DNA nanosphere loading method

Abstract

The invention relates to the technical field of biology, in particular to a DNA nanosphere loading method. The invention provides a method for improving the loading efficiency of DNA nanospheres and a method for loading the DNA nanospheres. According to the method disclosed by the invention, phi29 priming is added, so that phi29 is combined with the amino surface of the chip in advance, and the DNB loading efficiency is improved; the post-loading process is simplified, but the reservation of necessary steps is ensured, and the DNB post-loading quality is not reduced; by utilizing the biochemical principle of complementary pairing of bases, the process of primer hybridization is improved, and the hybridization efficiency is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a DNA nanoball loading method. Background Art

[0002] DNA nanoball sequencing (DNA nanoball, DNB sequencing) is a high-throughput sequencing technology that can be used to determine the arrangement and composition of the whole genome sequence of living organisms. The core of this technology is to circularize genomic DNA fragments into single-stranded circular DNA, and then use rolling circle amplification (RCA) technology to make the circular single-stranded DNA form multiple copies of single-stranded DNA connected end to end, and freely fold into a nanoball structure in the solution, namely DNA nanoball (DNB). Due to the mutual repulsion of the negative charges carried by DNA nanoballs, the interaction between DNB individuals can be reduced, making DNB individuals independent of each other. The matrix column sequencing technology based on DNA nanoballs makes the DNBs at each matrix point have at least several hundred copies, and these copies gather together to produce strong signals, so that the sequencing results of DNBs can be obtained by sequencing. However, the loading process in the sequencing process has many types of reagents, cumbersome steps, and long time consumption. The natural primer hybridization at room temperature is time-consuming and inefficient. Summary of the invention

[0003] In view of this, the DNA nanoball loading method provided by the present invention improves the DNB loading efficiency, simplifies the post-loading process without reducing the DNB post-loading quality, and improves the primer hybridization efficiency.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides DLB having a pH of 4.8 and a potassium ion concentration of 1531-1593 mM.

[0006] The present invention also provides the use of the DLB in improving the loading efficiency of DNA nanospheres.

[0007] The present invention also provides a method for loading DNA nanoballs, comprising: contacting a reagent containing phi29 with an amino-modified sequencing chip without incubation, and then contacting a solution containing DNA nanoballs with the sequencing chip, incubating for 5 minutes, and completing the loading;

[0008] The phi29-containing reagent includes the above-mentioned DLB;

[0009] The solution containing DNA nanospheres includes the above-mentioned DLB.

[0010] In some specific embodiments of the present invention, the reagent containing phi29 in the above method comprises 50 μl H2O, 50 μl DNB preparation buffer, 2 μl phi29 and 33 μl of the above DLB.

[0011] In some specific embodiments of the present invention, in the above method, after incubation for 5 minutes and before loading, a step of washing the sequencing chip is further included.

[0012] In some specific embodiments of the present invention, the step of cleaning the sequencing chip in the above method comprises:

[0013] Step (1): using PWB to clean the sequencing chip without incubation;

[0014] Step (2): taking DCB to clean the sequencing chip without incubation;

[0015] Step (3): taking SB1 to clean the sequencing chip;

[0016] and:

[0017] The step of taking out DCB multiple times to clean the sequencing chip is not included;

[0018] The step of taking REB to clean the sequencing chip is not included;

[0019] The step of taking PAW to clean the sequencing chip is not included;.

[0020] In some specific embodiments of the present invention, the above method comprises:

[0021] The volume of the PWB comprises 200 μl; and / or

[0022] The volume of DCB comprises 100 μl; and / or

[0023] The volume of SB1 comprises 250 μl; and / or

[0024] The flow rate of the wash comprised 100 μl / min.

[0025] In some specific embodiments of the present invention, in the above method, after cleaning the sequencing chip and before completing the loading, it also includes a step of combining the sequencing chip with primers.

[0026] In some specific embodiments of the present invention, in the above method, the binding conditions are 55°C for 1 min and 30°C for 1 min.

[0027] In some specific embodiments of the present invention, in the above method, the flow rate of the primers in the binding is 100 μl / min.

[0028] The DNA nanosphere loading method of the present invention has the following effects:

[0029] The new DLB formula determined in this scheme, as well as the DNB loading, post-loading and primer hybridization methods, can complete chip preparation within 20 minutes and meet data indicators such as BIC, FIT, and ESR.

[0030] 1. Add phi29 priming to pre-bind phi29 to the amino surface of the chip to improve DNB loading efficiency;

[0031] 2. Simplify the post-loading process, but ensure that the necessary steps are retained and the quality of DNB post-loading is not reduced;

[0032] 3. Utilize the biochemical principle of complementary base pairing to improve the primer hybridization process and increase hybridization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0034] Figure 1A to Figure 1E The results of various post-loading indicators after reducing the post-loading steps are shown; among them, Figure 1A It shows that the post-loading signal is stronger after reducing the post-loading step; Figure 1B It shows that the SNR of the second chain is higher after reducing the post-loading step; Figure 1C It shows that the FIT values ​​after reducing the post-loading step are roughly the same; Figure 1D It shows that the BIC values ​​after reducing the post-loading step are roughly the same; Figure 1E It shows that the ESR value after reducing the post-loading step is roughly the same;

[0035] Figure 2A to Figure 2C The heatmap of pH and salt ion concentration on loading efficiency is shown; Figure 2A Show BIC; Figure 2B Show FIT; Figure 2C Indicates ESR. DETAILED DESCRIPTION

[0036] The present invention discloses a method for loading DNA nanospheres, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0037] The DNB loading technology used in the present invention mainly utilizes the characteristics of phi29 that it can perform MDA (multiple strand displacement polymerization) and bind to the amino surface of the chip under specific conditions, so that the DNB obtained by RCA is bound to the chip to achieve position fixation, and then dNTP polymerization reaction is performed and signals are collected to achieve sequencing function.

[0038] Current DNB loading and post-loading technologies (see Table 1):

[0039] Loading: Add 1 / 3 volume of DLB ​​with pH 4.7 and 2 μl of phi29 to the prepared DNB, mix well and load onto the chip, incubate for 30 min;

[0040] Loading & Post-loading process (see Table 1);

[0041] Primer hybridization: 30 min at room temperature.

[0042] Table 1: Original DNB loading & post-loading process

[0043]

[0044] The present invention optimizes and improves the original scheme from the process and method of DNB loading and post-loading:

[0045] Adding phi29 priming step in loading process improves loading efficiency and improves AT separation of human library;

[0046] Post-loading step: eliminates a lot of unnecessary pumping steps and incubations, greatly reducing operation time;

[0047] Primer hybridization step: utilizes the biochemical principle of primer binding to template sequence, changes the hybridization temperature conditions, shortens the primer hybridization time, and improves the hybridization efficiency.

[0048] The present invention optimizes and improves the original scheme from the aspects of the process and method of DNB loading and post-loading and the DNB loading reagent:

[0049] Process Optimization:

[0050] DNB loading: Add phi29 priming, and then perform DNB loading without incubation. After loading, incubate for 5 minutes, and then enter the post-loading process. Reduce the DNB volume and increase the flow rate, reducing the original 33-minute DNB loading process to 7 minutes;

[0051] DNB post-loading: Remove the cumbersome steps (different pumping steps and incubations), retain only the necessary steps, use Wash Reagent 1 instead of REB, and reduce the post-loading process to PWB-DCB-SB1. Optimize the volume and flow rate. After the improvement, the post-loading time is reduced to 6 minutes.

[0052] Primer hybridization: The original incubation time of 30 min at RT was changed to 1 min at 55°C and 1 min at 30°C, which improved the primer binding efficiency and shortened the time, reducing the primer time from 32 min to 4 min.

[0053] The entire process was reduced from 100 minutes to 17 minutes, and the actual operation was completed within 20 minutes.

[0054] New loading & post-loading process (see Table 2).

[0055] Table 2: New DNB loading & post-loading scheme

[0056]

[0057] Reagent Optimization:

[0058] Original DNB loading buffer: pH 4.7, K + Concentration 1021mM, improved pH 4.8, K + The concentration was 1593 mM. After the improvement, the fast loading and post-loading ESR increased by about 2% compared with the control reagent.

[0059] Reagents:

[0060] The use of salt ions can deprive the protein of the outer hydration layer and increase the K+ concentration, making phi29 more reactive and improving DNB loading efficiency in the same process.

[0061] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0062] The present invention will be further described below in conjunction with embodiments:

[0063] Example 1

[0064] Equipment and materials:

[0065] BGI-SEQ500 DNB preparation kit, BGI-SEQ500 SE100 kit, PCR instrument, DNB loader, BGISeq500 sequencer, E160 library, Qubit3.0 fluorescence quantification instrument, Qubit TM ssDNA Assay Kit.

[0066] Priming reagent: 50μl H2O + 50μl DNB preparation buffer + 33μl DLB + 2μl phi29

[0067] DIY post-loading Kit: PWB DCB WB1 Primer

[0068] DNB preparation and loading, chip loading process:

[0069] ① Take 40fmol of ssCircle DNA, add 20μl DNB to prepare buffer, add H2O to make up the volume to 40μl, and mix well;

[0070] ②PCR reaction: 95℃ 1min, 65℃ 1min, 40℃ 1min, maintain at 4℃;

[0071] ③Take out the reaction product of step ②, add 40μl polymerase buffer and 2μl phi29, and mix well;

[0072] ④ Place in PCR instrument and incubate at 30℃ for 20min;

[0073] ⑤ Add 20μl stop buffer, 33μl DLB, 2μl phi29, and mix gently by pipetting 5-6 times with a 200μl pipette tip;

[0074] ⑥Using Qubit3.0 fluorescence quantification instrument, Qubit TMssDNA Assay Kit was used to measure the DNB concentration.

[0075] ⑦DNB loading: Use DIY DNB loading kit (or standard DNB post-loading V3.0KIT) and load DNB with a loading instrument.

[0076] ⑧ Prepare reagents on the machine as required and perform sequencing reactions.

[0077] Example 2: Testing the effect of reducing the DNB incubation time after priming

[0078] The preparation method and sequencing method of DNB are as shown in Example 1, and the test scheme is shown in Table 3, wherein:

[0079] Test group 1: phi29 priming, DNB incubation for 3 min;

[0080] Test group 2: phi29 priming, DNB incubation for 5 min;

[0081] Control group: No phi29 priming was performed and DNB was incubated for 30 min.

[0082] Post-loading process: fast post-loading

[0083] Table 3: Loading scheme

[0084] Chip No. Lane phi29 spiking phi29 priming DNB incubation CL100061799 L01 √ √ 3min Test Group 1 CL100061803 L01 √ √ 5min Test Group 2 L02 √ × 30min Control group

[0085] Compare the differences between phi29 priming and loading for 3min and 5min, and the control group without phi29 priming and loading for 30min.

[0086] The test results are shown in Table 4.

[0087] Table 4: Loading results under different conditions

[0088] Test Group 1 Test Group 2 Control group #BIC 0.963 0.967 0.970 #FIT 0.935 0.929 0.921 #ESR 0.949 0.956 0.966

[0089] The test group that underwent phi29 priming and DNB incubation for 5 minutes showed a 0.3% decrease in BIC, a 1% decrease in ESR, and a 0.8% increase in FIT compared to the control group. The overall results were very good.

[0090] However, the ESR and other indicators of the test group incubated with DNB for 3 minutes decreased significantly, so this condition was not considered for use.

[0091] The overall results show that phi29 priming can effectively improve DNB loading efficiency and reduce DNB incubation time.

[0092] Example 3: Post-loading effect test after reducing the post-loading step

[0093] PE100+10 sequencing was used to compare the stability of fast post-loading and standard post-loading chips in long cycle reactions.

[0094] Reagent conditions:

[0095] PE100+10

[0096] E320 library

[0097] RCA 25min

[0098] Test conditions (see Table 5): Comparison of the differences between conventional post-loading and fast post-loading for PE100+10 reaction, the loading process was conventional DNB 30min incubation.

[0099] Table 5: Test plan

[0100]

[0101] Test results (see Table 6):

[0102] Due to instrument failure, the second-strand primer was pumped incorrectly, so the overall ESR was low, but the overall results of PE100+10, fast post-loading (fast in Table 6) and standard post-loading (control in Table 6) were basically consistent, and Q30% was also 1.76% higher than standard.

[0103] Table 6: Test results

[0104]

[0105] Fast post-loading signal is stronger ( Figure 1A ), the second chain SNR is higher than standard post-loading ( Figure 1B ). FIT( Figure 1C ), BIC( Figure 1D ), ESR( Figure 1E ) are also better maintained in long cycle reactions.

[0106] Conclusion: The overall results show that fast post-loading can shorten the time, has no effect on sequencing quality, and may even promote it.

[0107] Example 4: Primer hybridization condition test

[0108] The preparation method and sequencing method of DNB are as shown in Example 1, and the primer hybridization efficiency under different conditions is tested (see Table 7):

[0109] Test group 1: 55℃ 1min + 30℃ 1min

[0110] Test group 2: RT, 1h

[0111] Test group 3: RT, 3 min

[0112] Test group 4: RT, overnight

[0113] Table 7: Primer hybridization conditions tested

[0114] Group No. Chip No. Lane Annealing condition Test Group 1 1st CL200040069 L01 55℃ 1min+30℃ 1min Test Group 2 CL200040068 L02 RT,1h Test Group 3 2nd CL200040068 L02 RT, 3min Test Group 4 CL200040069 L02 RT,1night

[0115] Four primer hybridization conditions were compared: room temperature for 3 min, 1 h, overnight, and 55°C for 1 min + 30°C for 1 min.

[0116] Reagent conditions:

[0117] Library: E320

[0118] Input:40fmol

[0119] RCA: 25min

[0120] Loading solution: Fast loading&post-loading

[0121] The results are shown in Table 8: The ESR of rapid primer hybridization (test group 1) was 0.09% higher than that of conventional room temperature hybridization for 1 hour (test group 2), 2.6% higher than that of room temperature hybridization for 3 minutes (test group 3), and slightly better than that of overnight hybridization (test group 4).

[0122] Table 8: Test results of different primer hybridization conditions

[0123]

[0124] This experiment determined that 55℃ 1min + 30℃ 1min is a more efficient primer hybridization condition.

[0125] Example 5: Effect of pH and salt ion concentration on loading efficiency

[0126] DLB preparation (see Table 9):

[0127] Preparation of high salt DLB: pH 4.7, K + 1531mM, pH4.8K + 1593mM, compared with commercial DLB pH4.7, K + 1021mM.

[0128] Table 9: DLB preparation

[0129] DLB Citric acid (mM) Tripotassium citrate (mM) <![CDATA[K + (mM)]]> pH 4.7 (high salt) 390 510 1531 pH 4.8 (high salt) 369 531 1593 pH 4.7 (commercial product) 260 340 1021

[0130] pH 4.7 (high salt) and pH 4.8 (high salt) were prepared and tested along with a commercial control.

[0131] Test scheme (see Table 10): fast loading and standard loading were performed with commercial DLB, and two controls were provided; fast loading was performed with two groups of high-salt DLB at pH 4.7 and pH 4.8. The difference between fast loading and standard loading, the difference between high salt and CTRL (commercial control) under fast loading conditions, and the difference between pH 4.7 and pH 4.8 were compared. The preparation method and sequencing method of DNB are shown in Example 1.

[0132] Table 10: DLB conditions and loading conditions

[0133] Slide Lane Condition <![CDATA[K + (mM)]]> CL175484 L01 CTRL-Fast loading 1021 L02 pH4.7-high salt 1531 CL175485 L01 pH4.8-high salt 1593 L02 CTRL-standard loading 1021

[0134] Control groups standard loading&post-loading and Fast loading&post-loading were set up for comparison with high salt conditions DLB pH4.7 and pH4.8 Fast loading&post-loading.

[0135] result:

[0136] From the heatmap ( Figure 2A to Figure 2C ), the ESR of the CTRL DLB+Standard loading group was the most uniform, and the mean value was not low. The pH4.8 group had some entrance loading problems. Overall, the CTRL DLB fast loading result was the worst ( Figure 2C ). The BIC value also shows a consistent trend ( Figure 2A ). The FIT values ​​are slightly different ( Figure 2B ), but the impact of this indicator is not significant.

[0137] From the results of the whole chip (see Table 11), the standard loading ESR is the highest, and the fast loading group pH4.7 high salt group has the highest ESR. The BIC values ​​of the four groups are basically close, and the FIT values ​​are not much different.

[0138] Table 11: Test results

[0139]

[0140] Conclusion: Among the three fast loading groups, the ESR of the pH4.7 high salt group was the highest. However, combined with the heatmap, the pH4.8 group had a lower ESR overall due to the outlet problem. Therefore, when the outlet problem was excluded, the pH4.8 group was better than the pH4.7 group. The pH4.7 group had the same buffer ratio as the CTRL group, so high salt was better than low salt, and pH4.8 was better than pH4.7.

[0141] Based on Example 2, Example 3, Example 4 and this example, all fast loading and fast post-loading conditions were compared with the currently used standard reagent formula, standard loading and post-loading groups, and the ESR of the fast and control groups was reduced by less than 1.6%.

[0142] Using pH 4.8 high salt formula, phi29 priming and 5min DNB incubation, fast post-loading, and fastprimer hybridization, a loading effect that is not much different from the control group can be obtained. In the long cycle and PE sequencing process, it performs better than conventional loading.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. DLB, characterized in that The pH was 4.8 and the potassium ion concentration was 1531-1593 mM.

2. Use of DLB ​​as claimed in claim 1 in improving the loading efficiency of DNA nanospheres.

3. A method for loading DNA nanospheres, characterized in that: include: The reagent containing phi29 is brought into contact with the amino-modified sequencing chip without incubation, and then the solution containing DNA nanospheres is brought into contact with the sequencing chip and incubated for 5 minutes to complete the loading; The phi29-containing reagent comprises the DLB as claimed in claim 1; The solution containing DNA nanospheres comprises the DLB as claimed in claim 1.

4. The method according to claim 3, characterized in that The phi29-containing reagent includes 50 μl H2O, 50 μl DNB preparation buffer, 2 μl phi29 and 33 μl DLB as described in claim 1.

5. The method according to claim 3 or 4, characterized in that After incubation for 5 minutes and before loading, the step of washing the sequencing chip is also included.

6. The method according to claim 5, characterized in that The step of cleaning the sequencing chip comprises: Step (1): using PWB to clean the sequencing chip without incubation; Step (2): taking DCB to clean the sequencing chip without incubation; Step (3): taking SB1 to clean the sequencing chip; and: The step of taking out DCB multiple times to clean the sequencing chip is not included; The step of taking REB to clean the sequencing chip is not included; The step of taking PAW to clean the sequencing chip is not included.

7. The method according to claim 6, characterized in that include: The volume of the PWB comprises 200 μl; and / or The volume of DCB included 100 μl; and / or The volume of SB1 comprises 250 μl; and / or The flow rate of the wash comprised 100 μl / min.

8. The method according to any one of claims 5 to 7, characterized in that After cleaning the sequencing chip and before loading, the method further includes a step of combining the sequencing chip with primers.

9. The method according to claim 8, characterized in that The binding conditions are 55°C for 1 min and 30°C for 1 min.

10. The method according to claim 8 or 9, characterized in that The flow rate of the primers in the binding was 100 μl / min.

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