CN112251500A

CN112251500A - Method capable of improving amplification efficiency of trace nucleic acid

Info

Publication number: CN112251500A
Application number: CN202011135366.1A
Authority: CN
Inventors: 涂景; 乔祎; 陆祖宏; 龙乃云; 罗雨菡
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-01-22

Abstract

The invention discloses a method capable of improving the amplification efficiency of trace nucleic acid, which is characterized by comprising the following steps: when an amplification solution system is configured before amplification begins, all trace nucleic acid substances are put into the reaction system at one time, and only part of other substances for amplification except nucleic acid are put into the reaction system, so that the initial state nucleic acid concentration is improved, and the low efficiency time of the initial reaction stage is shortened; after the amplification reaction is started, the remaining other substances for amplification are additionally added, and the nucleic acid amplification is continued after the additional addition, thereby reducing the concentration of the nucleic acid in the system and prolonging the efficient time of the reaction. The method improves the efficiency of nucleic acid amplification reaction and shortens the reaction time by changing the adding time of other reaction components except trace nucleic acid, is suitable for different amplification methods with different volumes and nucleic acid initial quantities, and can flexibly adjust the volume of each stage according to the using condition.

Description

Method capable of improving amplification efficiency of trace nucleic acid

Technical Field

The invention relates to a nucleic acid amplification method, in particular to a method capable of improving the amplification efficiency of trace nucleic acid.

Background

The amplification of trace nucleic acid has important significance in the fields of life science, forensic identification and the like. The level of trace nucleic acid molecules from a sample is often too low to allow direct correlation detection or efficient participation in biological reactions, and therefore amplification is required before downstream experimentation can be performed. However, since the amount of some samples (e.g., trace amounts of DNA, genome of a single cell) is very low, it is difficult to amplify them in large amounts in a short time even in a nucleic acid amplification reaction. Amplification reactions under these conditions often require a long wait to obtain a sample with a higher amplification factor. This greatly hinders the development of rapid detection in the fields of clinical and forensic sciences.

The amplification rate of a nucleic acid amplification reaction is not constant, and the rate depends mainly on the concentration of nucleic acid in the reaction when an excess of other substances is present. The concentration of nucleic acid in the initial state of the amplification reaction is low, but the amount of other components used for amplification (e.g., inorganic salts, water, single-stranded primers, oligonucleotides, fluorescent dyes, DNA polymerase, etc.) is high and in excess. While the excess components favor the forward direction of the reaction when the reaction enters a rapid stage, they occupy a large part of the volume of the reaction system in the initial stage, resulting in further dilution of the concentration of trace nucleic acid, which is unfavorable for the start of the reaction, resulting in inefficiency in the initial stage of amplification. In addition, in a nucleic acid amplification reaction, nucleic acid as a product is often also a template in subsequent amplification. Under this premise, after the nucleic acid is amplified to a higher concentration, the reaction is limited from proceeding forward, so that the amplification reaction at higher efficiency becomes inefficient.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method capable of improving the amplification efficiency of trace nucleic acid.

The technical scheme is as follows: a method for increasing the efficiency of amplification of a trace amount of nucleic acid, wherein when an amplification solution system is prepared before the start of amplification, all of a trace amount of nucleic acid material is put into a reaction system at one time, only a part of other material for amplification is put into the reaction system, the remaining other material for amplification is additionally put into the reaction system after the start of amplification, and nucleic acid amplification is continued after the additional input.

Further, the additional input is performed at least once or continuously by fluid injection, and the volume of each additional input is the same or different.

Further, the other substances for amplification include inorganic salts, water, single-stranded primers, oligonucleotides, fluorescent dyes, DNA polymerases, organic molecules not directly participating in the reaction.

Further, before additional charging of the other substances for amplification, the portion of the reaction system having a volume ratio of less than 99.9999999% is discarded, and the additional charging operation is performed.

Further, when additional input of the rest of the other substances for amplification is started, the concentration of the nucleic acid in the reaction system is already amplified to 1.001 to 1000 times the initial concentration.

Further, after each additional input of the remaining other substances for amplification, the nucleic acid concentration in the reaction system was decreased to 0.001 to 0.999 times that before the additional input.

Further, the ratio of the volume of a part of the reaction substances other than nucleic acid added at each addition to the total volume of all the reaction substances other than nucleic acid amplified at the time of the amplification was 10^-9-1. Further, the volume of the aqueous phase portion containing the reaction solution at the start of the reaction is 10fL to 100. mu.L, and the volume of the system at the end of the reaction is increased to 1.1 to 10 at the start of the reaction⁹And (4) doubling.

Further, the reaction system is a single-phase system composed of only the reaction solution or a two-phase system composed of the reaction solution and an immiscible other liquid or solid.

Further, the mass of the trace nucleic acid before the start of amplification was 10^-7-10⁵Within the range of pg.

Further, the amplification includes MDA (multiple strand displacement amplification), PCR (polymerase chain reaction), RCA (rolling circle amplification), DOP-PCR (degenerate oligonucleotide primer polymerase chain reaction), MALBAC (multiple annealing circular cycle amplification), LAMP (loop-mediated isothermal amplification).

In the above technical scheme:

the method improves the amplification efficiency of nucleic acid and shortens the reaction time by changing the adding time of other reaction components except trace nucleic acid. This method is characterized in that, when the amplification solution system is disposed, a small amount of the nucleic acid substance is all charged into the reaction system at one time, only a part of the other substance for amplification is charged, the other substance for amplification is additionally charged after the start of the amplification reaction, and the concentration of the nucleic acid in the system is decreased after the additional charging process, thereby amplifying the nucleic acid.

In this method, since the whole amount of the nucleic acid to be amplified is charged at the beginning of the reaction, but not all other reaction substances are charged, the concentration of the nucleic acid in the initial state is increased and the excess amount of other substances is low. Thus, the nucleic acid concentration determining the amplification reaction rate is higher, and the reaction can be carried out faster to a rapid stage. The other reaction substances added thereafter dilute the nucleic acid concentration which has become too high to be useful for amplification, and delay the reaction rate-decreasing process. The other substances for amplification to be added after the start of the amplification reaction include one or more of inorganic salts, water, single-stranded primers, oligonucleotides, fluorescent dyes, DNA polymerase, and organic molecules not directly involved in the reaction. This is because reducing the volume of any reactant other than nucleic acid during the initial phase of the reaction is beneficial to increasing the nucleic acid concentration, and although it is theoretically most effective to reduce the volume of all reactants other than nucleic acid template, in practice fine tuning is required to ensure that the reduction of reaction components does not reduce the amplification efficiency of the reaction from the mechanism of the biomacromolecule. Before additional feeding of the other substances for amplification, the additional feeding operation may be performed after discarding a portion of 0.001 to 0.999 in volume of the conventional reaction system. This can achieve the purpose of saving reagent. If existing nucleic acids have been extensively amplified, appropriate discarding procedures will only reduce the number of copies of the nucleic acid molecule, and will not lose the unique fragment.

Even with an increase in the initial nucleic acid concentration, the reaction may still take some time to reach a high lifetime. At this time, the concentration of the nucleic acid in the amplification system has changed, and when other reagents for amplification are supplied, the concentration of the nucleic acid in the system should have been amplified to 1.001 to 1000 times the initial concentration. After the other substances for amplification are additionally added, the concentration of the nucleic acid in the system is reduced to 0.99 to 0.001 times that before the additional addition process, so as to exhibit the dilution effect. In order to accurately quantify the change of the concentration of the nucleic acid, the concentration of the nucleic acid in the system can be measured by measuring the intensity of the emission light of the fluorescent dye and ultraviolet absorption spectroscopy during the reaction.

The method for sample adding in batches can be compatible with various reaction system configurations, sample adding volumes and times. The volume of the aqueous phase portion containing the reaction solution at the start of the reaction is 1fL to 100. mu.L, preferably 1pL to 1. mu.L, and the volume of the system at the end of the reaction is increased to 1.1 to 10 at the start of the reaction¹¹And (4) doubling. The reaction system may be a single-phase system composed of only the reaction solution or a two-phase system composed of the reaction solution and other liquid or solid which is not miscible. The additional charging process is performed at least 1 time, and may be performed 1 to 1000 times, respectively, or may be performed by continuous injection of a fluid. Namely, a small-volume aqueous phase amplification system can be dispersed in an immiscible continuous phase, and the additionally-added system sample adding is completed by means of a microfluidic chip, a capillary tube, a microneedle and the like in the amplification process; alternatively, the amplification reaction may be carried out in a larger volume of continuous aqueous phase, and additional system loading may be accomplished by means of a pipette, capillary, syringe, or the like. According to different sample adding modes, the finishing times can be freely adjusted, and the fluctuation range of the dilution multiple is larger. In some means, new reaction components not containing nucleic acid can be continuously injected during the reaction, and the number of times of sample injection should be counted as 1 time.

The mass of the trace nucleic acid material before the start of amplification was 10 according to the initial reaction volume and the sample source^-7-10⁵Within the range of pg. The amplification method can select MDA (multiple strand displacement amplification), PCR (polymerase chain reaction), RCA (rolling circle amplification), DOP-PCR (degenerate oligonucleotide primer polymerase chain reaction), MALBAC (multiple annealing circular amplification) and LAMP (loop-mediated isothermal amplification). Because the change of reaction components is not involved, the amplification method can be used by combining different amplification methods and reagent combinations, and the universality is strong.

Has the advantages that: the amplification method of the present invention reduces the total time required for amplification by 1/3 or more, compared to ordinary amplification without using the method. Meanwhile, the enlarged reaction system allows the amplification reaction to be further carried out in the forward direction, so that a larger amplification multiple is achieved, and the amplification efficiency is improved. The method is suitable for different amplification methods with different volumes and nucleic acid initial quantities, and the volume of each stage can be flexibly adjusted according to the use condition.

Drawings

FIG. 1 shows the change in volume of the amplification system in example 1;

FIG. 2 is a graph showing changes in the concentration of nucleic acid in the amplification system in example 1;

FIG. 3 is a photograph of a micro-droplet reaction chamber of example 2, in which an aqueous phase reaction solution is dispersed in a continuous HFE-7500 oil phase.

Detailed Description

Example 1:

principle verification experiment: configuring the difference between the two initial nucleic acid concentrations to be 10⁴The MDA reaction was carried out in 2 ng/. mu.l (group 1) and 0.2 pg/. mu.l (group 2) systems, respectively, and the specific formulations are shown in the following table.

The MDA amplification reaction was started by placing the group 1 and group 2 solutions in a 30 degree celsius environment. During the MDA amplification process, the system of the group 1 is diluted for 10 times⁴Double, so that its initial concentration can be equivalent to 0.2 pg/. mu.l, the same as in group 2. The diluted solution was the same concentration as each component in the MDA reaction system but without the template (see the above table for formulation), and the number of dilutions was set to 7. At each dilution, 2.2. mu.l of the reaction solution (the remainder was discarded) was taken out and 6. mu.l of the solution used for dilution group 1 was mixed, i.e., one dilution-3.727-fold. After 7 times of dilution operation, the dilution multiple reaches 9994 times, and approximately reaches the requirement of 10000 times of dilution. The dilution process for group 1 is shown in figure 1 as a black line; group 2 was not diluted, see grey line in figure 1. At the end of the reaction, the results in group 1 correspond to similar amounts of starting nucleic acid to those in group 2, since some template is discarded with each dilution.

The EVAgreen fluorescence increases with the increase of DNA concentration during amplification, and is measured using a fluorescence quantitative PCR instrument during MDA to characterize the change of DNA concentration. The fluorescence quantification of MDA is shown in FIG. 2, where a high initial concentration of nucleic acid (black curve in FIG. 2, group 1) appears as a significant "head-up" shortly after the start of the reaction, greatly reducing the time to inefficiency in the early stages of the reaction (compare the gray curve in FIG. 2, group 2).

After a significant, large increase in concentration (126 minutes after the start of the reaction) had begun, a stepwise dilution step was started, each dilution being indicated by an arrow in the figure. Each dilution was 16 minutes apart. The gray curve in FIG. 2 is a control 0.2 pg/. mu.l starting MDA reaction which is not diluted throughout the reaction, but when other samples are taken for dilution, this sample is also taken together, so that the reaction conditions between the groups are as consistent as possible. The fluorescence of the reaction system is in a period of high-speed increase between every two dilutions, which represents the dilution effect: so that the amplification reaction which is possible to slow down can be maintained at high speed for a longer time, and the aim of accelerating MDA is achieved.

In this experiment, the stepwise dilution method of rapid MDA only took 280 minutes to amplify nucleic acid to near saturation, while the low initial concentration MDA reaction, which was diluted at the time of preparation, took 1100 minutes to achieve similar effect.

Example 2:

a nucleic acid solution of 15kbp in DNA molecular length was used to prepare an MDA system with an initial nucleic acid concentration of 1.7 pg/. mu.l, and a nucleic acid-free system was prepared for subsequent input, and the specific formulation is shown in the following table.

The experimental group solution (2. mu.l) was then added to 20. mu.l of HFE-7500 oil phase using microfluidic confocal means, forming droplets with an average volume of 1pL (12.4 μm diameter) (see FIG. 3). The liquid is then placed in an environment of 30 ℃ to carry out the MDA reaction. Due to the poisson distribution, it can be calculated that 10% of all droplets contain nucleic acid template, whereas 90% do not, amplification cannot start, i.e. the effective initial MDA system volume is 0.2 μ Ι.

After the reaction is carried out for 1 hour, the emulsion breaking operation is carried out on all the liquid drops by using an alternating current electric field. The solution in the template-free droplet at this time diluted the amplified template-containing solution by a factor of 10. Meanwhile, the liquid drop system is changed into an oil-water layered system, and the water is connected into a whole. After further 30 minutes of MDA, 8. mu.l of the additional input solution was added to the aqueous phase, and the volume of the aqueous phase was changed from 2. mu.l to 10. mu.l, whereby the DNA in the system was diluted 5-fold. After MDA was carried out for another 20 minutes, 30. mu.l of the additional solution was added to the aqueous phase, and the volume of the aqueous phase was changed from 10. mu.l to 40. mu.l, whereby the DNA in the system was diluted 4-fold. After 40 minutes the amplification was terminated and the aqueous phase product was collected.

In this example, the volume of MDA containing the template in the initial state was 0.2. mu.l, the remaining additional substances used for amplification were added 3 times, and the volume of MDA solution containing DNA at the end was 40. mu.l, which was a 200-fold dilution ratio.

Example 3:

a PCR reaction system was prepared according to the following formulation.

The temperature cycling settings for the PCR reactions are as follows:

according to this formulation, the reaction substances other than nucleic acid at the beginning of the reaction were added only to 1/20 in total amount, and the initial concentration of nucleic acid was increased by 5 times. According to the reaction principle of quantitative PCR, the time for the PCR reaction with a high initial nucleic acid concentration to enter a significant amplification stage (i.e., "head-up time") is linearly shortened, which is expected to be shortened by 5 cycles. After the reaction was carried out for 10 temperature cycles, all of the remaining "additional input solution" was added, amplification was continued for 10 temperature cycles, and the amplification reaction was terminated.

The initial nucleic acid template with relatively high concentration has less base line time length with relatively low PCR efficiency, and after the reaction enters into fast amplification period, the nucleic acid concentration is increased exponentially. After the reaction is finished, compared with the traditional PCR system in which all reaction components are put in at one time, the PCR reaction with improved efficiency saves 5 temperature cycle times with lower initial efficiency, and increases the reaction efficiency.

Example 4:

mu.l of a standard MDA nucleic acid amplification system was prepared and 100pg of the starting nucleic acid template was added (i.e., the initial concentration reached 10 pg/. mu.l). The amplification system described above was used to generate a large number of microdroplets (i.e., about 2.7 μm in diameter) of 10fL in volume using a microfluidic chip. Subsequently, 1 individual droplet was picked up under the microscope using a capillary tube and injected into the clean HFE-7500 oil phase system, and mineral oil was added thereto to prevent evaporation of the water phase. At this time, this system contained only a 10fL aqueous phase volume and 10^-7The pg nucleic acid template ensures that only a very small number of random nucleic acid fragments exist in the initial stage, and the lower number of random nucleic acid fragments is convenient for the subsequent sequencing analysis of the amplification result and the detection of base pairing errors occurring in the amplification process. The temperature was adjusted to 30 degrees celsius to start amplification.

After 30 minutes of amplification, 10. mu.l of an MDA reaction solution containing no nucleic acid was added to the interface between the mineral oil and HFE-7500 (i.e.the layer on which the droplets were deposited) using a pipette gun, i.e.the volume was enlarged by 10⁹The reaction was terminated after amplification was carried out for 60 minutes. Standard library construction and sequencing procedures were followed.

Claims

1. A method capable of improving amplification efficiency of trace nucleic acid, comprising: when the amplification solution system is disposed before the initiation of amplification, a small amount of nucleic acid substances are all put into the reaction system at one time, only a part of the substances other than nucleic acid for amplification are put into the reaction system, the remaining substances for amplification are additionally put into the reaction system after the initiation of amplification reaction, and nucleic acid amplification is continued after the additional input.

2. The method according to claim 1, wherein the method comprises: the additional input is at least one time or is continuously injected by fluid, and the volume of each additional input is the same or different.

3. The method according to claim 1, wherein the method comprises: the other substances for amplification than nucleic acid include inorganic salts, water, single-stranded primers, oligonucleotides, fluorescent dyes, DNA polymerases, organic molecules not directly involved in the reaction.

4. The method according to claim 1, wherein the method comprises: before additional charging of the other substances for amplification, the current reaction system is discarded in a portion of less than 99.9999999% by volume, and the additional charging operation is performed.

5. The method according to claim 1, wherein the method comprises: adding other substances except the nucleic acid for amplification, wherein the concentration of the nucleic acid in the reaction system is already amplified to be 1.001-1000 times of the initial concentration; after each additional input of the other substances for amplification other than the nucleic acid, the concentration of the nucleic acid in the reaction system is reduced to 0.001 to 0.999 times that before the additional input.

6. The method according to claim 1, wherein the method comprises: in the arrangement before the start of amplification, the ratio of the volume of a part of the reaction substances other than nucleic acid added at each additional time to the total volume of all the reaction substances other than nucleic acid added at the time of amplification was 10^-9-1。

7. The method according to claim 1, wherein the method comprises: at the beginning of the reactionThe volume of the aqueous phase portion containing the reaction solution is 10fL to 100. mu.L, and the volume of the system increases to 1.1 to 10 at the beginning of the reaction at the end of the reaction⁹And (4) doubling.

8. The method according to claim 1, wherein the method comprises: the reaction system is a single-phase system composed of only the reaction solution or a two-phase system composed of the reaction solution and other liquid or solid which is not miscible.

9. The method according to claim 1, wherein the method comprises: the trace amount of nucleic acid had a mass of 10 before the start of amplification^-7-10⁵Within the range of pg.

10. The method according to claim 1, wherein the method comprises: the nucleic acid amplification includes MDA (multiple strand displacement amplification), PCR (polymerase chain reaction), RCA (rolling circle amplification), DOP-PCR (degenerate oligonucleotide primer polymerase chain reaction), MALBAC (multiple annealing circular amplification), LAMP (loop-mediated isothermal amplification).