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WO2012071985A1 - Procédé d'extraction d'adn à partir d'échantillons ffpe et leur utilisation - Google Patents

Procédé d'extraction d'adn à partir d'échantillons ffpe et leur utilisation Download PDF

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Publication number
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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Prior art keywords
dna
nucleic acid
ffpe
sample
fragment
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Chinese (zh)
Inventor
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

L'invention concerne un procédé d'extraction d'ADN à partir d'échantillons FFPE, un procédé de construction de banques d'acides nucléiques d'échantillons FFPE, des banques d'acides nucléiques d'échantillons FFPE et un procédé de détermination de séquences nucléotidiques d'échantillons FFPE. Selon l'invention, le procédé d'extraction d'ADN à partir d'échantillons FFPE comprend les étapes suivantes : déparaffinage des échantillons FFPE par l'utilisation d'un agent de déparaffinage, qui est au moins un choisi parmi le xylène et le D-limonène, pour obtenir des échantillons déparaffinés ; digestion des échantillons déparaffinés par l'utilisation d'un tampon de lyse et d'une protéase pour obtenir des produits digérés qui contiennent de l'ADN libéré ; incubation des produits digérés à 75-95 degrés centigrade pendant 30-60 minutes ; puis récupération et purification de l'ADN obtenu.
PCT/CN2011/082374 2010-12-02 2011-11-17 Procédé d'extraction d'adn à partir d'échantillons ffpe et leur utilisation Ceased WO2012071985A1 (fr)

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