45 seconds.
  • a further aspect of the invention provides a nucleic acid library of a FFPE sample constructed by a method of constructing a nucleic acid library of a FFPE sample according to an embodiment of the invention.
  • the invention provides a method of determining a nucleic acid sequence of a FFPE sample. According to an embodiment of the invention, the method comprises the following steps:
  • a nucleic acid library of a FFPE sample is constructed using a method of constructing a nucleic acid library of a FFPE sample according to an embodiment of the present invention.
  • the nucleic acid library of the obtained FFPE sample was sequenced to obtain sequencing results.
  • the method of sequencing the nucleic acid library of the obtained FFPE sample is not particularly limited, and according to a specific example, it is preferable to perform sequencing using a high-throughput sequencing technique, and more preferably, to use the Solexa sequencing technique for the FFPE sample.
  • the nucleic acid library was sequenced.
  • the nucleic acid sequence of the FFPE sample is determined.
  • the nucleic acid sequence of the FFPE sample can be accurately and efficiently determined using the method of determining the nucleic acid sequence of the FFPE sample according to an embodiment of the present invention.
  • the inventors have surprisingly found that the method of determining the nucleic acid sequence of a FFPE sample according to a method for determining a nucleic acid sequence of an FFPE sample according to an embodiment of the present invention can effectively reduce the bias of data production, and the repeatability is very good.
  • the FFPE-like sample can be determined according to the following steps. Sequence information of the target sequence of the present invention: First, a nucleic acid library of the FFPE sample is constructed by the method of constructing a nucleic acid library of the FFPE sample according to an embodiment of the present invention; secondly, at least one selected from the group consisting of solid phase hybridization and liquid phase hybridization techniques is utilized.
  • target sequence refers to a DNA sequence derived from the ORF sequence of the FFPE sample, the kind of which is not particularly limited, and includes, but is not limited to, an exon sequence. According to some specific examples of the present invention, the exon sequence information in the FFPE sample can be efficiently determined using the above method.
  • the present invention establishes a method for analyzing nucleic acid sequencing techniques and target sequence capture techniques for analyzing FFPE samples.
  • the method further comprises the steps of capturing a target sequence in a nucleic acid library of the FFPE sample and sequencing the captured target sequence.
  • the types of target sequences include, but are in no way limited to, exon sequences.
  • the target sequence capture referred to in the present invention refers to a technique of capturing a target sequence by a solid phase chip or liquid phase hybridization or the like.
  • the above operations can be performed by a commercially available method or means, for example, NimbleGen's Sequence Capture Micro arrays and Agilent's SureSelect Target Enrichment System can be used.
  • an exome sequencing technique is generated. Exon sequencing is an efficient technique for the selective determination of protein coding regions in the human genome to find new genes associated with rare or common diseases. (Sarah B Ng, et al, (2010) Exome sequencing identifies the cause of a mendelian disorder.
  • sequencing can be performed by any sequencing method, including but not limited to dideoxy chain termination High-throughput sequencing methods, including but not limited to second-generation sequencing techniques or single-molecule sequencing techniques.
  • the second generation sequencing technology (Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet.
  • the FFPE sample was cut into 5 microns thick each, and 10 pieces were placed in an Eppendorf tube (sometimes referred to herein as "EP tube”).
  • Eppendorf tube sometimes referred to herein as "EP tube”.
  • the frozen sample of human gastric cancer tissue was used as a control, and DNA was extracted from the frozen sample of human gastric cancer tissue by using the QIAGEN tissue DNA extraction kit and the method described by the manufacturer.
  • Fig. 3 shows the results of electrophoretic detection of DNA extracted from frozen samples of human gastric cancer tissues using the QIAGEN tissue DNA extraction kit and DNA extracted from FFPE samples of human gastric cancer tissues according to an embodiment of the present invention.
  • D2000 and ⁇ ⁇ ⁇ are molecular weight Marker
  • Frozen is the DNA lane of the frozen sample
  • FFPE is the DNA lane of the FFPE sample.
  • the DNA of the FFPE sample is degraded relative to the DNA extracted from the frozen sample, and the inventors speculated that the degradation was caused by the fabrication process of the FFPE sample. Comparing the lane glue holes of the FFPE sample with the frozen sample, it was found that the protein removal in the DNA of the FFPE sample was relatively complete, indicating that the method of extracting DNA from the FFPE sample according to the embodiment of the present invention, the deproteinization and decrosslinking reactions were sufficient.
  • Example 2 Construction of a FFPE sample nucleic acid library
  • the nucleic acid library of the FFPE sample was constructed according to the following procedure. And using the DNA of the frozen sample obtained in Example 1 as a control:
  • DNA fragmentation The DNA was fragmented using a Covaris ultrasonic interrupter. The size of the interrupt was 250-300 bp, and the parameters were Duty Cycle 10%, Intensity 5, Cycles per Burst 200, and two interrupts. The time was interrupted for 75 s each time to obtain a DNA fragment, and then the DNA fragment was purified using Ampure magnetic beads.
  • the 100 microliter end-repair system is a DNA fragment obtained by 77.4 microliters of the previous step, 10 microliters of lOx polynucleotide kinase buffer, 1.6 microliters of a 25 mM dNTP mixture, and 5 microliters of T4 DNA polymerase. 1 microliter of Klenow fragment and 5 microliters of T4 polynucleotide kinase,
  • the 50 microliter base-added system comprises: 5 microliters of lOxBlue buffer, 2 microliters of 5 mM dATP, 3 microliters of Klenow (3'-5' exo-), and 40 microliters of the previous step. The resulting end-repaired DNA fragment.
  • Linker The system of the adaptor was placed in Thermocycles and incubated overnight at 16 ° C to connect the DNA fragment having the sticky end A to the linker to obtain a ligation product, which was then purified using magnetic beads. The elution volume is 5 (H liter.
  • the linker here is the label PE Adapter), and the label on the tag linker is used to mix the nucleic acid library of the FFPE sample and the nucleic acid library of the frozen sample for exon. During the capture and sequencing process, two libraries were distinguished.
  • the Multiplexing Sample Preparation Oligonucleotide Kit contains: 5 ⁇ l of 10xT4 DNA ligase buffer ( Paired -End DNA Sample Prep Kit, IP- 102-1001, illumina ), 3 x L 40-Tole Binary Enzyme Kit (PE-400-1001, Illumina), 5 ⁇ l T4 DNA Ligase (Paired-End) DNA Sample Prep Kit, IP-102-1001, Illumina) and 37 ⁇ l of the DNA fragment with sticky tip A obtained in the previous step.
  • 10xT4 DNA ligase buffer Paired -End DNA Sample Prep Kit, IP- 102-1001, illumina
  • 3 x L 40-Tole Binary Enzyme Kit PE-400-1001, Illumina
  • 5 ⁇ l T4 DNA Ligase Pieraired-End
  • Fragment selection The junction product was selected by 2% agarose electrophoresis, and the length of the gel was obtained.
  • a 350-400 bp fragment of interest was then purified using a QIAGEN gel purification kit with an elution volume of 10 CH liters.
  • PCR amplification and purification of the amplified product The PCR reaction system is configured, and then the PCR reaction system is subjected to PCR amplification to obtain an amplification product, wherein the PCR program is 94 ° C for 5 min; 8 cycles of 94 ° C for 30 s. , 62 °C 45s, 72 °C 45s; 72 °C 10min.
  • the PCR reaction system is: 5 ⁇ l of Pfx buffer, 13.6 ⁇ l of ddH 2 0, 2 ⁇ l of 10 mM dNTP, 2 ⁇ l of 50 mM MgS0 4 , 2 ⁇ l of 10 ⁇ M upstream and downstream primers (Multiplexing) Sample Preparation Oligonucleotide Kit, PE-400-1001, Illumina), 0.4 ⁇ l of Pfx polymerase and 23 ⁇ l of the desired fragment from the previous step.
  • the amplified product was subjected to magnetic bead purification with an elution volume of 50 ⁇ l.
  • Fig. 4 shows the results of electrophoresis detection of a frozen sample and a nucleic acid library of a FFPE sample constructed by the method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention.
  • D2000 and ⁇ ⁇ ⁇ are molecular weights Marker
  • Frozen is the lane of the nucleic acid library of the frozen sample
  • FFPE is the lane of the nucleic acid library of the FFPE sample.
  • the FFPE sample nucleic acid library constructed by the method for constructing the FFPE sample nucleic acid library according to the embodiment of the present invention was not significantly different from the frozen sample nucleic acid library, and the library size and concentration were similar.
  • the DNA of the FFPE sample obtained in Example 1 was subjected to low-intensity and high-strength disruption treatment using a Covaris ultrasonic interrupter (Covaris, USA;).
  • the low-intensity treatment conditions are: Duty Cycle 10%, Intensity 5, Cycles per Burst 200, interrupted twice, and the interruption time is 110s and 80s respectively.
  • the high-intensity treatment conditions were: Duty Cycle 20%, Intensity 5, Cycles per Burst 200, interrupted 3 times, and the interruption times were 95s, 50s and 45s respectively.
  • Fig. 5 shows the results of electrophoretic detection of DNA fragments obtained by two kinds of disruption treatments according to an embodiment of the present invention.
  • D2000 and 50bp are molecular weight Marker
  • lane 1 is low intensity treatment (ie, Duty Cycle 10%, Intensity 5, Cycles per Burst 200, 2 interruptions, and the interruption time are 110s and 80s respectively).
  • the DNA fragment, Lane 2 was obtained by high-intensity treatment (Duty Cycle 20%, Intensity 5, Cycles per Burst 200, 3 interruptions, interruption time 95s, 50s and 45s, respectively).
  • the DNA fragment of lane 1 is relatively concentrated at the position of 250-300 bp, and there are many DNA fragments in lane 2 at the position of 750 bp, indicating that the Covaris ultrasonic interrupter is used for low-intensity treatment (ie, Duty Cycle 10%).
  • Intensity 5 Cycles per Burst 200 interrupted 2 times, interrupt time is 110s and 80s respectively, the DNA of the obtained FFPE sample is fragmented, and the desired DNA of 250-300 bp in length can be effectively obtained. Fragments; ⁇ using high-intensity treatment, the number of DNA fragments obtained from 250-300 bp in length is small, which cannot meet the needs of nucleic acid library construction.
  • Example 4 The importance of FFPE-like acid during the construction process
  • a method for constructing a nucleic acid library of a FFPE sample according to an embodiment of the present invention omitting a fragment selection step, directly performing PCR amplification of the ligated product, and recovering and purifying the amplified product to construct a FFPE sample nucleic acid library, as the FFPE constructed in Example 2 A control of the nucleic acid library of the sample.
  • FIG. 6 shows the results of electrophoretic detection of two nucleic acid libraries constructed using the method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention and the method of constructing a FFPE sample nucleic acid library omitting the fragment selection step. As shown in Fig.
  • D2000 is a marker
  • lane 1 is a nucleic acid library constructed by a method of constructing a FFPE sample nucleic acid library omitting a fragment selection step
  • lane 2 is constructed by a method of constructing a FFPE sample nucleic acid library according to an embodiment of the present invention. Nucleic acid library.
  • the step of selecting the ligated product that is, the step of selecting the fragment
  • the step of selecting the fragment is not necessary for the library construction of many common samples, but it is very important for the construction of the FFPE sample nucleic acid library, and the step of omitting the fragment selection is due to the PCR enzyme.
  • the small fragment is mainly amplified, and the amplification fragment of the target fragment, that is, the DNA fragment of 250-300 bp in length is low.
  • Example 5 Construction of an exome sequencing library of FFPE samples
  • the exon sequencing library of the FFPE sample was constructed by the following procedure using the nucleic acid library of the FFPE sample obtained in Example 2:
  • the nucleic acid library of the frozen sample and the nucleic acid library of the FFPE sample were each taken to 1.5 ⁇ g, respectively, with 400 ⁇ g of Cot-l DNA and 0.6 nmol of the closed linker sequence 1 (Hybridization Enhancing) and to the closed linker.
  • Sequence 2 Multixing Sample Preparation Oligonucleotide Kit, PE-400-1001, Illumina was mixed, then placed in SpeedVac at about 60 ° C for about 1 h, and evaporated to dryness.
  • Hybridization was carried out in accordance with NimbleGen Arrays User's Guide, Version 3.1, 7 Jul 2009, Roche NimbleGen, Inc. Among them, the sample was loaded with 35 ⁇ l, hybridized at 42 ° C for 64-72 h, then eluted with 90 CH 160 160 mM NaOH, and the eluted product was purified by QIAGEN MinElute PCR purification kit, and then 80 ⁇ m. The elution buffer is eluted to obtain an exon sequence fragment.
  • the PCR reaction system was configured as follows: 150 ⁇ l of Phusion Mix (Phusion Mix, F-531L, NEB), 4.2 liters of each of the upstream and downstream primers (multiplexing Sequencing primers and phix control kit, PE-400-1002, IUumina ), 8 (H is the same as the exon sequence fragment obtained in the previous step and 85: liter ddH 2 0 is mixed and packed into 6 tubes.
  • the 6 tubes PCR reaction system is simultaneously subjected to PCR amplification, wherein the number of cycles is 16 Among them, the amplification products were mixed and purified by Ampure magnetic beads, and the elution volume was 5 (H liter).
  • the nucleic acid library obtained in Example 2 and the exon sequencing library obtained in the present example were diluted to 1 ng/ ⁇ l, respectively, and the final volume was required to be >12 ⁇ l, according to on-chip 4
  • Example 6 Solexa sequencing
  • the FFPE sample and the frozen sample exon sequencing library obtained in Example 5 were tested and the library yield was obtained using an Agilent 2100 Bioanalyzer and Q-PCR. After the test was completed, the library was diluted to the corresponding concentration, and the bridge was bridged in the Cluster Station. PCR, each template in the library is clustered on the Flow cell, and sequenced according to the method of Hiuqia HiSeq2000 (HiSeq 2000 User Guide. Catalog # SY-940-1001 Part # 15011190 Rev B , IUumina ) The data of the sequencing quality control file are shown in Table 1 below.
  • Percentage of readings that can be mapped to the genome is 16558821 (85.7295%) 21150344 (88.1711%) Percentage of readings uniquely mapped to the genome 15875628 (82.2073%) 20286149 (84.5684%) Percentage of readings targeted to the target area 10354297 (53.6167%) 12996532 (54.1796%) Target area coverage mode 15.0000 21.0000 Target area average coverage 18.6100 23.3300
  • the FFPE sample used for exon sequencing has less reading, data yield and coverage of the target area than the frozen sample, and the uniformity of exon capture is slightly lower than that of the frozen sample.
  • the reading ratio of the reference sequence is similar to that of the frozen sample, and the target area coverage % of at least 1 reading is also very similar.
  • SNP single nucleotide polymorphism
  • embodiments of the present invention successfully established techniques for enriching exons of FFPE samples and performing Solexa sequencing, indicating that FFPE samples can be effectively applied to exon capture sequencing.
  • the method for extracting DNA from FFPE samples, the method for constructing a nucleic acid library of FFPE samples, the nucleic acid library of FFPE samples, and the method for determining nucleic acid sequences of FFPE samples of the present invention can be applied to high-throughput sequencing platforms and target sequences of FFPE samples. Capture sequencing technology can be effectively applied to in-depth genomics studies of FFPE samples.

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Abstract

提供了从FFPE样本中提取DNA的方法、构建FFPE样本的核酸文库的方法、FFPE样本的核酸文库以及确定FFPE样本的核酸序列的方法。其中，从FFPE样本中提取DNA的方法包括以下步骤：利用脱蜡剂将FFPE样本进行脱蜡，以便获得脱蜡的样本，其中，该脱蜡剂为选自二甲苯和右旋柠檬烯的至少一种；利用裂解液和蛋白酶对脱蜡的样本进行消化处理，以便获得消化产物，其中消化产物中含有释放的DNA；将消化产物在75-95摄氏度下孵育30-60分钟；以及回收纯化所得的DNA。

Description

从 FFPE样本中提取 DNA的方法及其用途 优先权信息
本申请请求 2010 年 12 月 2 日向中国国家知识产权局提交的、 专利申请号为 201010571180.0的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域
本发明涉及基因组学领域， 特别是涉及 FFPE样本的基因组学研究领域， 具体地， 本发明涉及从 FFPE样本中提取 DNA的方法及其用途。更具体地，本发明提供了从 FFPE 样本中提取 DNA的方法、 构建 FFPE样本的核酸文库的方法、 FFPE样本的核酸文库以及 确定 FFPE样本的核酸序列的方法。 背景技术
组织样品多釆用 FFPE ( formalin-fixed paraffin-embedded, 福尔马林固定石蜡包埋） 样品的形式进行保存。 FFPE方法在临床和科研领域的应用已有一个世纪。 数量巨大的 归档 FFPE样本为回顾性研究， 阐明疾病机制， 发现治疗靶标和指示预后提供宝贵的资 源， 但石蜡包埋的医疗样本十分珍贵， 每个样本总量都很有限且不可替代， 而且组织样 品在离体后就开始发生降解，福尔马林的固定会使组织中的核酸发生不同程度的降解和 分子间的交联， 石蜡的高温渗入过程进一步加速核酸的降解，保存的时间及环境对样品 中的核酸也有巨大的影响， 因此， 现阶段对 FFPE样本的研究难度很大。
由此， 目前对 FFPE样本的基因组学研究方法仍有待改进。
发明内容
本发明是基于发明人的下列发现而完成的： 目前，有较多商品化的 FFPE样本 DNA 提取试剂盒，如 Ambion公司的全回收总核酸分离试剂盒（ Recover All Total Nucleic Acid Isolation Kit ) , QIAGEN公司的 QIAamp® DNA FFPE Tissue试剂盒等， 但利用这些试 剂盒或者按照试剂盒所釆用的操作方法从 FFPE样本中提取的 DNA, 不能有效地用于 某些分子水平的研究。 此外， 由于 FFPE样本的特殊性， 其可否用于外显子捕获测序目 前尚未有的论证， 并且也没有相应方法的报道。
本发明旨在至少解决现有技术中存在的技术问题之一。 由此， 为了从有限的且容易 发生核酸降解的 FFPE样本中成功提取 DNA,并利用外显子捕获测序技术对 FFPE样本 进行基因组学研究， 发明人经过大量的实验和研究， 完成了本发明。 根据本发明的实施 例， 本发明提供了从 FFPE样本中提取 DN Α的方法及其用途。
根据本发明的一个方面， 本发明提供了一种从 FFPE样本中提取 DNA的方法。 根据 本发明的实施例， 该方法包括以下步骤： 利用脱蜡剂将所述 FFPE样本进行脱蜡， 以便获得 脱蜡的样本， 其中， 所述脱蜡剂为选自二甲苯和右旋柠檬烯的至少一种； 利用裂解液和蛋 白酶对所述脱蜡的样本进行消化处理， 以便获得消化产物， 所述消化产物中含有释放的 DNA; 将所述消化产物在 75-95摄氏度下孵育 30-60分钟； 以及回收纯化所述 DNA。 利用 根据本发明实施例的从 FFPE样本中提取 DNA的方法， 能够有效地从 FFPE样本中提取 DNA, 并且获得的 DNA能够有效地应用于后续的分子水平的研究如外显子捕获测序研究。
根据本发明的再一方面， 本发明提供了一种构建 FFPE样本的核酸文库的方法。根据本 发明的实施例， 该方法包括以下步骤： 利用根据本发明实施例的从 FFPE样本中提取 DNA 的方法， 从所述 FFPE样本提取 DNA; 将所述 DNA进行片段化， 以便获得 DNA片段； 将 所述 DNA片段进行末端修复及 3'末端添加碱基 A,以便获得具有粘性末端 A的 DNA片段； 将所述具有粘性末端 A的 DNA片段与接头相连， 以便获得连接产物； 将所述连接产物进行 片段选择， 以便获得目的片段； 以及将所述目的片段进行 PCR扩增， 以便获得扩增产物， 所述扩增产物构成所述 FFPE样本的核酸文库。 利用根据本发明实施例的构建 FFPE样本的 核酸文库的方法， 能够有效地构建 FFPE样本的核酸文库， 并且该核酸文库能够有效地应用 于后续处理， 例如用于高通量测序平台或外显子捕获测序技术。 另外， 发明人发现， 上述 方法过程简单， 极易操作， 操作流程易标准化， 易于推广。 除此之外， 发明人还惊奇地发 现， 当针对相同的 FFPE样本， 基于上述方法构建多个核酸文库时， 对各个核酸文库进行高 通量测序所得到的测序数据结果的稳定性和可重复性非常好， 表明本发明实施例的构建 FFPE样本的核酸文库的方法有效可靠。
根据本发明的又一方面， 本发明提供了一种 FFPE样本的核酸文库。根据本发明的实施 例， 该 FFPE样本的核酸文库是通过才艮据本发明实施例的构建 FFPE样本的核酸文库的方法 构建的。根据本发明实施例的 FFPE样本的核酸文库， 能够有效地应用于高通量测序平台及 外显子捕获测序研究。
根据本发明的另一方面， 本发明提供了一种确定 FFPE样本的核酸序列的方法。根据本 发明的实施例， 该方法包括下列步骤： 利用才艮据本发明实施例的构建 FFPE样本的核酸文库 的方法， 构建所述 FFPE样本的核酸文库； 将所述 FFPE样本的核酸文库进行测序， 以便获 得测序结果； 以及基于所述测序结果， 确定所述 FFPE样本的核酸序列。 利用才艮据本发明实 施例的确定 FFPE样本的核酸序列的方法， 能够准确有效地确定 FFPE样本的核酸序列。 另 夕卜， 发明人发现， 釆用根据本发明实施例的确定 FFPE样本的核酸序列的方法确定 FFPE样 本的核酸序列， 能够有效地减少数据产出的偏向性， 可重复性非常好。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：
图 1 : 显示了根据本发明实施例的从 FFPE样本中提取 DNA的方法的流程示意图； 图 2: 显示了 #居本发明实施例的构建 FFPE样本核酸文库的方法的流程示意图； 图 3: 显示了釆用 QIAGEN组织 DNA提取试剂盒从人胃癌旁组织的冷冻样品中提取 的 DNA以及根据本发明实施例的从人胃癌旁组织的 FFPE样本中提取的 DNA的电泳检测 结果；
图 4: 显示了根据本发明实施例的构建 FFPE样本核酸文库的方法构建的冷冻样本及 FFPE样本的核酸文库的电泳检测结果；
图 5:显示了根据本发明实施例的经过两种打断处理所得的 DNA片段的电泳检测结果； 以及
图 6： 显示了利用根据本发明实施例的构建 FFPE样本核酸文库的方法及省略片段选择 步骤的构建 FFPE样本核酸文库的方法构建的两种核酸文库的电泳检测结果。 发明详细描述
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。
从 FFPE样本中提取 DNA的方法
根据本发明的一个方面， 本发明提供了一种从 FFPE样本中提取 DNA的方法。 根据 本发明的实施例， 参考图 1 , 该方法包括以下步骤：
首先， 利用脱蜡剂将 FFPE样本进行脱蜡， 以便获得脱蜡的样本， 其中， 该脱蜡剂为选 自二甲苯和右旋柠檬烯的至少一种。根据本发明的实施例， FFPE样本的厚度不受特别限制， 根据具体示例， 优选 FFPE样本的厚度为 2-10微米。 发明人发现， 当将 FFPE样本切片， 使 得 FFPE样本的厚度为 2-10微米时， 使得脱蜡处理易于进行， 脱蜡效果非常好， 且样本不 会受损， 但当 FFPE样本的厚度大于 10微米时， 不利于脱蜡， 小于 2微米时则容易使样本 受损。 根据本发明的一些具体示例， 利用脱蜡剂将所述 FFPE样本进行脱蜡， 可以进一步包 括通过乙醇洗涤去除脱蜡剂的步骤， 其中该乙醇优选无水乙醇， 由此， 能够有效地去除脱 蜡剂， 易于后续处理。 在本文中所使用的术语 "FFPE样品"， 有时在本文中也称为 "FFPE 样本"，含义都是通过福尔马林固定石蜡包埋进行处理的样品，福尔马林固定石蜡包埋的 方法可以釆用本领域中常规的方法和手段进行。 例如， FFPE样品的制作程序可以包括下 列步骤： 将组织样品用 4-10%甲醛固定 14-20小时， 通过彻底脱水后， 利用石蜡包埋。
其次， 利用裂解液和蛋白酶对脱蜡的样本进行消化处理， 以便获得消化产物， 其中该 消化产物中含有释放的 DNA。 根据本发明的实施例， 该裂解液可以含有： 10-50 mmol/L Tris-HCl, H 7.4； 100-500 mmoVL NaCl; 5-20 mmol/L EDTA, pH 8.0； 以及 1重量％-2 重量％SDS。才艮据本发明的具体示例， 优选地， 该裂解液含有： 10mmol/L Tris-HCl, pH 7.4； 150mmol/L NaCl ； lOmmol/L EDTA, pH 8.0； 以及 1.5重量％ SDS。 发明人惊奇地发现， 当釆用上述优选的裂解液对脱蜡的样本进行裂解消化处理时， 能够使脱蜡的样本充分裂解， 且不会损坏样本中的 DNA。 根据本发明的实施例， 对脱蜡的样本进行裂解消化处理所釆用 的裂解液的量不受特别限制， 根据一些具体示例， 优选每 10mg FFPE样品釆用 200-500微 升前面所述的裂解液， 更优选地， 每 10mg FFPE样品釆用 300微升该裂解液， 由此， 能够 在不损坏样本中的 DNA的情况下， 使脱蜡的样本充分裂解。
根据本发明的一些实施例， 对脱蜡的样本进行消化处理所釆用的蛋白酶， 优选为蛋白 酶 K。 根据本发明的实施例， 对脱蜡的样本进行消化处理所釆用的蛋白酶的量不受特别限 制， 根据具体示例， 优选每 10mg FFPE样品釆用 l-3mg蛋白酶， 更优选地， 每 10mg FFPE 样品釆用 1.5mg蛋白酶。 由此， 能够在不损坏样本中的 DNA的情况下， 使脱蜡的样本中的 蛋白质被充分消化， 便于 DNA的释放。
根据本发明的实施例， 对脱蜡的样本进行消化处理的温度和持续时间不受特别限制， 根据本发明的具体示例， 在 50-60摄氏度（在本文中有时也表示为 " °C " ) 下进行消化处理 15-20小时， 优选 16小时， 更优选地， 在 56摄氏度下进行消化处理 16小时。 发明人发现， 当在 56摄氏度下对脱蜡的样本进行消化处理 16小时时， 能够使脱蜡的样本充分裂解和消 化， 从而能够显著减少蛋白污染， 有效地提高 DNA的纯度以及 DNA参与的后续反应的效 率。
根据本发明的实施例， 利用裂解液和蛋白酶对脱蜡的样本进行消化处理， 可以进一步 包括在消化处理过程中添加额外的蛋白酶，根据一些具体示例，优选额外添加 2-4次蛋白酶， 发明人惊奇地发现， 当釆用上述操作， 即在蛋白酶消化处理的过程中， 添加 2-4次蛋白酶， 能够使脱蜡的样本中的蛋白质消化完全， 从而能够有效地提高 FFPE样本中 DNA的产率和 纯度， 并能够有效解除蛋白质与 DNA的交联， 有利于后续反应的进行， 另外，发明人发现， 添加蛋白酶的次数不宜过多， 否则会使得所得到的 DNA不适合后续操作。 因而， 根据本发 明， 最优选釆用额外添加 2-4次蛋白酶。
接下来， 将消化产物在 75-95摄氏度下孵育 30-60分钟。 根据本发明的实施例， 将消化 产物进行孵育的温度和时间不受特别限制， 优选在 75-95摄氏度下孵育 30-60分钟， 更优选 地， 在 90摄氏度下孵育 45分钟。 由此， 能够帮助 DNA恢复分子交联， 并使消化产物充分 地释放 DNA, 且 DNA结构不易受损。
然后， 回收纯化该 DNA。 根据本发明的实施例， 回收纯化 DNA的方法不受特别限制， 根据本发明的一些具体示例， 可以利用商品化的纯化试剂盒来回收纯化 DNA。
利用根据本发明实施例的从 FFPE样本中提取 DNA的方法， 能够有效地从 FFPE样本 中提取 DNA, 并且所得 DNA能够有效地应用于后续的对 FFPE样本的分子水平研究， 例如 外显子捕获、 核酸测序文库构建、 高通量测序以及外显子序列信息分析的研究。
FFPE样本的核酸 L^及其构建方法
根据本发明的再一方面， 本发明提供了一种构建 FFPE样本的核酸文库的方法。根据本 发明的实施例， 参考图 2, 该方法包括以下步骤：
首先， 利用根据本发明实施例的从 FFPE样本中提取 DNA的方法， 从 FFPE样本提取 DNA。
其次， 将所得的 DNA进行片段化， 以便获得 DNA片段。 根据本发明的实施例， 将所 得的 DNA进行片段化的方法不受特别限制， 根据一些具体示例， 可以通过选自雾化、 超声 打断法、 HydroShear以及酶切处理的至少一种进行片段化， 优选使用 ovaris 超声打断仪将 FFPE样本 DNA片段化。根据本发明的一些实施例，使用 ovaris 超声打断仪进行片段化时， 可以将 Ovaris超声打断仪的模式选择为 Frequency Sweeping mode, 打断参数为 Duty Cycle 10% , Intensity 5, Cycles per Burst 200, 共打断 2-3次， 每次打断时间为 60-110秒， 优选 75 秒。 由此， 能够减少 DNA的损失， 有效地获得所需的 DNA片段。 根据本发明的实施例， 片段化处理后所得的 DNA片段的长度为 200-300bp, 优选 250-300bp, 由此， 获得的 DNA 片段能够有效地用于核酸文库的构建及后续处理。
接着， 将 DNA片段进行末端修复及 3'末端添加碱基 A, 以便获得具有粘性末端 A的 DNA片段。 根据本发明的实施例， 可以利用 Klenow片段、 T4 DNA聚合酶和 T4多核苷酸 激酶将 DNA片段进行末端修复， 其中， 该 Klenow片段具有 5 '→ 3 '聚合酶活性和 3 '→ 5 '聚 合酶活性， 但缺少 5'→3'外切酶活性， 由此， 能够有效地将 DNA片段进行末端修复。 根据 本发明的一些具体示例，可以利用 Klenow (3'-5' exo-)，即具有 3'→5'外切酶活性的 Klenow, 将经过末端修复的 DNA片段的 3'末端添加碱基 A, 由此， 能够有效地获得具有粘性末端 A 的 DNA片段。
接下来， 将具有粘性末端 A的 DNA片段与接头相连， 以便获得连接产物。根据本发明 的实施例， 可以利用 T4 DNA连接酶将具有粘性末端 A的 DNA片段与接头相连， 由此， 能 够有效地获得连接产物。 根据本发明的实施例， 该接头中可以进一步包含标签， 由此可以 方便地同时构建多个 FFPE样本的核酸文库，并能够有效地应用于外显子捕获技术及高通量 测序平台， 从而通过测序， 能够准确地获得核酸文库、 外显子及标签的序列信息， 基于标 签的序列信息， 就能够准确地区分多个 FFPE样本的核酸序列信息以及外显子序列信息， 由 此， 能够充分地利用高通量测序平台， 有效地节省时间、 降低测序成本。
然后， 将连接产物进行片段选择， 以便获得目的片段。 根据本发明的实施例， 可以利 用 2%琼脂糖电泳对连接产物进行片段选择， 由此， 能够方便有效地获得目的片段。 根据本 发明的一些具体示例， 目的片段的长度为 350-400bp, 由此， 所得的目的片段能够有效地应 用于后续的 PCR扩增， 显著增强扩增效率， 提高所构建的 FFPE样本的核酸文库的浓度， 从而核酸文库能够有效应用于后续研究。
最后， 将所得的目的片段进行 PCR扩增， 以便获得扩增产物， 该扩增产物构成 FFPE 样本的核酸文库。 根据本发明一些实施例， 将目的片段进行 PCR扩增前， 可以进一步包括 纯化目的片段的步骤。 根据本发明的实施例， 纯化目的片段的方法不受特别限制， 根据具 体示例， 优选利用纯化试剂盒将目的片段进行纯化， 由此， 获得的目的片段纯度非常高， 易于进行后续处理。 根据本发明的实施例， PCR扩增的退火及延伸时间和循环数不受特别 限制， 根据本发明的一些实具体示例， 优选 PCR扩增的退火及延伸时间为 40-60s, 循环数 为 6-10个， 更优选地， 退火及延伸时间为 45s, 循环数为 8个， 由此， 能够显著地提高 PCR 扩增的效率， 从而能够有效地提高 FFPE样本的核酸文库的浓度。
利用根据本发明实施例的构建 FFPE样本的核酸文库的方法， 能够有效地构建 FFPE样 本的核酸文库， 并且该核酸文库能够有效地应用于对 FFPE样本的后续基因组学研究， 例如 能够应用于外显子捕获测序研究， 也能够直接应用于高通量测序平台， 以确定 FFPE样本的 核酸序列信息， 从而能够有效地应用于后续研究。 而且， 发明人发现， 根据本发明实施例 的构建 FFPE样本的核酸文库的方法过程简单， 操作流程易标准化， 实施简便， 易于推广。 此外， 发明人惊奇地发现， 当针对相同的 FFPE样本， 基于上述方法重复构建核酸文库时， 文库构建稳定性和可重复性非常好。
根据本发明的实施例，根据本发明实施例的构建 FFPE样本的核酸文库的方法， 可以进 一步包括利用选自固相杂交和液相杂交技术的至少一种，对所述 FFPE样本的核酸文库进行 目标序列捕获， 以便获得所述目标序列的测序文库。 根据本发明的具体示例， 优选地， 使 用 NimbleGen的 2.1M外显子捕获芯片进行上述固相杂交， 对所述 FFPE样本的核酸文库进 行目标序列捕获， 由此， 能够方便有效地获得 FFPE样本的外显子测序文库， 从而通过对外 显子测序文库进行高通量测序， 能够准确有效地确定 FFPE样本的外显子序列信息。
根据本发明的又一方面， 本发明提供了一种 FFPE样本的核酸文库。根据本发明的实施 例， 该 FFPE样本的核酸文库是通过根据本发明实施例的构建 FFPE样本的核酸文库的方法 构建的。 根据本发明实施例的 FFPE样本的核酸文库， 能够有效地应用于高通量测序平台， 基于测序结果， 能够准确地获得 FFPE样本的 DNA序列信息， 从而能够有效地应用于后续 的分子水平的研究。 此外， 发明人惊奇地发现， 根据本发明实施例的 FFPE样本的核酸文库 还能够有效地应用于目标序列如外显子的捕获测序研究， 具体地， 通过利用选自固相杂交 和液相杂交技术的至少一种， 对 FFPE样本的核酸文库进行目标序列如外显子捕获， 能够方 便有效地获得该目标序列如外显子， 通过测序技术， 能够准确地确定 FFPE样本的目标序列 如外显子序列的信息。 进一步， 根据本发明的实施例， 本发明提供了一种 FFPE样本核酸文库及其构建方法。 本发明的一个方面提供了一种 FFPE样本核酸文库的构建方法， 其包括以下的步骤： 步骤一 FFPE样本核酸的提取；
步骤二核酸片段化
片段化的方法包括雾化、 超声片段化、 HydroShear或酶切处理， 从而将核酸打断为大 小为 200 - 300bp的片段；
步骤三 DNA片段末端修复及 3'端连接碱基 A;
步骤四 接头的连接
将末端连接碱基 A的 DNA随机片段末端连接序列已知的接头；
步骤五 片段选择
将连接产物进行琼脂糖电泳， 切胶纯化长度为 350-400bp的连接产物， 作为目的片段； 步骤六 PCR扩增
根据已知的接头序列设计引物， 进行 PCR扩增， 即得到 FFPE样本核酸文库。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中，步骤一所述核酸的提取 包括以下步骤：
将 FFPE样本切片， 然后将切片浸于二曱苯或右旋柠檬烯中以去除石蜡， 振荡混勾后离 心去除上清； 用无水乙醇洗涤以去除二曱苯或右旋柠檬烯， 离心去除上清； 每 10mg组织加 200-500 升加裂解緩冲液和 l-3mg蛋白酶 K,在消化期间添加 2-4次蛋白酶 K, 50-60 °C消 化 15-20小时； 消化后 75-95 °C孵育 30-60分钟； 对所得 DNA进行纯化； 其中， 所述裂解緩 冲液成分： 10-50 mmol/L Tris-HCl, H 7.4； 100-500 mmol/LnaCl; 5-20 mmol/LEDTA, H 8.0； 1-2 %重量 SDS。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中，步骤一所述核酸的提取 包括以下步骤：
将 FFPE样本切片为 2-10微米， 然后将切片浸于二甲苯或右旋柠檬烯中去除石蜡， 振 荡混匀后离心去除上清； 用无水乙醇洗涤以去除二甲苯或右旋柠檬烯， 离心去除上清； 每 10mg组织加 300 升加裂解緩冲液和 1.5mg蛋白酶 K, 在消化期间添加 2-4次蛋白酶 K, 56°C消化 16小时； 消化后 90 °C孵育 45分钟； 所得 DNA进行纯化； 所述裂解緩冲液成分： 10mmol/LTris-HCl, pH 7.4； 150mmol/LnaCl; 10mmol/L EDTA, pH 8.0； 1.5%重量 SDS。
现有的 FFPE核酸提取试剂盒提供的从 FFPE样本中提取核酸的方法， 其消化时间为 1 小时， 但发明人发现， 由于 FFPE样本中核酸与蛋白发生交联， 1小时的消化处理无法使与 核酸交联的蛋白消化， 导致提取的核酸中有蛋白污染， 核酸纯度低， 从而影响后续的反应。 因为核酸纯度的高低直接影响下游分子反应的效率， 由此发明人将消化时间延长至 15-20 小时， 从而能够有效地提高核酸的纯度及其参与的后续反应的效率。
现有的 FFPE核酸提取试剂盒中提供的方法在消化时只加一次蛋白酶 K,发明人惊奇地 发现， 在大部分情况下， 只加一次不足以使蛋白消化完全， 而在一次中加过量的蛋白酶 K, 也不能达到完全消化的效果， 而根据本发明实施例的多次添加蛋白酶 K的方法， 能够使蛋 白消化完全。
发明人发现， 本发明釆取的分次添加蛋白酶 K, 并釆用较长的消化时间， 能够有效提 高 FFPE样本的 DNA的产率和纯度， 并能够解除蛋白质与 DNA的交联， 从而有利于下游 反应的顺利进行。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中，步骤二所述片段化使用 超声的方法， 超声使用低强度， 多次打断。 一般常用的超声打断仪都会有低强度超声和高 强度超声的选择。 例如使用 Covaris 超声打断仪打断， 可以选择模式 Frequency Sweeping mode, 打断参数设置为 Duty Cycle 10% , Intensity 5 , Cycles per Burst 200, 共打断 2-3次， 每次打断时间为 60-110秒， 优选 75秒。
本发明通过控制超声强度使打断片段长度尽量集中在 250-300bp,以减少 DNA的损失， 从而利于文库构建； 并且在进行 PCR扩增前增加一步将长度为 350-400bp的连接产物， 即 目的片段进行切胶纯化的步骤， 因为对连接产物进行切胶处理， 对许多普通样品来说是可 有可无的步骤， 但是对 FFPE样品而言， 如果省略这一步直接进行 PCR扩增， 由于 PCR酶 主要扩增小片段， 则会导致对目的片段的扩增效率降低。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中，步骤二所述打断后的片 段化 DNA在包括但不限于 T4 DNA聚合酶、 Klenow片段和 T4多聚核苷酸激酶等酶的作用 下， 进行末端修复， 形成平末端的 DNA随机片段， 然后在包括但不限于 Klenow (3 '-5' exo-) 酶的作用下， 在平末端化的 DNA随机片段的 3'末端连接碱基 A。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中， 步骤六所述的 PCR扩 增， 其退火及延伸时间为 40-60秒， 循环数为 6-10个。 发明人发现， 在普通样品建库流程 的相应 PCR步骤中， PCR的退火时间一般为 10-30秒，延伸时间为每 500bp 30秒，而在 FFPE 样品核酸文库的建库过程中使用这些时间会使文库扩增成功率较低， 发明人推测可能是由 于样品的原因导致酶反应效率降低， 根据本发明的实施例， 退火及延伸时间均延长至 40-60 秒， 优选为 45秒， 从而能够显著提高建库成功率， 得到大小和浓度合适的文库。
本发明提供的 FFPE样本核酸文库的构建方法的一个实施例中， 步骤六所述 PCR扩增， 其退火及延伸时间为 45秒。
本发明的又一方面提供了一种 FFPE样本的核酸文库，其是通过根据本发明实施例的构 建 FFPE样本的核酸文库的方法构建的。
确定 FFPE样本的核酸序列的方法
根据本发明的另一方面， 本发明提供了一种确定 FFPE样本的核酸序列的方法。根据本 发明的实施例， 该方法包括下列步骤：
首先， 利用根据本发明实施例的构建 FFPE样本的核酸文库的方法， 构建 FFPE样本的 核酸文库。
其次， 将所得的 FFPE样本的核酸文库进行测序， 以便获得测序结果。 根据本发明的实 施例， 将所得的 FFPE样本的核酸文库进行测序的方法不受特别限制， 根据具体示例， 优选 利用高通量测序技术进行测序， 更优选地， 利用 Solexa测序技术对 FFPE样本的核酸文库 进行测序。
然后， 基于测序结果， 确定 FFPE样本的核酸序列。
利用根据本发明实施例的确定 FFPE样本的核酸序列的方法，能够准确有效地确定 FFPE 样本的核酸序列。发明人惊奇地发现， 釆用根据本发明实施例的确定 FFPE样本的核酸序列 的方法确定 FFPE样本的核酸序列， 能够有效地减少数据产出的偏向性， 可重复性非常好。
发明人惊奇地发现，上述确定 FFPE样本的核酸序列的方法还可以进一步用于确定 FFPE 样本中目标序列的序列信息的研究，根据本发明的具体示例， 可按照以下步骤确定 FFPE样 本中目标序列的序列信息： 首先， 利用根据本发明实施例的构建 FFPE样本的核酸文库的方 法， 构建 FFPE样本的核酸文库； 其次， 利用选自固相杂交和液相杂交技术的至少一种， 对 FFPE样本的核酸文库进行目标序列捕获， 以便获得目标序列的测序文库； 将目标序列的测 序文库进行测序， 以便获得测序结果； 以及基于测序结果， 确定 FFPE样本中目标序列的序 列信息。 本领域的技术人员可以理解， 这里所使用的术语 "目标序列"是指来源于 FFPE样 本基因组序列的 DNA序列， 其种类不受特别限制， 包括但不限于外显子序列。 根据本发明 的一些具体示例， 釆用上述方法能够有效地确定 FFPE样本中的外显子序列信息。
进一步， 根据本发明的实施例， 本发明建立了将核酸测序技术和目标序列捕获技术用 于分析 FFPE样本的方法。
本发明提供的分析 FFPE样品的方法的一个实施例中， 还包括捕获 FFPE样品核酸文库 中的目标序列， 并对所捕获的目标序列进行测序的步骤。 目标序列的种类包括但绝不限于 外显子序列。
本发明所指目标序列捕获是指利用固相芯片或液相杂交等方法对目标序列进行捕获的 技术。 可以利用商品化的方法或者手段进行上述操作， 例如， 可以使用 NimbleGen的序列 捕获芯片 ( Sequence Capture Micro arrays )和 Agilent液相杂交序列捕获技术( SureSelect Target Enrichment System )。 当序列捕获的目标为外显子， 并进一步结合测序技术时， 产生了外显 子测序技术。 外显子测序是一种选择性地测定人类基因组中蛋白编码区的高效技术， 以此 找到与罕见或普通疾病相关的新基因。（ Sarah B Ng,等人， (2010) Exome sequencing identifies the cause of a mendelian disorder. Nature Genetics 42, 30 - 35. )其中， 所述测序可通过任何测 序方法进行， 包括但不限于双脱氧链终止法； 优选高通量的测序方法， 包括但不限于第二 代测序技术或者是单分子测序技术。 所述第二代测序技术 （ Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010 Jan;ll(l):31-46 ) 包括但不限于 Solexa、 Solid和 454 (焦磷酸测序）测序技术（平台）； 所述单分子测序技术（单分子测序平台） 包 括但不限于 Helicos公司的 True Single Molecule DNA sequencing技术, Pacific Biosciences 公司的 the single molecule, real-time (SMRT.TM.) 技术, 以及 Oxford Nanopore Technologies 公司的纳米孔测序技术等（Rusk, Nicole (2009-04-01). Cheap Third-Generation Sequencing. Nature Methods 6 (4): 244-245 )。
需要说明的是， 根据本发明实施例的从 FFPE样本中提取 DNA的方法及其用途， 是本 申请的发明人经过艰苦的创造性劳动和优化工作完成的。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社）或者按照产品说明书进行。 所用试剂或仪器未注 明生产厂商者， 均为可以通过市购获得的常规产品， 例如可以釆购自 Illumina公司。 实施例 1: 人胃癌旁组织 FFPE样品 DNA提取
按照下列步骤从人胃癌旁组织 FFPE样品中提取 DNA, 并且釆用人胃癌旁组织冷冻样 品作为对照：
将 FFPE样品切成每片 5微米厚，取 10片置于 Eppendorf管（在本文中有时也称为 "EP 管"）中备用； 向 EP管中加入 1毫升二甲苯， 振荡混匀后用最大速度离心 3min, 去除上清， 以便对 FFPE样本进行脱蜡处理； 向 EP管中加入 1毫升无水乙醇， 振荡混匀后最大速度离 心 3min, 去除上清， 于室温下挥发剩余乙醇， 以便洗涤去除二甲苯； 向 EP管中加入 180 微升裂解緩冲液和 20微升 20mg/ml蛋白酶 K, 于 50°C下进行消化 16小时， 其中在消化期 间向 EP管中添加三次 20mg/ml的蛋白酶 K, 每次添加 20微升， 以便获得消化产物， 其中 消化产物中包含释放的 DNA；将所得的消化产物于 80°C下孵育 lOmin, 以便逆转福尔马林 交联；利用 0.6倍体积的 Ampure磁珠纯化 DNA,并去除小片段降解的 DNA,以便获得 FFPE 样本的 DNA, 备用。
以人胃癌旁组织的冷冻样品为对照， 釆用 QIAGEN组织 DNA提取试剂盒， 参照厂家 说明的方法， 从人胃癌旁组织的冷冻样品中提取 DNA, 备用。
利用琼脂糖凝胶电泳检测釆用 QIAGEN组织 DNA提取试剂盒从人胃癌旁组织的冷冻 样品中提取的 DNA以及根据本发明实施例的从人胃癌旁组织的 FFPE样本中提取的 DNA。 图 3显示了釆用 QIAGEN组织 DNA提取试剂盒从人胃癌旁组织的冷冻样品中提取的 DNA 以及根据本发明实施例的从人胃癌旁组织的 FFPE样本中提取的 DNA的电泳检测结果。 如 图 3所示， D2000与 λ Ηή ΠΙΙ均为分子量 Marker, Frozen为冷冻样本的 DNA泳道， FFPE 为 FFPE样本的 DNA泳道。
由图 3可知，相对于从冷冻样品中提取的 DNA而言， FFPE样品的 DNA存在降解现象， 发明人推测降解是由 FFPE样品的制作过程造成的。 将 FFPE样品的泳道胶孔与冷冻样品的 进行对比， 可知 FFPE样品的 DNA中蛋白去除较完全， 表明根据本发明实施例的从 FFPE 样本中提取 DNA的方法， 去蛋白及去交联反应充分。
实施例 2: FFPE样本核酸文库的构建
利用实施例 1所得的 FFPE样本的 DNA, 按照下列步骤构建 FFPE样本的核酸文库， 并且釆用实施例 1所得的冷冻样本的 DNA作为对照：
(1) DNA 片段化： 釆用 Covaris 超声打断仪将 DNA进行片段化， 打断目的大小为 250-300bp, 打断参数为 Duty Cycle 10% , Intensity 5 , Cycles per Burst 200, 共打断两次， 每 次打断时间为 75s , 以便获得 DNA片段， 然后用 Ampure磁珠将 DNA片段进行纯化。
(2) 末端修复： 配置 100 升的末端修复体系， 将末端修复体系置于 Thermocycles中于
20°C下孵育 30 min, 以便获得经过末端修复的 DNA片段， 然后利用 Ampure磁珠将其进行 纯化。 其中， 该 100微升的末端修复体系是通过 77.4微升上一步所得的 DNA片段， 10微 升 lOx多核苷酸激酶緩冲液， 1.6微升 25mM的 dNTP混合物， 5微升 T4 DNA聚合酶， 1 微升 Klenow片段以及 5微升 T4多核苷酸激酶，
(3) 添加碱基 A: 配置 50 升的添加碱基 A 的体系， 将添加碱基 A 的体系置于
Thermocycles中于 37 °C下孵育 30 min, 以便获得具有粘性末端 A的 DNA片段， 然后利用 磁珠将其进行纯化。 其中， 该 50微升的添加碱基 A的体系包含： 5微升 lOxBlue緩冲液， 2 微升 5 mM的 dATP, 3微升 Klenow (3'-5' exo-)以及 40微升上一步所得的经过末端修复的 DNA片段。
(4) 连接接头： 将连接接头的体系置于 Thermocycles中于 16°C下孵育过夜， 以便将具 有粘性末端 A的 DNA片段与接头相连， 获得连接产物， 然后利用磁珠将其进行纯化， 其中 洗脱体积为 5(H敫升。 这里的接头为标签接头 （Index PE Adapter ), 标签接头上的标签是用 于在后续的将 FFPE样本的核酸文库和冷冻样本的核酸文库混合进行外显子捕获及测序过 程中， 区分两个文库。 其中， 该 50 ^：升接头连接体系 （Multiplexing Sample Preparation Oligonucleotide Kit , PE-400-1002 , Illumina ) 包含： 5 微升 10xT4 DNA 连接酶緩冲液 ( Paired-End DNA Sample Prep Kit, IP- 102-1001 , illumina ), 3 微升 40 摩标签接头 ( Multiplexing Sample Preparation Oligonucleotide Kit, PE-400-1001 , Illumina ), 5微升 T4 DNA连接酶（Paired-End DNA Sample Prep Kit, IP- 102-1001 , Illumina ) 以及 37微升上一 步所得的具有粘性末端 A的 DNA片段。
(5) 片段选择： 利用 2%琼脂糖电泳将连接产物进行片段选择， 切胶获得长度为
350-400bp的目的片段， 然后利用 QIAGEN切胶纯化试剂盒将目的片段进行纯化，其中洗脱 体积为 10CH敫升。
(6) PCR扩增及纯化扩增产物： 配置 PCR反应体系， 然后将 PCR反应体系进行 PCR扩 增， 以便获得扩增产物， 其中 PCR程序为 94°C 5min; 8个循环的 94 °C 30s, 62 °C 45s, 72 °C 45s; 72 °C 10min。 其中 PCR反应体系为： 5微升 Pfx緩冲液， 13.6微升 ddH20, 2微 升 10mM的 dNTP, 2微升 50mM的 MgS04, 各 2微升 10微摩的上下游引物 (Multiplexing Sample Preparation Oligonucleotide Kit, PE-400-1001 , Illumina ), 0.4微升 Pfx聚合酶以及 23 微升上一步所得的目的片段。
然后， 将扩增产物进行磁珠纯化， 洗脱体积为 50微升。
利用琼脂糖凝胶电泳对 FFPE样本的核酸文库和冷冻样本的核酸文库进行检测。图 4显 示了根据本发明实施例的构建 FFPE样本核酸文库的方法构建的冷冻样本及 FFPE样本的核 酸文库的电泳检测结果。 如图 4所示， D2000与 λ Ηή ΠΙΙ均为分子量 Marker, Frozen为 冷冻样本的核酸文库的泳道， FFPE为 FFPE样本的核酸文库的泳道。
由图 4可知， 利用根据本发明实施例的构建 FFPE样本核酸文库的方法所建的 FFPE样 品核酸文库与冷冻样品核酸文库， 没有显著差别， 文库大小和浓度相近。
实施例 3: 打断条件对 FFPE样品的影响
使用 Covaris 超声打断仪 ( Covaris, 美国；)， 分别将实施例 1所得 FFPE样本的 DNA进 行低强度和高强度的打断处理。 其中， 低强度的处理条件为： Duty Cycle 10% , Intensity 5 , Cycles per Burst 200,共打断 2次，打断时间分别为 110s和 80s。 高强度的处理条件为： Duty Cycle 20% , Intensity 5 , Cycles per Burst 200,共打断 3次，打断时间分别为 95s、 50s和 45s。
利用琼脂糖凝胶电泳对经过两种打断处理所得的 DNA片段进行检测。 图 5显示了根据 本发明实施例的经过两种打断处理所得的 DNA片段的电泳检测结果。 如图 5所示， D2000 和 50bp为分子量 Marker,泳道 1为低强度处理（即 Duty Cycle 10% , Intensity 5 , Cycles per Burst 200下共打断 2次， 打断时间分别为 110s和 80s ) 所得的 DNA片段， 泳道 2 为高强度处理 （ Duty Cycle 20% , Intensity 5 , Cycles per Burst 200下共打断 3次， 打断 时间分别为 95s、 50s和 45s ) 所得的 DNA片段。
由图 5可知， 泳道 1的 DNA片段相对集中在 250-300bp位置， 而泳道 2中还有许多 DNA片段处于 750bp的位置，表明使用 Covaris 超声打断仪，在低强度处理（即 Duty Cycle 10% , Intensity 5 , Cycles per Burst 200下共打断 2次， 打断时间分别为 110s和 80s )下， 将所得 FFPE样本的 DNA进行片段化， 能够有效地获得所需的长度为 250-300bp的 DNA 片段； 釆用高强度处理， 获得的长度为 250-300bp的 DNA片段较少， 无法满足核酸文库构 建的需要。
实施例 4: FFPE样 酸 构建过程中片 ¾1择步躁的重要性
将根据本发明实施例的构建 FFPE样本的核酸文库的方法， 省略片段选择步骤， 直接将 连接产物进行 PCR扩增及扩增产物回收纯化而构建的 FFPE样本核酸文库， 作为实施例 2 构建的 FFPE样本的核酸文库的对照。
利用琼脂糖凝胶电泳对利用根据本发明实施例的构建 FFPE样本核酸文库的方法及省 略片段选择步骤的构建 FFPE样本核酸文库的方法构建的两种核酸文库进行检测。图 6显示 了利用根据本发明实施例的构建 FFPE 样本核酸文库的方法及省略片段选择步骤的构建 FFPE样本核酸文库的方法构建的两种核酸文库的电泳检测结果。 如图 6所示， D2000 为 marker, 泳道 1 为利用省略片段选择步骤的构建 FFPE样本核酸文库的方法构建的核酸文 库， 泳道 2为利用根据本发明实施例的构建 FFPE样本核酸文库的方法构建的核酸文库。
由图 6可知， 对连接产物进行切胶处理， 即片段选择的步骤， 对于许多普通样品的文 库构建并非必须， 但是对于 FFPE样品核酸文库的构建却非常重要， 省略片段选择的步骤会 由于 PCR酶主要扩增小片段，而导致目的片段即长度为 250-300bp的 DNA片段的扩增效率 较低。
实施例 5: FFPE样本的外显子测序文库的构建
以实施例 2所得的冷冻样本的核酸文库为对照，利用实施例 2所得的 FFPE样本的核酸 文库， 按照下列步骤构建 FFPE样本的外显子测序文库：
(1) 将杂交仪（ 12-Bay Hybridization Systems, Nimblegen, #05223695001 ) 电源打开， 预热 3h以上，使得杂交仪温度稳定至 42 °C。将 2个干浴器打开，分别将其调至 70°C和 95 °C。
(2)根据 NanoDrop定量，将冷冻样本的核酸文库及 FFPE样本的核酸文库各取 1.5微克, 分别与 400微克的 Cot-l DNA及 0.6nmol 的封闭接头序列 1 ( Hybridization Enhancing )和 去于闭接头序列 2( Multiplexing Sample Preparation Oligonucleotide Kit, PE-400-1001 , Illumina ) 混合， 然后置于 SpeedVac中于 60摄氏度下 lh左右， 蒸干备用。
(3) 向蒸干的样品中加入 11.24敫升纯水， 震荡混匀后置于离心机上全速离心 3-30秒。 将离心后的样品转移至 70°C的干浴器中放置 10分钟， 使 DNA充分溶解。
(4) 将上一步所得的样品取出， 震荡后置于离心机上全速离心 30秒， 然后分别加入以 下两种试剂：
2xSC 杂交緩冲液（SC Hybridiation Buffer, 来自序列捕获杂交试剂盒， Nimblegen, 5340721001 ) 18.5微升
SC杂交组合物 A( SC Hybridiation Component A ,来自序列捕获杂交试剂盒， Nimblegen ,
5340721001 ) 7.3微升
(5) 将上一步所得的样品震荡混匀后置于离心机上全速离心 3-30 秒。 将离心后的样品 转移至 95 °C干浴器（美国 Labnet AccuBlock, 型号 D1100-230V ) 中放置 10分钟， 使 DNA 变性。
(6) 将样品取出， 震荡后置于离心机上全速离心 30 秒， 置于杂交仪器 （ 12-Bay
Hybridization Systems, Nimblegen, #05223695001 ) 的离心管放置位置上， 于 42 °C下准备杂 交。
(7) 参照 NimbleGen公司的芯片杂交方法（ NimbleGen Arrays User 's Guide, Version 3.1, 7 Jul 2009, Roche NimbleGen, Inc. )进行杂交。 其中， 样品上样量为 35微升， 于 42°C下杂交 64-72h, 然后用 90CH敫升 160mM NaOH洗脱， 用 QIAGEN MinElute PCR纯化试剂盒将洗脱 产物进行纯化， 然后再用 80微升洗脱緩冲液进行洗脱， 以便获得外显子序列片段。
(8) 配置 PCR反应体系， 利用 PCR反应体系将上一步所得的外显子序列片段进行 PCR 扩增， 以便获得扩增产物， 然后将扩增产物混合并用 Ampure磁珠进行纯化， 该扩增产物构 成外显子测序文库。 其中， 按照下列步骤配置 PCR反应体系： 将 150微升 Phusion混合物 (Phusion Mix, F-531L, NEB) ,各 4.2 升的上下游引物 （multiplexing Sequencing primers and phix control kit, PE-400-1002, IUumina) , 8(H敫升上一步所得的外显子序列片段以及 85 : 升 ddH20混合分装成 6管即可。将 6管 PCR反应体系分别同时进行 PCR扩增， 其中循环数 为 16。 其中， 将扩增产物混合并用 Ampure磁珠进行纯化后， 洗脱体积为 5(H敫升。
(9)根据 Nanodrop定量检测浓度， 将实施例 2所得的核酸文库与本实施例所得的外显 子测序文库分别稀释至 1纳克 /微升， 要求终体积 >12微升， 根据芯片上 4个探针的扩增引 物进行 Q-PCR计算外显子富集度， 富集度 =(ef) A Ct, 其中 ef为每对扩增引物的扩增效率， △ Q为捕获前与捕获后文库 Q值之差。 由计算结果可知， 本实施例的外显子测序文库对外 显子序列的平均富集度大于 60。 实施例 6: Solexa测序
使用 Agilent 2100 Bioanalyzer和 Q-PCR,对实施例 5所得的 FFPE样本和冷冻样本的外 显子测序文库进行检测和文库产量， 检测合格后， 将文库稀释到相应浓度后， 在 Cluster Station中进行桥式 PCR, 使文库中的各个模板成簇固定在 Flow cell上， 参照 IUumina公司 HiSeq2000的操作方法（ HiSeq 2000 User Guide. Catalog # SY-940-1001 Part # 15011190 Rev B , IUumina )进行测序， 所得的测序质控文件数据见下表 1。
表 1 胃癌旁组织冷冻样本及 FFPE样本的外显子测序文库上机测序质控数据 样品 FFPE样品 冷冻样品
总读数 19311700 23987852
数据产量 (Mb) 1419.41 1763.11
可定位至基因组的读数百分率 16555821(85.7295%) 21150344(88.1711%) 唯一定位至基因组的读数百分率 15875628(82.2073%) 20286149(84.5684%) 定位至目标区域的读数百分率 10354297(53.6167%) 12996532(54.1796%) 目标区域覆盖度众数 15.0000 21.0000 目标区域平均覆盖度 18.6100 23.3300
目标区域覆盖度差异 368.5900 316.3100
标准偏差 19.2000 17.7900
至少 1个读数的目标区域覆盖率％ 31728305(93.3054%) 32248910(94.8363%) 至少 10个读数的目标区域覆盖率％ 22279624(65.5191%) 25212015(74.1425%) 至少 20个读数的目标区域覆盖率％ 13464513(39.5959%) 17839217(52.4609%) 至少 40个读数的目标区域覆盖率％ 3020310(8.8820%) 5997159(17.6362%) 少于 5个读数的目标区域覆盖率％ 6866654(20.1932%) 4989829(14.6739%) 总有效读数 (M) 15.88 20.29
总有效产率 (Mb) 1165.91 1490.43
平均读长 (bp) 73.44 73.48
外显子组中的平均读长 (bp) 73.45 73.47
可定位至外显子组区域的有效读数百 65.22% 64.07%
分率
外显子组区域的错配率 0.2901% 0.2805%
有效数据的总错配率 0.2885% 0.2766%
外显子捕获均一性 42.35% 45.50%
从表 1可知， 相对于冷冻样本， FFPE样本用于外显子测序的读数、 数据产量及目标区 域的覆盖度较少， 且外显子捕获的均一性稍低于冷冻样本， 而可定位到参考序列的读数比 例与冷冻样本的相近， 至少 1个读数的目标区域覆盖率％也很相近。
另外， 发明人经过实验证明， 在单碱基测序深度达到 20x时， 胃癌旁组织的冷冻样本及 FFPE样本的单核苷酸多态性（SNP )位点的差异并不显著， 发明人推测， 这些不显著的差 异出现可能是由 FFPE样品中 DNA的损伤造成的， 具体表现为嘌呤与嘌呤之间及嘧啶与嘧 淀之间的转换。
由此， 本发明的实施例成功地建立了富集 FFPE样本的外显子并进行 Solexa测序的技 术， 表明 FFPE样本能够有效地应用于外显子捕获测序。 工业实用性
本发明的从 FFPE样本中提取 DNA的方法、 构建 FFPE样本的核酸文库的方法、 FFPE 样本的核酸文库以及确定 FFPE样本的核酸序列的方法，能够应用于高通量测序平台及 FFPE 样本的目的序列捕获测序技术， 进而能够有效地应用于对 FFPE样本的深入的基因组学研 究。
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims

权利要求书
1、 一种从 FFPE样本中提取 DNA的方法， 其特征在于， 包括以下步骤：
利用脱蜡剂将所述 FFPE样本进行脱蜡， 以便获得脱蜡的样本， 其中， 所述脱蜡剂为选 自二甲苯和右旋柠檬烯的至少一种；
利用裂解液和蛋白酶对所述脱蜡的样本进行消化处理， 以便获得消化产物， 所述消化 产物中含有释放的 DNA;
将所述消化产物在 75-95摄氏度下孵育 30-60分钟； 以及
回收纯化所述 DNA。
2、 根据权利要求 1所述的方法， 其特征在于， 所述 FFPE样本的厚度为 2-10微米。
3、 根据权利要求 1所述的方法， 其特征在于， 利用脱蜡剂将所述 FFPE样本进行脱蜡 进一步包括通过无水乙醇洗涤去除所述脱蜡剂的步骤。
4、 根据权利要求 1所述的方法， 其特征在于， 所述裂解液含有：
10-50 mmol/L Tris-HCl, H 7.4；
100-500 mmol/L NaCl, ;
5-20 mmol/L EDTA, pH 8.0； 以及
1重量％-2重量％ SDS。
5、 根据权利要求 4所述的方法， 其特征在于， 所述裂解液含有：
10mmol/L Tris-HCl, pH 7.4；
150mmol/L NaCl ；
lOmmol/L EDTA, pH 8.0； 以及
1.5重量％ SDS。
6、 根据权利要求 1所述的方法， 其特征在于， 每 10mg FFPE样品釆用 200-500微升所 述裂解液， 优选 300 升所述裂解液。
7、 根据权利要求 1所述的方法， 其特征在于， 所述蛋白酶为蛋白酶 K。
8、根据权利要求 1所述的方法， 其特征在于， 每 10mg FFPE样品釆用 l-3mg所述蛋白 酶， 优选 1.5mg所述蛋白酶。
9、 根据权利要求 1所述的方法， 其特征在于， 利用裂解液和蛋白酶对所述脱蜡的样本 进行消化处理， 进一步包括在所述消化处理过程中添加额外的蛋白酶， 优选额外添加 2-4 次蛋白酶。
10、 根据权利要求 1所述的方法， 其特征在于， 在 50-60摄氏度下进行所述消化处理
15-20小时， 优选 16小时。
11、 根据权利要求 10所述的方法， 其特征在于， 在 56摄氏度下进行所述消化处理 16 小时。
12、 根据权利要求 1所述的方法， 其特征在于， 将所述消化产物在 90摄氏度下孵育 45 分钟。
13、 一种构建 FFPE样本的核酸文库的方法， 其特征在于， 包括以下步骤：
利用根据权利要求 1-12任一项所述的方法， 从所述 FFPE样本提取 DNA;
将所述 DNA进行片段化， 以便获得 DNA片段；
将所述 DNA片段进行末端修复及 3'末端添加碱基 A,以便获得具有粘性末端 A的 DNA 片段；
将所述具有粘性末端 A的 DNA片段与接头相连， 以便获得连接产物；
将所述连接产物进行片段选择， 以便获得目的片段； 以及
将所述目的片段进行 PCR扩增， 以便获得扩增产物， 所述扩增产物构成所述 FFPE样 本的核酸文库。
14、 根据权利要求 1 所述的方法， 其特征在于， 所述片段化是通过选自雾化、 超声打 断法、 HydroShear以及酶切处理的至少一种进行的。
15、 根据权利要求 14所述的方法， 其特征在于， 使用 ovaris 超声打断仪将 FFPE样本 DNA片段化。
16、 根据权利要求 15所述的方法， 其特征在于， 所述 Ovaris超声打断仪的模式选择 Frequency Sweeping mode,打断参数为 Duty Cycle 10% , Intensity 5, Cycles per Burst 200, 共 打断 2-3次， 每次打断时间为 60-110秒， 优选 75秒。
17、 根据权利要求 1所述的方法， 其特征在于， 所述 DNA片段的长度为 200-300bp, 优选 250-300bp。
18、 根据权利要求 1所述的方法， 其特征在于， 将所述 DNA片段进行末端修复是利用 Klenow片段、 T4 DNA聚合酶和 T4多核苷酸激酶进行的， 其中， 所述 Klenow片段具有 5' →3'聚合酶活性和 3'→5'聚合酶活性， 但缺少 5'→3'外切酶活性。
19、 根据权利要求 1所述的方法， 其特征在于， 将所述经过末端修复的 DNA片段的 3' 末端添加碱基 A是利用 Klenow (3'-5' exo-)进行的。
20、根据权利要求 1所述的方法， 其特征在于， 将所述具有粘性末端 A的 DNA片段与 接头相连是利用 T4 DNA连接酶进行的。
21、 根据权利要求 1所述的方法， 其特征在于， 利用 2%琼脂糖电泳对所述连接产物进 行片段选择。
22、 根据权利要求 1所述的方法， 其特征在于， 所述目的片段的长度为 350-400bp。
23、 根据权利要求 1所述的方法， 其特征在于， 将所述目的片段进行 PCR扩增前， 进 一步包括纯化所述目的片段的步骤。
24、 根据权利要求 1 所述的方法， 其特征在于， 所述 PCR扩增的退火及延伸时间为 40-60s, 循环数为 6-10个， 优选所述退火及延伸时间为 45s, 循环数为 8个。
25、 根据权利要求 1 所述的方法， 其特征在于， 进一步包括利用选自固相杂交和液相 杂交技术的至少一种， 对所述 FFPE样本的核酸文库进行目标序列捕获， 以便获得所述目标 序列的测序文库。
26、 根据权利要求 24所述的方法， 其特征在于， 使用 NimbleGen的 2.1M外显子捕获 芯片进行所述固相杂交， 以便获得所述 FFPE样本的外显子测序文库。
27、 一种 FFPE样本的核酸文库， 其是通过根据权利要求 13-26中任一项所述的方法构 建的。
28、 一种确定 FFPE样本的核酸序列的方法， 其特征在于， 包括下列步骤： 利用根据权利要求 13-26中任一项所述的方法， 构建所述 FFPE样本的核酸文库； 将所述 FFPE样本的核酸文库进行测序， 以便获得测序结果； 以及
基于所述测序结果， 确定所述 FFPE样本的核酸序列。
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