KR20160110902A - A method for isolating circulating cell-free nucleic acid - Google Patents
A method for isolating circulating cell-free nucleic acid Download PDFInfo
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Abstract
The present invention relates to a method for separating circulating free nucleic acid from a sample containing circulating cell-free nucleic acid, a circulating free nucleic acid separating kit and a lysis buffer for circulating free nucleic acid isolation.
Description
The present invention relates to a method for separating circulating free nucleic acid from a sample containing circulating cell-free nucleic acid, a circulating free nucleic acid separating kit and a lysis buffer for circulating free nucleic acid isolation.
Cancer-specific extracellular DNA fragments and mRNA present in blood, or circulating cell-free nucleic acid (cfNA) such as fetal nucleic acid present in maternal blood, It is known that DNA is present in the plasma in the form of DNA of 1000 bp or less, or RNA of less than 100 nt. In particular, it is known that nucleic acid liberated by cancer necrosis or dead cells during the development of cancer is present in plasma. Kamat et al (Cancer Biol Ther. 2006 Oct; 5 (10): 1369-74.) Injected cancer cells into nude mice and then measured the amount of cfDNA, the amount of cfDNA reflected well the size of the cancer, And the response to subsequent treatment is well reflected. Thus, it is known that cfDNA is variously expressed in various cancers and shows the development, progression, and characteristics of metastatic cancer. Therefore, in order to utilize the cfDNA for cancer research and diagnosis, the necessity of effective separation of circulating free nucleic acid is emphasized have.
As a method of separating circulating free nucleic acid, a kit and a method for separating circulating free nucleic acid using a column are known (QIAamp Circulating Nucleic Acid Kit). However, there is still a need to develop an improved separation kit and method for circulating free nucleic acid compared to previously known methods.
The present inventors have made intensive efforts to develop kits and methods capable of effectively obtaining circulating free nucleic acids, and as a result, they have found that a sample containing circulating free nucleic acid is dissolved, and a solution containing a salt and PEG and a magnetic bead Thereby obtaining a circulating free nucleic acid from the magnetic beads to which the free glass nucleic acid is bound. Using the above-described method, it was confirmed that the circulating free nucleic acid can be efficiently separated from the sample containing the circulating free nucleic acid, and the present invention has been completed.
It is an object of the present invention to provide a method for separating circulating free nucleic acid from a sample containing circulating cell-free nucleic acid.
Another object of the present invention is to provide a pharmaceutical composition comprising a lysis buffer; A solution comprising a salt and PEG; And a magnetic bead. The present invention also provides a circulating glass nucleic acid separation kit.
It is another object of the present invention to provide a lysis buffer for circulating glass nucleic acid isolation comprising a chaotropic salt, a chelating agent, a nonionic surfactant and Tris-Cl .
One specific aspect of the present invention for solving the above problems is
(a) adding a solution and a magnetic bead containing a salt and PEG (polyethylene glycol) to a separated sample containing a circulating free nucleic acid; And
(b) obtaining a magnetic bead from the sample to which the magnetic bead is added, and separating the circulating free nucleic acid from the magnetic bead, and separating the circulating free nucleic acid from the sample containing the circulating free nucleic acid. The method of separating the nucleic acid may also be referred to as PREP.
As used herein, the term "circulating cell-free nucleic acid " refers to DNA that circulates in the blood and is present. The circulating free nucleic acid specifically includes circulating cell-free DNA (cfDNA), circulating free RNA, and the like. Specifically, it may be circulating free DNA, but is not limited thereto. The circulating free nucleic acid generally has a length of 1000 bp or less (DNA) or 100 nt or less (RNA) in plasma or serum, but is not limited thereto. There has been a demand for development of a method for obtaining the circulating free nucleic acid in a small amount in blood and acquiring it in a high yield.
In the present invention, a sample containing a circulating free nucleic acid is referred to as a magnetic bead; And a salt and a PEG, and separating the circulating free nucleic acid bound to the magnetic beads. In the case of using a solution containing both a salt and a PEG-containing solution in reacting the magnetic beads and the circulating free nucleic acid, the magnetic beads can capture the circulating free nucleic acid more effectively, ≪ / RTI > Furthermore, the present invention has developed a technique capable of effectively separating circulating free nucleic acids in a reproducible manner by optimizing the preparation of the dissolution buffer and plasma in the above method.
Hereinafter, the steps of the present invention and each configuration will be described in more detail.
In the method of the present invention, step (a) is a step of adding a solution containing a salt and PEG and a magnetic bead to a separated sample containing a circulating free nucleic acid so that the circulating free nucleic acid binds to the magnetic bead.
In the present invention, if the separated sample containing the circulating free nucleic acid is a separated sample containing circulating free nucleic acid, the kind thereof is not particularly limited, but it may be plasma or serum. Specifically, it may be, but is not limited to, plasma or serum, more specifically plasma, isolated from an individual in need of analysis or obtaining circulating free nucleic acid.
The separated sample containing the circulating free nucleic acid may be prepared by dissolving plasma or serum with lysis buffer.
Accordingly, the method of the present invention comprises a first step of adding a lysis buffer to a separated plasma or serum sample containing circulating free nucleic acid; A second step of adding a solution containing a salt and PEG and a magnetic bead to the sample containing the circulating free nucleic acid dissolved in the first step; And a third step of obtaining a magnetic bead in the sample to which the magnetic bead is added and separating the circulating free nucleic acid from the magnetic bead, but the present invention is not particularly limited thereto.
The lysis buffer used for the dissolution of plasma or serum may specifically include chaotropic salt, chelating agent, nonionic surfactant and Tris-Cl, But is not limited thereto.
According to one embodiment of the present invention, when a lysis buffer having the above composition is used, as compared with the case where another buffer solution including a lysis buffer composed of EDTA as a chelating agent, SDS as a chaotropic salt, and Tris-Cl is used, It was confirmed that separation was easy and / or yield was high.
In the present invention, the chaotropic salt includes a substance capable of interfering with the hydrogen bonding between water molecules. The chaotropic salt causes a change in the structure of the protein, weakens the binding force between the protein and DNA, So as to increase the degree of separation.
Examples of the chaotropic salts include guanidinium salts, lithium salts, magnesium salts, sodium dodecyl sulfate (SDS), thiourea, urea, butanol or ethanol, It is not limited.
Examples of the guanidinium salt include guanidinium chloride. Examples of the lithium salt include lithium perchlorate and lithium acetate. Examples of the magnesium salt include magnesium chloride. It is not. In one embodiment of the present invention, guanidinium chloride was used as the chaotropic salt.
In the present invention, the chelating agent is Mg 2+ and Ca can be used to isolate the divalent cation, such as + 2, and therefore can protect the DNA from the enzyme to decompose it, but is not limited thereto.
Examples of the chelating agent may include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and N, N-bis (carboxymethyl) glycine It is not limited. EDTA was used as a chelating agent in one embodiment of the present invention.
The EDTA may be, but is not limited to, an EDTA moiety in an EDTA compound (e.g., K 2 EDTA, K 3 EDTA, or Na 2 EDTA).
In the present invention, a nonionic surfactant is a substance having hydrophilic and hydrophobic moieties together in one molecule, and exhibits nonionic properties at dissociation.
An example of the nonionic surfactant that can be used in the present invention includes, but is not limited to, triton X-100.
Wherein the lysis buffer comprises 2 to 6 M chaotropic salts; 1 to 50 mM chelating agent; 0.1 to 5% (w / v) nonionic surfactant; And / or 10 to 100 mM Tris-Cl.
Here, the pH of the dissolution buffer may be pH 7.0 to 8.5.
More specifically, the lysis buffer may include, but is not limited to, guanidinium chloride, EDTA, Triton X-100, and Tris-Cl.
Further, when the plasma or serum sample is dissolved with the above-mentioned dissolution buffer, proteolytic enzyme may be added together.
As the protease, various proteases commonly used for nucleic acid isolation may be used. For example, proteinase K may be used.
The separated plasma sample may include a first centrifugal separation step for separating plasma from a separated blood sample; And separating the plasma from the first centrifuged sample and subjecting the separated plasma to a second centrifugal separation, but the present invention is not limited thereto.
In the present invention, in the case of producing a plasma sample in a blood sample, when primary centrifugation is carried out at a low speed and then secondary centrifugation is carried out at high speed, cfDNA without contamination of DNA derived from white blood cells (WBC) Can be separated more purely.
Specifically, the first centrifugation may be performed at a condition of 1900 to 2000 x g, and the second centrifugation may be performed at a condition of 12000 to 18000 x g. However, the present invention is not limited thereto. In addition, the above-described centrifugal separation may be performed at a temperature of about 4 캜, but is not limited thereto.
In the present invention, the solution containing the magnetic beads and the salt and PEG may be added to the sample in sequence or simultaneously, but is not limited thereto.
In the case of performing sequentially, the above method may be performed by adding the solution after the addition of the magnetic beads, adding the solution, and then adding the magnetic beads.
In the present invention, the solution containing the salt and the PEG added together with the magnetic beads can assist in capturing small nucleic acid fragments of 100 bp or 100 nt or less, although not particularly limited thereto.
In the solution containing the salt and PEG (polyethylene glycol), the kind of the salt is not particularly limited, and it may be, for example, sodium chloride.
The PEG of the solution may be PEG having an average molecular weight of 6,000 to 10,000 Da, but is not limited thereto.
More specifically, the solution comprising the salt and PEG may include, but is not limited to, a 1-4 M salt and 10-60% (w / v) PEG.
The magnetic beads in the present invention refer to particles or beads that react to a magnetic field. Generally, a magnetic bead refers to a material that does not have a magnetic field, but forms a magnetic dipole that is exposed to a magnetic field. For example, it refers to a substance which can be magnetized under a magnetic field but does not have magnetism in the absence of a magnetic field. The magnetism used in the present invention includes, but is not limited to, paramagnetic or superparamagnetic materials.
For the purpose of the present invention, the magnetic beads are preferably beads having a property of binding to a nucleic acid, and examples thereof include, but are not limited to, a form having a functional group binding to a nucleic acid such as -COOH group.
In the present invention, the step (b) is a step of obtaining a magnetic bead in the sample to which the magnetic bead is added and separating the circulating free nucleic acid therefrom.
Specifically, the step (b) separates the circulating free nucleic acid attached to the magnetic beads added in the step (a) from the magnetic beads.
And washing the magnetic beads before obtaining the magnetic beads in the sample to which the magnetic beads are added.
At this time, washing of the magnetic beads may be performed using a 50 to 95% (v / v) ethanol solution, specifically, an ethanol solution of 80 to 90% (v / v).
Such a washing process can be carried out by placing the magnetic beads under a magnetic stand, collecting the magnetic beads, removing the supernatant, and adding washing buffer thereto. In addition, such a washing step can be carried out more than once.
The circulating free nucleic acid can be separated from the magnetic beads by selectively performing the washing process, separating the magnetic beads, and adding an elution buffer to the separated magnetic beads.
Another specific embodiment of the present invention relates to a method for preparing a pharmaceutical composition comprising: a lysis buffer; A solution comprising a salt and PEG; And a magnetic bead.
The solution containing the dissolution buffer, the salt and PEG, and the magnetic beads are as described above.
The circulating-glass nucleic acid separation kit of the present invention is not limited to its origin, and the cfDNA can be isolated, so that the cfDNA can be extracted from the cancer patient or the mother. Therefore, the cfDNA extracted from the blood of a cancer patient can be used to diagnose a specific genetic mutation of the cancer patient. In addition, cfDNA derived from maternal blood can be used for non-invasive prenatal diagnosis of fetus because maternal plasma contains a large amount of cfDNA derived from fetus. That is, the circulating-glass nucleic acid separation kit of the present invention can be used for cancer diagnosis or prenatal diagnosis, and the use thereof is not particularly limited as long as it can utilize cfDNA.
In addition, the kit may further include other apparatuses, solutions, etc. generally used for nucleic acid separation, and may include instructions for separation of circulating free nucleic acids, but the present invention is not limited thereto.
In addition, the kit may further include, but is not limited to, a magnetic bead wash buffer, and a magnetic bead separation device (e.g., a magnetic stand).
Another specific embodiment of the present invention is a lysis buffer for circulating free nucleic acid separation, comprising a chaotropic salt, a chelating agent, a nonionic surfactant and Tris-Cl.
The chaotropic salt, the chelating agent, the nonionic surfactant, the circulating free-flowing nucleic acid and the lysis buffer are as described above.
According to the circulating-glass nucleic acid separation kit and method of the present invention, a circulating free nucleic acid can be obtained in a high yield within a short period of time in a sample containing circulating free nucleic acid separation, particularly a plasma or serum sample.
Figure 1 graphically shows the amount of cfDNA obtained from 200 [mu] l of plasma in normal and lung cancer patients. Here, gray rhombus shows experimental results on plasma separated from normal persons, and black rhombus shows experimental results on plasma separated from lung cancer patients. The y-axis represents the amount (ng) of cfDNA.
Fig. 2 shows the result of confirming reproducibility after performing
FIGS. 3A and 3B show results of analysis of a pattern of cfDNA isolated from plasma of ovarian cancer patients by the Bioanalyzer according to the method of the present invention. Plasma cfDNA is known to be fragmented in units of nucleosomes. The results show that the size of the cfDNA obtained according to the method of the present invention is fragmented to about 180 bp, which is a nucleosome unit. .
FIG. 4 shows the result of confirming whether PCR product of ~100 BP and 237 BP size was amplified using cfDNA isolated according to the method of the present invention as a template. As shown in FIG. 4, the PCR product was well amplified, indicating that the purified cfDNA according to the method of the present invention has high purity, which does not affect the enzymatic reaction of the DNA polymerase.
FIG. 5A shows the results of confirming the amount of DNA obtained by performing the cfDNA PREP after separating plasma from normal human blood using the one-step centrifugation method and the two-step centrifugal separation method. The x-axis represents the number of each plasma sample, and the y-axis represents the amount of cfDNA (unit: ng).
FIG. 5B shows the result of analyzing the separated cfDNA with a Bioanalyzer after separating plasma from normal human blood using a one-step centrifugation method and a two-step centrifugation method, performing cfDNA PREP, and then separating the cfDNA.
Fig. 6 is a diagram showing the results of separation of cfDNA using the method according to the present invention (named CCGD_cfDNA) and the Qiagen Kit, respectively. The number on the bar graph represents the amount of DNA (in ng).
Figure 7 shows the results of performing the cfDNA PREP using Comparative Buffer 1 (including chaotropic salts, chelating agents and Tris-Cl) and the working buffer according to the present invention. The number on the bar graph represents the amount of DNA (in ng).
Figure 8 shows the results of performing cfDNA PREP using Comparative Buffer 2 (including chelating agent, nonionic surfactant and Tris-Cl) and the buffer according to the present invention. The number on the bar graph represents the amount of DNA (in ng).
FIG. 9 shows the results obtained by using the Picogreen quantitation method (PG) and the RQ-PCR quantitation method for the yield of cfDNA isolated using the method according to the present invention (named as CCGD_cfDNA) and the Qiagen Kit in plasma separated from 10 mothers .
FIG. 10 shows the results of analysis of correlation coefficient between Picogreen quantitative value (PG) and RQ-PCR quantitative value.
Fig. 11 shows the quality of cfDNA isolated using the method according to the present invention (named CCGD_cfDNA) and Qiagen Kit, respectively, in plasma separated from 10 mothers.
Fig. 12 shows the purity of cfDNA separated by using the method according to the present invention (named as CCGD_cfDNA) and Qiagen Kit, respectively, in plasma separated from the mother.
Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited by the following examples.
Example 1: Preparation of plasma samples
Fresh blood samples were centrifuged at 4 ° C and 1,900 x g for 10 minutes to separate the plasma. Then, the separated plasma was centrifuged again at 16,000 x g for 10 minutes at 4 ° C, and the supernatant was separated. Then, the plasma samples thus obtained were stored at -20 캜 before DNA separation. Plasma prepared using the above method was used in the following examples, except where specifically described.
Example 2: Isolation of cfDNA
Frozen plasma samples were dissolved before DNA isolation and 200 μl of plasma samples were placed in tubes. Then, 400 μl of plasma dissolution buffer (5.5 M guanidine HCl, 50 mM Tris-Cl, pH 8.0, 20 mM EDTA, pH 8.0, 1.3% Triton X-100) and 10 μl of proteinase K Were added and mixed.
Then, 100 μl of magnetic beads (AMPure XP, Backman Coulter) and 1100 μl of solution A (2.5 M NaCl, 20% PEG-8000) were added to each tube and mixed. The tube was then incubated in a magnetic stand and the supernatant of the tube was removed.
Then, 1 mL of wash buffer (85% EtOH) was added and reacted, then the ethanol supernatant was removed on a magnetic stand and the washing procedure was repeated several times.
The beads were then dried, the reaction tubes were removed from the magnetic stand and elution buffer (purely isolated tertiary distilled water or 10 mM TE buffer) was added. Then, the reaction tube was placed on a magnetic stand, and the eluate was separated to obtain a cfDNA sample.
Example 3: Obtaining cfDNA samples from plasma of normal and lung cancer patients
An experiment was conducted to compare the amount of cfDNA extracted between a normal person and a patient using the cfDNA separation method of Example 2 above.
Specifically, cfDNA was isolated using 200 쨉 l of plasma obtained from 31 normal samples and 49 lung cancer patients, respectively, and the results are shown in Fig. The sample was distributed at the BRC (Bio Resource Center) of Asan Medical Center in Seoul.
As a result, when comparing the amounts of cfDNA obtained from normal individuals and lung cancer patients, much more cfDNA could be obtained in patients with lung cancer, which was expected from the contents known in the art. In particular, the amount of cfDNA obtained in a 200 [mu] l plasma sample of a normal individual was 4.4 ng (maximum value: 9.2 ng, minimum value: 0.8 ng) 32.0 ng (maximum value: 268.0 ng, minimum value: 4.78 ng), indicating that cfDNA can be efficiently isolated even in a 200 μl plasma sample.
Example 4 Confirmation of reproducibility of the cfDNA PREP method of Example 2
The reproducibility of the cfDNA separation method of Example 2 was examined.
Specifically, the amount of cfDNA obtained in each experiment was compared after repeating two experiments using 200 μl of plasma obtained from 49 lung cancer patients, and the results are shown in FIG.
As a result, as shown in Fig. 2, the reproducibility was excellent as the correlation between the two values was close to 1.
Example 5: cfDNA pattern analysis of the sample obtained by the cfDNA PREP method of Example 2
The cfDNA PREP method of Example 2 was used to analyze the properties of PREF cfDNA using a BioAnalyzer
First, the size of PREP cfDNA from plasma samples of lung cancer patients was confirmed, and the results were shown in Figs. 3A and 3B.
As a result of the size confirmation, it was confirmed that the DNA ladder size pattern is well known for the characteristics of cfDNA produced by apoptosis.
In addition, since cfDNA is truncated in nucleosome units, a size of about 180 bp is the smallest unit, and the sizes of 180 * 2, 180 * 3, 180 * 4bp in nucleosomes of 2, (Fig. 3B).
However, DNA of larger size than expected was also seen, because the DNA was not completely removed when preparing plasma from blood, or gDNA extracted from hemolyzed white blood cells (WBC) was extracted together. This is believed to be due to the fact that the experiment was carried out using the plasma of lung cancer patients who had been prepared and stored long ago and that it can be solved by preparing fresh blood using the fresh blood and centrifuging in two steps as in Example 1 Respectively.
Example 6: Analysis of the quality of cfDNA obtained by the cfDNA PREP method of Example 2
In order to confirm the quality of PREF cfDNA using the cfDNA PREP method of Example 2, PCR was performed using primer pairs capable of amplifying 100 bp and 237 bp products. As the template DNA, 1 to 5 cfDNA samples isolated from lung cancer patients and two control gDNAs (genomic DNA PREP in Beas2B and H1975 cell lines) were used. The results are shown in Fig. 4 (in Fig. 4, the cfDNA sample is represented by 1 to 5).
The PCR conditions were as follows.
Specifically, 2 μl of the purified cfDNA, 1 μl of the primer pair (rs1952996 primer pair or RASSF1A primer pair (each primer pair concentration is 5 μM for each of the corresponding F and R) shown in Table 1, dNTP mix (2.5 mM) 1 , 0.1 μl of HotStar Taq DNA polymerase (5 U / μl) from Qiagen, 1 μl of 10 × PCR buffer, and finally 10 μl of final distilled water. After reacting with HotStarTaq polymerase at 94 ° C for 15 minutes, [94 ° C (20 sec) / 60 ° C (30 sec) / 72 ° C (30 sec)]] The reaction was repeated 35 times and the reaction was terminated at 72 ° C for 3 minutes.
The primer information used is as follows.
As a result, it was confirmed that amplification of PCR product was performed well in all cfDNA used. That is, it was confirmed that there was no problem in the quality of PREP cfDNA.
Example 7: Optimization of plasma production process
The process for preparing plasma used in the cfDNA PREP method of the present invention was optimized as follows.
Specifically, the effect of preparing the plasma by performing the two-step centrifugation at the cfDNA PREP was compared with the effect of the one-step centrifugation. For this purpose, fresh blood from normal subjects was used. In the first step centrifugation, fresh blood obtained from an individual is centrifuged at 1,900 xg and 4 ° C for 10 minutes to obtain a plasma sample. In the two-step centrifugation method, fresh blood obtained from an individual is centrifuged at 1,900 xg and 10 Followed by centrifugation for 10 minutes at 16,000 xg and 4 ° C to obtain a plasma sample.
The plasma samples obtained using the above-described one-step centrifugation method and two-step centrifugal separation method were subjected to the separation of cfDNA by the method of Example 2, and the amounts and characteristics thereof were compared. The results are shown in Figs. 5A, 2 and 5B, respectively.
As shown in Fig. 5A and Table 2 below, the amount of cfDNA isolated from the plasma obtained using the two-step centrifugation method was about 1/3 of the amount of cfDNA isolated from the plasma obtained by the one-step centrifugation method Respectively. In the case of the one-step centrifugation method, the amount of extracted cfDNA varied greatly and was higher than expected.
(1,900 g,
4 ℃
,
10 min
), total ng
(1,900 g,
4 ℃
,
10 min
-> 16,000 g, 4 ° C, 10 min),
total ng
Further, as shown in FIG. 5B, the analysis of the characteristics of cfDNA obtained from the plasma prepared using the two-step centrifugation method and the one-step centrifugal separation method showed that, in the case of the DNA obtained by the two-step centrifugation method, and the minimum nucleosome unit size of about 180 bp characteristic of cfDNA was confirmed. On the other hand, the DNA extracted by the first step centrifugation method showed a pattern of mixed genomic DNA (gDNA). That is, when loading the BioAnalyzer, intact gDNA larger than the upper marker and fragmented gDNA appearing as a smear pattern are displayed.
Example 8: Comparison between the cfDNA PREP method of Example 2 and the commercially available kit
In order to compare the effects of the cfDNA PREP method according to the present invention with the conventional commercialized kits, the following experiment was conducted.
Specifically, we compared the performance with the Qiagen circulating tumor DNA prep kit, which is currently used most frequently in cfDNA PREP. For this, experiments were performed using plasma of patients with ovarian cancer, and the results are shown in Table 3 and FIG. 6, the method of cfDNA PREP (Example 2) according to the present invention is expressed as 'CCGD_cfDNA'. The plasma was distributed at the BRC of Asan Medical Center in Seoul.
As a result, as shown in Table 3 and FIG. 6, cfDNA was isolated using each method. As a result, when the method of the present invention was used, cfDNA in an amount of 4 to 5 times higher than that in the case of using Qiagen Kit ≪ / RTI >
(ng / mu l)
(ng / mu l)
Example 9: Comparison of effect according to the type of buffer
The optimal conditions of the lysis buffer which can be effectively used in the cfDNA PREP method of the present invention are as follows.
First, buffer (buffer for comparison) containing a chaotropic salt, a chelating agent and Tris-Cl (comparative buffer 1) and a buffer solution containing a chaotropic salt, a chelating agent, a nonionic surfactant and Tris-Cl The cfDNA was isolated by the method of Example 2. At this time, experiments were conducted using plasma of ovarian cancer patients, and two centrifuged samples as in Example 1 were used.
The specific composition of the
0.1 mM EDTA (pH 8.0)
0.5% SDS
50 mM Tris-Cl (pH 8.0)
20 mM EDTA (pH 8.0)
1.3% Triton X-100
As a result, as shown in the following Table 5 and FIG. 7, it was confirmed that a much larger amount of cfDNA could be obtained when the buffer according to the present invention was used, as compared with the case of using the
Sample
(ng / mu l)
(ng / mu l)
In addition, the effect of cfDNA PREP on the presence or absence of chaotropic salts in the lysis buffer was compared. Specifically, DNA was isolated by the method of Example 2 using the buffer and buffer according to the present invention having the composition shown in Table 6 below. At this time, 200 ng of highly degraded gDNA was added to the plasma from two normal subjects to compare the DNA acquisition rates according to the conditions of the lysis buffer. The results are shown in Table 7 and FIG.
gDNA (ng)
ratio (%)
gDNA (ng)
ratio (%)
As a result, in all of the results of using plasma of two normal individuals, the highest recovery was obtained when the buffer according to the present invention was used.
Example 10: Isolation of cfDNA from Plasma of Maternal
The cfDNA prep method of the present invention can obtain cfDNA not only in lung cancer patients but also normal individuals. That is, cfDNA can be obtained from isolated plasma without limitation of the state of an individual. Thus, in order to further analyze the characteristics of the cfDNA prep method of the present invention, cfDNA was isolated from the mother in the following examples and analysis was performed. Further, in Example 2, 200 μl of plasma sample was used, but in order to apply the cfDNA prep method of the present invention to a larger volume sample, the separation conditions were appropriately changed as follows (Table 8).
(volume)
(volume)
Specifically, fresh blood samples collected from 10 mothers were centrifuged at 1,900 x g for 10 minutes at 4 ° C to separate plasma. Then, the separated plasma was centrifuged again at 16,000 x g for 10 minutes at 4 ° C, and the supernatant was separated. Then, using 500 μl of the separated plasma, the cfDNA prep method of the present invention and the Qiagen circulating tumor DNA prep kit, which is a commercially available kit, were compared.
Example 11: Comparison of the yield between the cfDNA PREP method of Example 10 and the commercially available kit
The yields of the cfDNA preparative method (CCGD_cfDNA) and cfDNA separated by the Qiagen kit of the present invention were compared using the Picogreen quantitation method (PG) and the RQ-PCR quantitative method (FIG. 9).
As a result, it was confirmed that the cfDNA prep method of the present invention showed similar or higher yields than the Qiagen kit, irrespective of the quantification method. In particular, the yields of the samples separated from maternal 4 and maternal 6 were two times or more, confirming that the cfDNA prep method of the present invention had an excellent effect in terms of yield as compared with the Qiagen kit.
On the other hand, when the respective quantitative methods were compared, it was confirmed that the Qiagen kit showed a large difference between the PG quantitation value and the RQ-PCR quantitative value, whereas the cfDNA prep method of the present invention showed a large difference in the two quantitative methods I did. In order to clarify this, the agreement rate between the PG quantitation value and the RQ-PCR quantitation value was confirmed (FIG. 10).
As a result, when the Qiagen kit was used, it was confirmed that all of the samples except the two high concentration samples were present at a very low concentration as shown in FIG. In addition, the correlation coefficient between the PG quantitation value and the RQ-PCR quantitation value was 0.9245 in the case of using the Qiagen kit and 0.9676 in the case of the cfDNA prep method of the present invention, indicating that the cfDNA prep method of the present invention had a higher correlation coefficient Respectively.
In the case of the Qiagen kit, a large amount of tRNA is present in the extracted cfDNA because the tRNA was used as a DNA carrier in the kit to increase the extraction efficiency of the small fraction of cfDNA. Therefore, when the cfDNA is isolated using the Qiagen kit, the quantitative value using the Picogreen shows the over-measurement due to the tRNA, which shows that the correlation coefficient is as described above. That is, 8 samples with yields on the order of 10 ng are expected to have many of the values measured by tRNA contamination. On the other hand, since the present invention does not use tRNA, a high level of correlation coefficient is exhibited in the two kinds of assays.
Example 12: Example 10 of cfDNA PREP Method of separating a commercially available kit cfDNA Comparing quality and purity
In order to analyze the quality of the cfDNA extracted using the cfDNA preparation method of the present invention and the Qiagen kit of the present invention from 10 mothers, the size of the extracted cfDNA using the Bioanalyzer was analyzed. As a result, as expected, it was confirmed that a peak signal showing a cfDNA size (~180 bp) in almost all specimens was very high when using the cfDNA prep of the present invention (FIG. 11), compared with the case using the Qiagen kit.
Next, it was tried to confirm whether the purity of the cfDNA extracted by the above two methods is as high as possible for the enzyme reaction. The cfDNA extracted from three mothers (8 mothers, 9 mothers and 10 mothers) was subjected to RQ-PCR to amplify amplicons of 110 bp in size. Under the same conditions, the amount of cfDNA extracted from each mother was increased by 1, 2 and 4 μl, and the Ct value was determined by RQ-PCR, and the resultant was converted into the equivalent amount (1/2 ^ Ct) The purity of the extracted cfDNA was reaffirmed by confirming the proportion of the relative mass identified by RQ-PCR according to the amount.
As a result, it was confirmed that the relative mass (RQ-PCR value, 1/2 ^ Ct) was increased in proportion to the amount of cfDNA used in all three cfDNA extracted from three mothers, Was 0.9574, and in the case of the cfDNA prep method of the present invention was 0.9983, it was confirmed that the cfDNA prep method of the present invention had a higher correlation coefficient (FIG. 12).
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
<110> THE ASAN FOUNDATION University of Ulsan Foundation for Industry Cooperation <120> A method for isolating circulating cell-free nucleic acid <130> KPA141272-KR-P1 <150> KR 10-2015-0034686 <151> 2015-03-12 <160> 4 <170> KoPatentin <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs1952966-F <400> 1 ggctctggtt acaacagctt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs1952966-R <400> 2 agaagtttgc ttggctgaag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RASSF1A-F <400> 3 gtggggaccc tcttcctcta 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RASSF1A-R <400> 4 ggaaggagct gaggagagc 19
Claims (33)
(b) obtaining a magnetic bead in the sample to which the magnetic bead is added, and separating the circulating free nucleic acid from the magnetic bead, and separating the circulating free nucleic acid from the sample containing the circulating free nucleic acid.
Wherein the lysis buffer comprises a chaotropic salt, a chelating agent, a nonionic surfactant and Tris-Cl.
Wherein the lysis buffer comprises 2 to 6 M chaotropic salts; 1 to 50 mM chelating agent; 0.1 to 5% (w / v) nonionic surfactant; And 10 to 100 mM Tris-Cl.
Wherein the chaotropic salt is selected from the group consisting of guanidinium salts, lithium salts, magnesium salts, sodium dodecyl sulfate (SDS), thiourea, urea, butanol and ethanol How, one.
Wherein the nonionic surfactant is triton X-100.
A first centrifugation to separate plasma from the separated blood sample; And
Separating the plasma from the first centrifuged sample and second centrifuging the separated plasma.
Wherein the lysis buffer comprises a chaotropic salt, a chelating agent, a nonionic surfactant and Tris-Cl.
Wherein the lysis buffer comprises 2 to 6 M chaotropic salts; 1 to 50 mM chelating agent; 0.1 to 5% (w / v) nonionic surfactant; And 10 to 100 mM Tris-Cl.
Wherein the chaotropic salt is selected from the group consisting of guanidinium salts, lithium salts, magnesium salts, sodium dodecyl sulfate (SDS), thiourea, urea, butanol and ethanol The kit, which is.
Wherein the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and N, N-bis (carboxymethyl) .
Wherein the nonionic surfactant is triton X-100.
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| CN110144345B (en) * | 2019-05-10 | 2022-07-15 | 上海交通大学 | Method for extracting cfDNA from follicular fluid |
| CN112322615B (en) * | 2020-11-18 | 2022-08-19 | 威高集团有限公司 | Nucleic acid preservation solution, nucleic acid extraction preservation solution, blood collection tube and method for extracting nucleic acid |
| CN112410327A (en) * | 2020-12-11 | 2021-02-26 | 福建和瑞基因科技有限公司 | Kit and method for extracting RNA |
| CN113621606B (en) * | 2021-04-25 | 2023-12-12 | 北京全式金生物技术股份有限公司 | Kit and method for extracting plasma free DNA |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | Magnetotherapeutic bead method pathogen nucleic acid extraction kit and use method thereof |
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