CN105274090B - A kind of paraffin DNA purification process and its application in genomics - Google Patents
A kind of paraffin DNA purification process and its application in genomics Download PDFInfo
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Abstract
The present invention provides the DNA method of purification in a kind of paraffin embedding sample, and the method includes (1) to dewax to sample；(2) formaldehyde in sample is removed；(3) cell cracking；(4) DNA is extracted；(5) adhesion of DNA and protein are removed；(6) DNA is purified, wherein the step (1) includes being dewaxed with dimethylbenzene to sample.The DNA purity is high that benefit is obtained by the present invention, DNA sample of the sequencing quality close to flesh tissue.
Description
Technical field
The present invention relates to a kind of application of paraffin DNA purification process and this method in genomics, more particularly, to
The method that DNA is purified from the human tissue sample of paraffin embedding, belongs to field of genetic engineering.
Background technique
The fast development of genomics is that accurate medicine brings opportunity.By taking tumor individual therapy as an example, hospital pathology department
The tumour paraffin specimen of preservation is to find drug target, realizes the premise of the accurate medical treatment of cancer patient.However, paraffin specimen is being made
Treatment process (such as dipped into formalin, paraffin embedding) experienced is the master for causing paraffin DNA height to be degraded during work
Want reason.Paraffin DNA after degradation can only be limited be applied to cancer genomics study ----DNA of poor quality (260/280
Ratio is higher or relatively low, and impurity is more etc.), DNA requirement is big (3-10 microgram), and PCR cycle number is big (12-18), the sequencing of two generations
It is followed by by low (the 20-50%) (W.N, etc. High-throughput detection of actionable genomic of ratio
2 (2012) 82-93. and G.H of alterations in clinical tumor, Cancer Discov, etc. Genome-
scale DNA methylation mapping of clinical samples at single-nucleotide
resolution,Nat Methods 7(2010)133-136.).These factors greatly hinder the hair of the accurate medicine of tumour
Exhibition.Therefore, the process and purifying for being highly desirable to extract for paraffin sample DNA optimize, to realize genomics power-assisted
Accurate medicine provides abundant material foundation.
The processing step of hospital pathology department paraffin embedding tumor tissues is as follows: 1, fixed；2, it washes；3, it is dehydrated；4, transparent；
5, waxdip；6, it embeds；7, it is sliced.Pathology department doctor according to the HE dyeing in paraffin section source, immunohistochemistry, immunofluorescence etc. most
The property and degree of disease are judged eventually.In this series for the treatment of process, it is embedded in tumor tissues DNA in paraffin mass not
It is evitable to experienced multiple damages；This is also the basic reason why paraffin DNA highly degrades.
The scheme that there is now the extraction of some document reports paraffin sample DNA is generally based on the extraction process of silicon absorption.
In the stage of fumbling of our early periods, find these schemes extract paraffin DNA there are low output, ropy popular features
(Fassunke,J.,et al.,Utility of different massive parallel sequencing
platforms for mutation profiling in clinical samples and identification of
pitfalls using FFPE tissue.Int J Mol Med,2015).Currently, there has been no complete for paraffin section production
The specific aim scheme of process come improve DNA extraction yield and quality.Thus, we are comprehensive emphatically on subsequent layout strategy
Consider tumour paraffin sample film-making process experienced, degradation of these processes to DNA is targetedly released or reduce, to reach
To the final purpose for improving paraffin sample DNA yield and quality.
Summary of the invention
The object of the present invention is to provide the DNA purification scheme in a kind of paraffin embedding sample, make that it is suitable for extensive bases
Because of a group research, to realize that the accurate medicine of tumor patient establishes material foundation.
For this purpose, the present invention the following technical schemes are provided:
A kind of DNA method of purification in paraffin embedding sample, the method includes (1) to dewax to sample；(2) it removes
Formaldehyde in sample；(3) cell cracking；(4) DNA is extracted；(5) adhesion of DNA and protein are removed；(6) DNA is purified, wherein
The step (1) includes being dewaxed with dimethylbenzene to sample.
Preferably, it in the step (1), is repeatedly dewaxed with dimethylbenzene to sample.
It is furthermore preferred that dimethylbenzene carries out 2-3 dewaxing to sample.
It preferably, include that formaldehyde is reduced to methanol using sodium borohydride in the step (2).
It is furthermore preferred that the concentration of the sodium borohydride is 0.1-1.0M.
Preferably, in the step (5) include adhesion using crosslinking agent is removed to remove DNA and protein.
It is furthermore preferred that described go crosslinking agent to include but are not limited to: SDS and NaHCO3.
It preferably, further include that Proteinase K is used in combination to remove the adhesion of DNA and protein in the method step (5).
It is furthermore preferred that the Proteinase K additive amount is 20-40 μ l.More preferably, Proteinase K is in 60-65 ° of lower water-bath.
The present invention also provides a kind of application of any of the above-described method in genomics.
The constraint and damage that can suffer from using method of the invention for the DNA of paraffin embedding, change accordingly
It is kind.Our strategy: based on DNA damaged points in paraffin specimen production process, corresponding scheme is pointedly designed.Utilize the present invention
The genome of the DNA sample that is prepared of method close to fresh sample show.Specifically, the solution have the advantages that:
1, by increasing the number of dimethylbenzene dewaxing, the content of paraffin is reduced, to reduce damage and constraint of the paraffin to DNA；2, it uses
Formaldehyde is reduced to methanol by sodium borohydride, and is evaporated, and is relieved formaldehyde to cyto-architectural fixation, is reduced formaldehyde to the pact of cell
Beam, to improve the yield of paraffin DNA；3, it in the preferred scheme, using crosslinker solution is removed, reduces and just mentions DNA and albumen
Crosslinking, thorough released dna improve paraffin DNA mass；4, joint increases Proteinase K dosage, improves bath temperature, promotes albumen
Degradation reduces the pollution to DNA.By multi-scheme, prolonged optimization and improve, the base for the paraffin specimen DNA that we extract
Because a group performance is significantly stronger than existing program, it is in close proximity to the genomics performance of fresh sample.In starting paraffin on an equal basis
It material and extracts under process simultaneously, it is bigger that our scheme can extract quantity, the higher paraffin DNA of quality.More importantly
The application of subsequent gene group further demonstrates the sequencing quality of these paraffin DNA comparable to fresh sample.It is swollen to carry out on a large scale
The accurate medicine place mat of tumor patient material foundation stone.
Detailed description of the invention
The DNA electrophoresis result figure of Fig. 1 embodiment 1；
Fig. 2: the DNA electrophoresis result figure of embodiment 2；
Fig. 3: the DNA electrophoresis result figure of embodiment 3；
Fig. 4: the gene order-checking Quality Map of embodiment 4.
Specific embodiment
Technical solution of the present invention is further described combined with specific embodiments below.It is understood that
The particular implementation of this description indicates by way of example, is not intended as limitation of the present invention.It is not deviating from
In the case where the scope of the invention, main feature of the invention can be used for various embodiments.Those skilled in the art will
Recognize or be able to confirm that, routine experiment is only used only, and many equivalents can be applied in particular step described herein.
These equivalents are considered place within the scope of the present invention, and are covered by claim.
The embodiment of the present invention is related to kit shown in table 1
Table 1: kit information
Embodiment 1
1. (20-25 °) of room temperature of kidney paraffin sample (paraffin bits) is placed 2 hours.
2. weighing≤25mg paraffin considers tissue to be worth doing, it is put into 2ml EP pipe.
3. 1200ul dimethylbenzene is added；Concussion mixes 10 seconds；Put gyroscope 10 minutes, room temperature.
4. centrifugation, 10000G, 70 seconds, room temperature.
5. abandoning supernatant.
6. it is primary to repeat step 3-5.
7. 100% ethyl alcohol of 1200ul is added, concussion is mixed 15 seconds.
8. centrifugation, 11000G, 70 seconds, room temperature.
9. abandoning supernatant (not remove any precipitating).
10. it is primary to repeat step 7-9.
11.37 ° water-bath 2ml EP pipe 30 minutes.
12. adding 100ul 0.1M, 0.2M, 0.5M 1.0M sodium borohydride solution (is set to the 2nd, 3,4,5 group).
13.50 ° water-bath 2ml EP pipe 30 minutes.(control group does not add sodium borohydride, and no step 12 and 13 is set as the 1st
Group)
14. adding 180ul ATL, 40ul Proteinase K；Concussion mixes 20 seconds.
15.61.5 ° water-bath 48-72 hours, until liquid-transparent (period takes out EP and manages and shake 3-5 times).
16. adding 200ul AL, concussion is mixed 20 seconds.
17.70 ° water-bath 10 minutes.
18. adding 200ul dehydrated alcohol, concussion is mixed 20 seconds, is slightly centrifuged.
19. EP, which is managed interior all mixtures, is transferred to QIAampMini spin column (covering in 2ml collecting pipe).
20. centrifugation, 6000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
21. adding 500ul AW1；Centrifugation, 7000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
22. adding 500ul AW2；Centrifugation, 8000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
23. centrifugation, 9000g, 1 minute；Abandon efflux.
24. pillar is uncapped, it is placed at room temperature for 10 minutes；Pillar is put into new 1.5ml EP pipe later.
25. 60 ° of preheating nuclease-free waters of 50ul are added, room temperature 1 minute.
26. centrifugation, 12000g, 1 minute.
27. it is primary that repetition step plays 25-26；Obtain eluent of the 100ul containing DNA.
28. preliminary quantitative: with NanoDrop and 1% gel electrophoresis figure (see Fig. 1).As shown in Figure 1, the DNA of experimental group 2-5
Band is significantly more than control group 1, and concentration is also above control group.DNA quantitative result is shown in Table 2.As shown in table 2, the reality of NaBH4 is added
It tests the DNA concentration that group (including 2-5 group) obtains and is apparently higher than control group, there is significant statistical difference, and each experiment
The DNA mass that group obtains is also significantly better than control group.
Table 2: control group and experimental group DNA quantitative result
| Electrophoresis sequence+sample names | Concentration (ng/ul) | Volume | A260/280 |
| 1. control group | 8.3 | 100 | 2.26 |
| 2. adding 0.1M NaBH4 | 17.3 | 100 | 1.96 |
| 3. adding 0.2M NaBH4 | 35.1 | 100 | 1.78 |
| 4. adding 0.5M NaBH4 | 31.5 | 100 | 2.05 |
| 5. adding 1.0M NaBH4 | 12.4 | 100 | 2.11 |
Embodiment 2
1. (20-25 °) of room temperature of kidney paraffin sample (paraffin bits) is placed 2 hours.
2. weighing≤25mg paraffin considers tissue to be worth doing, it is put into 2ml EP pipe.
3. 1200ul dimethylbenzene is added；Concussion mixes 15 seconds；Put gyroscope 10 minutes, room temperature.
4. centrifugation, 10000G, 70 seconds, room temperature.
5. abandoning supernatant.
6. repeating step 3-5 bis- times.
7. 100% ethyl alcohol of 1200ul is added, concussion is mixed 15 seconds.
8. centrifugation, 11000G, 70 seconds, room temperature.
9. abandoning supernatant (not remove any precipitating).
10. it is primary to repeat step 7-9.
11.37 ° water-bath 2ml EP pipe 30 minutes.
12. 100ul 0.2M sodium borohydride solution is added.
13.50 ° water-bath 2ml EP pipe 30 minutes.
14. adding 180ul ATL, 40ul Proteinase K；Concussion mixes 20 seconds.
15.61.5 ° water-bath 48-72 hours, until liquid-transparent (period takes out EP and manages and shake 3-5 times).
16. adding 200ul AL, concussion is mixed 20 seconds.
17.70 ° water-bath 10 minutes.
18. adding 200ul dehydrated alcohol, concussion is mixed 20 seconds, is slightly centrifuged.
19. EP, which is managed interior all mixtures, is transferred to QIAampMini spin column (covering in 2ml collecting pipe).
20. centrifugation, 6000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
21. adding 500ul AW1；Centrifugation, 7000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
22. adding 500ul AW2；Centrifugation, 8000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
23. centrifugation, 9000g, 1 minute；Abandon efflux.
24. pillar is uncapped, it is placed at room temperature for 10 minutes；Pillar is put into new 1.5ml EP pipe later.
25. 60 ° of preheating nuclease-free waters of 50ul are added, room temperature 1 minute.
26. centrifugation, 12000g, 1 minute.
27. it is primary that repetition step plays 25-26；Obtain eluent of the 100ul containing DNA.
28. preliminary quantitative: using NanoDrop and Qubit.
29. 10ul 5M sodium chloride solution is added, concussion is mixed 20 seconds.
30. adding 90ul composite solution (1%SDS, 100mM NaHCO3), concussion is mixed 20 seconds
31. 80 ° of water-bath, 1 hour.
32. adding Proteinase K 40ul or 20ul (being respectively set as the 2nd and 3 group).
33. 60 ° of water-bath, 48 hours.(control group does not have step 30-33, is set as the 1st group).
34. adding 3-4 times of volume ChIP DNA Binding Buffer, concussion is mixed 20 seconds.
35. EP pipe content is transferred to Zymo-SpinTM IC-XL Column(Zymo Research).
36. centrifugation, 6000g, 30 seconds；Abandon effluent.
37. add 200ul DNA Wash Buffer. centrifugation, 7000g, 30 seconds；Abandon effluent.It is repeated 2 times.
38. pillar is uncapped, and room temperature 5 minutes；Pillar is put into new 1.5ml EP pipe later.
39. 60 ° of preheating nuclease-free waters of 25ul are added, room temperature 1 minute.
40. centrifugation, 12000g, 1 minute.
41. it is primary to repeat step 39-40；Obtain the DNA that 50ul is finally purified.
42. quantitative: using Qubit and 1% gel electrophoresis figure (referring to fig. 2)；As shown in Fig. 2, the DNA band of experimental group 2-3
Significantly more than control group 1, concentration is also above control group.Standby subsequent gene group is applied.DNA quantitative result is shown in Table 3.Such as 3 institute of table
Show, the DNA concentration that each experimental group is compared with control group is obviously high, and the two has statistical significant difference.
Table 3: control group and experimental group DNA quantitative result
Embodiment 3
1. liver cancer, lymph node, prostate cancer, hyperplasia of prostate sample (paraffin bits) (being respectively set as experimental group 1-4) room
(20-25 °) of temperature is placed 2 hours.
2. weighing≤25mg paraffin considers tissue to be worth doing, it is put into 2ml EP pipe.
3. 1200ul dimethylbenzene is added；Concussion mixes 15 seconds；Put gyroscope 10 minutes, room temperature.
4. centrifugation, 10000G, 70 seconds, room temperature.
5. abandoning supernatant.
6. it is primary to repeat step 3-5.
7. 100% ethyl alcohol of 1200ul is added, concussion is mixed 15 seconds.
8. centrifugation, 11000G, 70 seconds, room temperature.
9. abandoning supernatant (not remove any precipitating).
10. it is primary to repeat step 7-9.
11.37 ° water-bath 2ml EP pipe 30 minutes.
12. 100ul 0.2M sodium borohydride solution is added.
13.50 ° water-bath 2ml EP pipe 30 minutes.
14. adding 180ul ATL, 40ul Proteinase K；Concussion mixes 20 seconds.
15.61.5 ° water-bath 48-72 hours, until liquid-transparent (period takes out EP and manages and shake 3-5 times).
16. adding 200ul AL, concussion is mixed 20 seconds.
17.70 ° water-bath 10 minutes.
18. adding 200ul dehydrated alcohol, concussion is mixed 20 seconds, is slightly centrifuged.
19. EP, which is managed interior all mixtures, is transferred to QIAampMini spin column (covering in 2ml collecting pipe).
20. centrifugation, 6000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
21. adding 500ul AW1；Centrifugation, 7000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
22. adding 500ul AW2；Centrifugation, 8000g, 1 minute；Efflux is abandoned, 2ml collecting pipe is changed.
23. centrifugation, 9000g, 1 minute；Abandon efflux.
24. pillar is uncapped, it is placed at room temperature for 10 minutes；Pillar is put into new 1.5ml EP pipe later.
25. 60 ° of preheating nuclease-free waters of 50ul are added, room temperature 1 minute.
26. centrifugation, 12000g, 1 minute.
27. it is primary that repetition step plays 25-26；Obtain eluent of the 100ul containing DNA.
28. preliminary quantitative: using NanoDrop and Qubit.
29. 10ul 5M sodium chloride solution is added, concussion is mixed 20 seconds.
30. adding 90ul composite solution (1%SDS, 100mM NaHCO3), concussion is mixed 20 seconds.
31. 80 ° of water-bath, 1 hour.
32. adding 3-4 times of volume ChIP DNA Binding Buffer, concussion is mixed 20 seconds.
33. EP pipe content is transferred to Zymo-SpinTM IC-XL Column(Zymo Research).
34. centrifugation, 6000g, 30 seconds；Abandon effluent.
35. add 200ul DNA Wash Buffer. centrifugation, 7000g, 30 seconds；Abandon effluent.It is repeated 2 times.
36. pillar is uncapped, and room temperature 5 minutes；Pillar is put into new 1.5ml EP pipe later.
37. 60 ° of preheating nuclease-free waters of 25ul are added, room temperature 1 minute.
38. centrifugation, 12000g, 1 minute.
39. it is primary to repeat step 37-38；Obtain the DNA that 50ul is finally purified.
40. quantitative: using Qubit and 1% gel electrophoresis figure (referring to Fig. 3)；As shown in figure 3, being added using method of the invention
After adding sodium borohydride and removing crosslinking agent, other tissue samples are equally applicable to, the DNA band of extraction is all very more, concentration
It is high.Standby subsequent gene group is applied.DNA quantitative result is shown in Table 4.As shown in table 4, the DNA concentration of each sample is all very high.
Table 4: each tissue samples DNA quantitative result
| Electrophoresis sequence+sample names | Concentration (ng/ul) | Volume |
| 1. liver cancer | 51.2 | 50 |
| 2. lymph node | 194.5 | 50 |
| 3. prostate cancer | 176.1 | 50 |
| 4. hyperplasia of prostate | 45.6 | 50 |
Meanwhile invention is also individually by taking liver cancer tissue as an example, compares without using going crosslinking agent (the i.e. not no step of the present embodiment
Rapid 30-31, is set as control group, the 1st group) and use crosslinking agent (i.e. the above method of the present embodiment, is set as experimental group, the 2nd group)
In the case where DNA purification effect, the results are shown in Table 5: the DNA concentration of experimental group is apparently higher than control group, and the two has system
Count the significant difference learned.
Table 5: control group and experimental group DNA quantitative result
| Electrophoresis sequence+sample names | Concentration (ng/ul) | Volume |
| 1. control group | 24.8 | 50 |
| 2. removing crosslinking agent | 51.2 | 50 |
4 paraffin DNA full genome of embodiment sets up library
According to the paraffin DNA of 2 purification of embodiment, we start full genome and set up library (according to NEBUltraTM
DNA Library Prep Kit for#E7370L).It is enterprising in the Hiseq-2000 platform of Illunima company
Row sequencing.Crucial sequencing quality parameter is shown in Fig. 4: as shown in figure 4, A schemes: after fresh sample DNA sequencing > and 90% reads base
Quality is higher than 30 (base mass range 0-40；The higher the better).B figure: after paraffin sample DNA (our improved methods) sequencing >
80% reads base quality is higher than 30.C figure: only 40% reads base quality is higher than 30 after the sequencing of paraffin sample DNA.Such as
Shown in table 6, the MappingB ratio for the paraffin sample B group that our method obtains is up to 70-85%, close to the fresh sample of A group
This.
Table 6: the Mapping ratio of various samples
| Sample source | Mapping ratio |
| A. fresh sample | 85-95% |
| B. paraffin sample (our improved plans) | 70-85% |
| C. paraffin sample (Qiagene scheme) | 20-50% |
The above result shows that the sequencing quality of paraffin DNA can be greatly improved with our improved plans, close to fresh sample
This, is allowed to be completely adapted to the accurate medicine needs of tumor patient.
Claims (3)
1. the DNA method of purification in a kind of paraffin embedding sample, the method includes (1) to dewax to sample；(2) sample is removed
Formaldehyde in this；(3) cell cracking；(4) DNA is extracted；(5) adhesion of DNA and protein are removed；(6) DNA, feature are purified
It is, the step (1) includes being dewaxed with dimethylbenzene to sample, includes using sodium borohydride by first in the step (2)
Aldehyde is reduced to methanol, and the concentration of the sodium borohydride is 0.1-1.0M, include in the step (5) be used in combination crosslinking agent and
Proteinase K is to remove the adhesion of DNA and protein, wherein described to remove crosslinking agent be SDS and NaHCO3.
2. the method as described in claim 1, which is characterized in that in the step (1), repeatedly with dimethylbenzene to sample into
Row dewaxing.
3. application of any method of claim 1-2 in genomics.
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| CN102115743A (en) * | 2010-12-21 | 2011-07-06 | 天津医科大学总医院 | Novel method and kit for extracting high-quality RNA (Ribose Nucleic Acid) from paraffin-embedded tissue |
| CN102181431A (en) * | 2011-03-11 | 2011-09-14 | 大连医科大学 | Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues |
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| CN102115743A (en) * | 2010-12-21 | 2011-07-06 | 天津医科大学总医院 | Novel method and kit for extracting high-quality RNA (Ribose Nucleic Acid) from paraffin-embedded tissue |
| CN102181431A (en) * | 2011-03-11 | 2011-09-14 | 大连医科大学 | Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues |
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| 福尔马林固定石蜡包埋组织中DNA提取;谭卓毅 等;《法医学杂志》;20061231;第22卷(第6期);正文1.1-1.2、2.1部分 * |
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