CN112359097A - Amplification method and kit for whole genome of single cell - Google Patents

Abstract

The invention provides a method and a kit for amplifying a whole genome of a single cell, which comprises the steps of separating and extracting the single cell; extracting whole genome DNA of a single cell; diluting whole genome DNA with a diluent to obtain a DNA template, mixing the DNA template with a random primer octamer to obtain a mixed solution A, mixing 10 Xphi 29 Buffer, dNTPs, dUTP, Phi29 Polymerase and water to obtain a mixed solution B, adding the mixed solution A and the mixed solution B into a reaction tube in equal volume, mixing to form a reaction solution, and adding mineral oil to cover the reaction solution to perform multiple displacement amplification reaction; and purifying the amplified product to obtain purified DNA.

Description

Amplification method and kit for whole genome of single cell

The application is filed as11/28/2014Application No. is201410709576.5The invention is named asA kind of Method for whole genome amplification based on MDADivisional application of the patent application.

Technical Field

The invention relates to the technical field of DNA amplification, in particular to a method and a kit for amplifying a whole genome of a single cell.

Background

Over the last twenty years, with the increasing level of gene sequencing technology and the successive development of major international cooperative projects such as the thousand people genome project, the cancer genome project, and the Meta-Hit project, the genome research is gradually pushed to the high tide. However, the sequencing materials used to date are, without exception, mixed DNA samples of millions or more cells. This method allows to obtain genome-wide sequence information, but the results of its study are only the mean of the signals in a population of cells, or only the information of the number of cells that predominate, the unique characteristics of the individual cells being overlooked. For example, it is almost impossible for scientists to find out which mutations are present in which cells, and mutations that are present only in a few cells (e.g., early cancer cells) are also substantially masked. On the other hand, some samples are rare and cannot be cultured in the laboratory, and the sample amount is not sufficient for whole genome analysis, such as tumor circulating cells, tissue microarrays, early-developing embryonic cells, and the like. These are all challenges encountered with whole genome sequencing.

The single cell whole genome sequencing technology is a new technology for amplifying and sequencing whole genome at single cell level. The principle is that the separated trace whole genome DNA of a single cell is amplified, a complete genome with high coverage rate is obtained, and then the complete genome is captured through an exon, so that high-throughput sequencing is used for revealing cell population difference and cell evolution relation.

Whole genome amplification techniques are largely divided into two types: one is PCR-based amplification techniques based on thermal cycling, such as degenerate oligonucleotide primer PCR (DOP-PCR), ligation mediated PCR (LM-PCR), pre-amplification primer extension reaction (PEP), etc.; one is isothermal reaction-based amplification techniques that are not based on PCR, such as Multiple Displacement Amplification (MDA) and primer-enzyme based whole genome amplification (pWGA).

In both types, PCR amplification is classical, but the efficiency of PCR amplification varies considerably for different sequences, subject to amplification bias problems: such as non-random loss of CG-rich DNA sequences and corresponding loci, non-random loss of alleles, and size-related deviations of DNA fragments (tending to amplify more short fragments), especially when applied to single cells, the large number of short fragments resulting in loss of sequence between fragments, thereby limiting their application

MDA is the best single cell genome amplification technology recognized at present, can carry out high-fidelity uniform amplification on a whole genome, can amplify fragments with the size of 10-100 kb, and can provide a large amount of uniform and complete whole genome sequences. However, when the sample size is reduced to a single cell, accurate amplification cannot be obtained, and a technical barrier exists for the single cell genome amplification requirement at present. The method of MALBAC has the defects of complex operation, low coverage rate, error introduced by PCR and the like. For clinical application, there are some problems including complicated procedures, low coverage, errors, etc.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an MDA-based whole genome amplification method, which can effectively amplify whole genomes of single cells and provide sufficient DNA for subsequent detection. The method overcomes the problem of low coverage rate, reduces the error rate compared with the PCR method, is simple and convenient to operate, and is more convenient to adopt for clinical detection.

The technical scheme of the invention is as follows: a method for MDA-based whole genome amplification comprising the steps of,

1) separating and extracting single cells;

2) extracting genomic DNA of the single cell; there are many methods for extracting genome DNA of a single Cell, such as Cell Search method and microfluidic chip extraction method. The method for obtaining the single cell genome DNA can also comprise the steps of adding cell lyase and lysate, standing for 10 minutes, and obtaining the genome DNA by cell lysis;

3) diluting the single cell genome DNA obtained in the step 2) with a diluent to obtain a DNA template, and performing multiple displacement amplification by using a Haplox method single cell whole genome amplification kit

4) Purifying the product obtained in the step 3) to obtain purified DNA.

Further comprising the step 5) of sequencing the resulting purified DNA.

The specific steps of the Haplox method single cell whole genome amplification kit for carrying out multiple displacement amplification are as follows:

preparing a mixed solution A: adding a random primer octamer and a DNA template into an amplification system, wherein the concentration of the random primer octamer is 40 mu M, and obtaining a mixed solution A;

preparing a mixed solution B: mix freshly 10 Xphi 29 Buffer,1mM dNTPs,0.1mM dUTP, H2O, and Phi29 Polymerase (10U/. mu.L, Enzymatics) to give mixture B; storing at 4 deg.C;

the reaction proceeds as follows: mixing the mixed solution A and the mixed solution B in equal volume, adding the mixed solution A and the mixed solution B into a reaction tube, reacting for 4-10 hours at 30 ℃, and heating to 65 ℃ to inactivate enzyme;

test DNA concentration: subsequently 50uL of H was added to the reaction tube₂O, the DNA concentration was tested to 300-3000 ng DNA using a Qubit 2.0 fluorometer.

Preferably, when the reaction is carried out, the mixed solution A and the mixed solution B are mixed in equal volume and added into the reaction tube, then mineral oil is added to cover the reaction solution, and then the reaction is carried out at 30 ℃.

Preferably, after the reaction is finished, heating to 65 ℃ to inactivate the enzyme, then adding 50uL of H2O into the reaction tube, uniformly mixing, quickly centrifuging, transferring the mixed solution onto a clean and sterile sealing film, wherein the liquid phase is droplet and the oil phase is dispersed on the sealing film; the droplets were pipetted into a new sterile cuvette using a pipette and the DNA concentration was tested to 300-3000 ng DNA using a Qubit 2.0 fluorometer.

The method for purifying DNA selects Agencour AMPure XP kit or Qiagen Mini PCR column of Backman.

The invention has the beneficial effects that: the MDA-based whole genome amplification method can effectively amplify the whole genome of a single cell and provide enough DNA for subsequent detection. Moreover, the method overcomes the problem of low coverage, and reduces the error rate compared with the PCR method. The method is simple and convenient to operate and more convenient for clinical detection.

Drawings

FIG. 1 shows the results of comparison of the sequencing coverage of MDA amplification, MALBAC amplification and general PCR amplification.

FIG. 2 shows the results of comparing the sequencing error rates of MDA amplification, MALBAC amplification and general PCR amplification.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following examples are intended to further illustrate specific ways of carrying out the invention and should not be construed as limiting the invention. Modifications of the invention will occur to those skilled in the art without departing from the spirit and principles of the invention, which are intended to be covered by the following claims.

Examples

In the present application, the practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA and immunology, which are conventional and well within the skill of the art. These techniques are described in detail in the following documents: molecular Cloning, described by Sambrook et al, Alaboratory Mannual, second edition (1989); nucleic Acid Hybridization (b.d. hames and s.j. higgin, eds., 1984); immunochemical Methods in Cell And Molecular Biology (Academic Press, London).

Example 1:

a method for MDA-based whole genome amplification comprising the steps of,

1) separating and extracting single tumor cells;

2) extracting the genomic DNA of the single tumor Cell by using the method of Cell Search;

3) taking the single cell genome DNA obtained in the step 2) as a template, and respectively carrying out Multiple Displacement Amplification (MDA) by using a Haplox method single cell whole genome amplification kit, specifically comprising the following steps:

preparing a mixed solution A: adding a random primer octamer and a genome DNA template into an amplification system, wherein the concentration of the random primer octamer is 40 mu M, and obtaining a mixed solution A;

preparing a mixed solution B: mix freshly 10 Xphi 29 Buffer,1mM dNTPs,0.1mM dUTP, H2O, and Phi29 Polymerase (10U/. mu.L, Enzymatics) to give mixture B; storing at 4 deg.C;

the reaction proceeds as follows: mixing the mixed solution A and the mixed solution B in equal volume, adding into a reaction tube, adding mineral oil to cover the reaction solution, reacting at 30 ℃ for 4-10 hours, and heating to 65 ℃ to inactivate enzyme;

test DNA concentration: followed byAdding 50uL H into the reaction tube₂O, uniformly mixing, quickly centrifuging, transferring the mixed solution onto a clean and sterile sealing film, wherein the liquid phase can present water droplet drops, and the oil phase can be dispersed onto the sealing film; the droplets were pipetted into a new sterile cuvette using a pipette and the DNA concentration was tested to 300-3000 ng DNA using a Qubit 2.0 fluorometer.

4) Purifying the products obtained in the step 3) respectively to obtain purified DNA;

5) the resulting purified DNA was sequenced separately, the sequencing results were compared to the template DNA sequence, the sequencing coverage and error rates were calculated, the data were recorded as in Table 1, and the data in Table 1 were plotted as in FIG. 1 and FIG. 2.

TABLE 1 comparison of the Haplox method Single cell Whole genome amplification coverage and error Rate

The comparative sequencing result shows that the Multiplex Displacement Amplification (MDA) is carried out by using the Haplox method single cell whole genome amplification kit, the sequencing coverage rate is higher than 95%, while the sequencing coverage rate of the amplification by the MALBAC method is 90-93%, and the sequencing coverage rate of the amplification by the common PCR is not higher than 85%.

The comparative sequencing result shows that the Multiplex Displacement Amplification (MDA) is carried out by using a Haplox method single cell whole genome amplification kit, and the sequencing error rate is 1X10^-6The sequencing error rate of the amplification by the MALBAC method and the amplification by the common PCR method is 1X10^-4And 1x10^-5。

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the present invention pertains, the architecture form can be flexible and varied without departing from the concept of the present invention, and a series of products can be derived. But rather a number of simple derivations or substitutions are made which are to be considered as falling within the scope of the invention as defined by the appended claims.

Claims (7)

1. A method for amplifying the whole genome of a single cell, which is an amplification method capable of amplifying the whole gene of a single cell to provide a sufficient amount of DNA for detection, comprising: separating and extracting single cells, wherein the single cells are tumor cells; extracting whole genome DNA of the single cell; diluting the whole genome DNA with a diluent to obtain a DNA template, mixing the DNA template with a random primer octamer to obtain a mixed solution A, mixing 10 xPhi 29 Buffer, dNTPs, dUTP, Phi29 Polymerase and water to obtain a mixed solution B, adding the mixed solution A and the mixed solution B into a reaction tube in the same volume to form a reaction solution, adding mineral oil to cover the reaction solution to perform multiple displacement amplification reaction to obtain an amplification product, wherein in the reaction solution, the concentration of the random primer octamer is 20 muM, the concentration of the dNTPs is 0.5mM, the concentration of the dUTP is 0.05mM, and the concentration of the Phi29 Polymerase is 5U/muL, the conditions of the amplification displacement reaction are that the reaction is carried out at 30 ℃ for 4-10 hours, after the multiple displacement amplification reaction is completed, the reaction solution is heated to 65 ℃ to inactivate the enzyme, then 50 muL of water is added into the reaction tube, the mixture is uniformly mixed and quickly centrifuged, transferring the reaction solution to a sealing film to disperse the mineral oil into the sealing film, then sucking the reaction solution into a small test tube, and then testing by using a Qubit 2.0fluorometer to obtain the DNA concentration of the amplification product; and purifying the amplification product to obtain purified DNA.

2. The amplification method according to claim 1,

the method for purifying DNA selects Agencour AMPure XP kit or Qiagen Mini PCR column of Backman.

3. The amplification method according to claim 1,

the method for extracting the whole genome DNA of the single Cell is a Cell Search method or a microfluidic chip extraction method.

4. The amplification method according to claim 1,

the method for extracting the whole genome DNA of the single cell comprises the following steps: adding cell lyase and a lysis solution to the single cell, standing for 10 minutes, and obtaining whole genome DNA by cell lysis.

5. The amplification method according to claim 1,

the purified DNA was also subjected to sequencing analysis in which the sequencing coverage was higher than 95% and the sequencing error rate was 1X10^-7To 1X10^-6。

6. The amplification method according to claim 1,

the DNA concentration is 300 to 3000 ng.

7. A kit for amplifying the whole genome of a single cell,

use of the amplification method of any one of claims 1 to 6.

Images