CN107944221B - Splicing algorithm for parallel separation of nucleic acid fragments and application thereof - Google Patents

Splicing algorithm for parallel separation of nucleic acid fragments and application thereof Download PDF

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CN107944221B
CN107944221B CN201711168549.1A CN201711168549A CN107944221B CN 107944221 B CN107944221 B CN 107944221B CN 201711168549 A CN201711168549 A CN 201711168549A CN 107944221 B CN107944221 B CN 107944221B
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吕华
张吉华
陈功俊
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Nanjing Superyears Gene Technology Co ltd
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Abstract

The invention relates to the field of medical diagnosis, in particular to a splicing algorithm for separating nucleic acid fragments in parallel and application thereof. The method comprises the following steps: selecting a channel i, carrying out peak identification on the internal standard signal, and recording the position of each peak; arranging the peak positions of the internal standard signals in the order from small to large; selecting corresponding peak positions according to the signal characteristics of each channel, thereby determining the signal extraction range of each channel; extracting the rest signals except the internal standard in the range corresponding to the channel i; calculating the electrophoresis voltage V of channel iiAnd channel 1 electrophoretic voltage V1Ratio k ofi(ii) a The extracted signal is according to the ratio kiScaling to make channel i coincide with the signal of channel 1; repeating the steps until the results of all the channels after expansion and contraction are obtained; and splicing the results stored in each channel according to the sequence of the channels to obtain complete gene fragment information. The method can splice the segmented nucleic acid segments, thereby greatly accelerating the detection speed.

Description

Splicing algorithm for parallel separation of nucleic acid fragments and application thereof
Technical Field
The invention relates to the field of medical diagnosis, in particular to a splicing algorithm for separating nucleic acid fragments in parallel and application thereof.
Background
Capillary Electrophoresis (CE) is a new electrophoresis technology in which ions or charged particles are separated efficiently and rapidly by using a high-voltage electric field as a driving force and a capillary as a separation channel according to the difference in mobility or distribution coefficient between components in a sample. The instrument device comprises a high-voltage power supply, a capillary tube, a detector and two liquid storage bottles which are used for inserting the two ends of the capillary tube and are connected with the power supply. The separation process of capillary electrophoresis is typically a differential motion process. During the migration process of the mixture, each sample molecule will gradually divide into different zones, i.e. before the fast one and after the slow one, due to the different speeds of the sample molecules. The longer the time, the smaller the zones, the greater the number, the further apart the distance, i.e. the better the separation. A detector is installed at the end point of the separation channel, and the condition that the molecules pass through the end point is recorded, so that the electrophoretogram can be obtained. Taking the most common capillary zone electrophoresis as an example, the capillary and the electrode cell are filled with a background electrolyte solution of the same composition and the same concentration. The sample is introduced from one end (sample introduction end) of the capillary, and when a certain voltage is applied to the two ends of the capillary, the charged substance moves towards the electrode direction with the opposite polarity of the charge. Meanwhile, an electric double layer is formed at the interface of the inner wall of the capillary tube and the buffer solution, so that the solution in the capillary tube integrally moves towards one direction under the action of an external electric field, namely, the electroosmotic flow phenomenon. Since the speed of electroosmotic flow is 5-7 times faster than the electrophoresis speed, capillary electrophoresis can move positive, negative ions and neutral molecules together in one direction by using electroosmotic flow, and the migration speed of ions or charged particles is the vector sum of the electrophoresis and electroosmotic flow speeds. Due to the difference of migration speeds among the components of the sample, after a certain time, the components flow out of the capillary tube in sequence according to the speed of the components to reach a detection end for detection, and an electrophoresis spectrogram distributed according to time is obtained. Carrying out qualitative analysis by using the migration time of a spectrum peak; and carrying out quantitative analysis according to the height or peak area of the spectrum peak.
The rapid development of life sciences has put increasing demands on the separation and analysis of biological samples. Because biological samples are various in types, complex in structure, small in sample amount, difficult to prepare, concentrate and separate and large in analysis workload, people urgently hope to find an analysis method with high flux, high sensitivity and high efficiency. Capillary electrophoresis plays an important role in the analysis of biological samples due to the advantages of high separation efficiency, high analysis speed, small sample consumption, easy realization of automation and the like. Capillary electrophoresis has now been applied to many aspects of DNA analysis, such as sequencing, gene mutation analysis, DNA fragment or PCR product determination, gene expression, DNA damage analysis, disease diagnosis, and the like. Direct analysis of mRNA is relatively difficult, but can also be performed by reverse transcription into complementary DNA (cDNA).
However, in the prior art, in the method of analyzing DNA fragments, a method of collecting signals of all base lengths of gene fragments in one channel and analyzing the signals is generally used. Since the migration rate of the large fragment is significantly lower than that of the small fragment, it takes a long time (generally 30 minutes) to analyze the large fragment in the DNA fragment, which affects the detection time. The inventors of the present invention have found that the detection speed can be greatly increased by dividing a nucleic acid to be detected into a plurality of nucleic acid fragments and detecting the nucleic acid fragments using a multichannel capillary electrophoresis apparatus, and limiting the voltage of each channel to thereby achieve the effect of breaking the whole into parts. However, no feasible algorithm exists in the prior art when splicing signals.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a splicing algorithm for separating nucleic acid fragments in parallel, which can splice the segmented nucleic acid fragments together with a detection method for segmenting nucleic acid to be detected into a plurality of nucleic acid fragments and detecting by using a multi-channel capillary electrophoresis apparatus, thereby greatly accelerating the detection speed.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention relates to a splicing algorithm for separating nucleic acid fragments in parallel, which comprises the following steps:
step 120: selecting a channel i, carrying out peak identification on the internal standard signal, and recording the position of each peak;
step 130: arranging the peak positions of the internal standard signals in the order from small to large;
step 140: selecting corresponding peak positions according to the signal characteristics of each channel, thereby determining the signal extraction range of each channel;
step 150: extracting the rest signals except the internal standard in the range corresponding to the channel i;
step 160: calculating the electrophoresis voltage V of channel iiAnd channel 1 electrophoretic voltage V1Ratio k ofi；
Step 170: the signal extracted in step 150 is according to the ratio kiScaling to make channel i coincide with the signal of channel 1;
step 180: repeating the step 120 and the step 170 to obtain the results of all the channels after expansion and contraction;
step 190: and splicing the results stored in each channel according to the sequence of the channels to obtain complete gene fragment information.
According to the relation between the migration velocity v of electrophoresis and the electric field intensity E of electrophoresis:
v＝k VE
wherein the proportionality coefficient k is Q/(e)aM+b6. eta. L), Q is the net charge of the fragment, a and b are coefficients of the relationship between molecular weight and molecular radius, eta is the viscosity of the electrophoretic gel, and L is the capillary length. As can be seen from the formula, the migration rate of the gene fragment has a direct relationship with the electrophoretic voltage and the molecular weight of the fragment.
In the prior art, the method for detecting nucleic acid fragments is to apply the same voltage to separate the fragments of 100-500bp size in the same capillary. According to the above formula, the migration rate of the large segment is significantly smaller than that of the small segment, so the prior art method slows down the overall detection time due to the low migration rate of the large segment.
Different electrophoresis voltages are applied to two ends of different capillary array channels, so that each capillary channel has a separation effect on fragments with different lengths and has approximately the same migration rate (because the CCD detects the exciting light signals of each channel at the same time when detecting signals, the closer the migration rate is, the shorter the electrophoresis time is); after electrophoresis is finished, the electrophoresis separation data of each channel is spliced through the splicing algorithm provided by the invention, and the complete information of the gene fragment can be finally obtained.
According to one aspect of the invention, the invention also provides the use of a splicing algorithm for the parallel separation of nucleic acid fragments as described above for detecting the presence or absence of a specific locus and for STR typing.
Compared with the prior art, the invention has the beneficial effects that:
in the existing gene fragment analysis, for the analysis of fragments about 500bp, the time for obtaining a final result usually takes 30-40 minutes, and the method can shorten the time from 30-40 minutes to about 10 minutes, thereby greatly improving the analysis efficiency.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram illustrating the specific steps of a stitching algorithm employed in an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method provided by the present invention;
FIG. 3 is a schematic diagram of an apparatus for performing analysis using an 8-channel capillary array according to an example;
FIG. 4 is a graph of electrophoretic data before stitching in the example;
FIG. 5 is a diagram showing electrophoretic data after completion of splicing in the example.
Detailed Description
The invention relates to a splicing algorithm for separating nucleic acid fragments in parallel, which comprises the following steps:
step 120: selecting a channel i, carrying out peak identification on the internal standard signal, and recording the position of each peak;
step 130: arranging the peak positions of the internal standard signals in the order from small to large;
step 140: selecting corresponding peak positions according to the signal characteristics of each channel, thereby determining the signal extraction range of each channel;
step 150: extracting the rest signals except the internal standard in the range corresponding to the channel i;
step 160: calculating the electrophoresis voltage V of channel iiAnd channel 1 electrophoretic voltage V1Ratio k ofi；
Step 170: the signal extracted in step 150 is according to the ratio kiScaling to make channel i coincide with the signal of channel 1;
step 180: repeating the step 120 and the step 170 to obtain the results of all the channels after expansion and contraction;
step 190: and splicing the results stored in each channel according to the sequence of the channels to obtain complete gene fragment information.
Preferably, the algorithm for splicing nucleic acid fragments separated in parallel as described above further comprises, before step 120, step 110: and denoising and smoothing the internal standard signals in each channel.
Preferably, the algorithm for splicing nucleic acid fragments separated in parallel as described above, the method for separating nucleic acid fragments in parallel comprises:
dividing nucleic acid to be detected into fragment compositions containing a plurality of nucleic acid fragments, wherein the fragment compositions are marked by fluorescent dyes, carrying out multichannel simultaneous detection on the fragment compositions by using a multichannel capillary array electrophoresis apparatus, and adjusting the voltage at two ends of each channel according to the preset length interval of the nucleic acid fragment to be detected of each channel so as to enable the migration rate of electrophoresis of the preset nucleic acid fragment to be detected in each channel to be approximately the same; and collecting and splicing electrophoresis result information of each channel to obtain complete information of the nucleic acid to be detected.
In the existing gene fragment analysis, the time for obtaining the final result usually needs 30-40 minutes, and the time consumption can be shortened to about 10 minutes from 30-40 minutes by adopting the method, so that the analysis efficiency is greatly improved.
During detection, a molecular weight internal standard DNA fragment is added into each channel.
Specifically, the operation flow chart of the nucleic acid analysis method based on capillary electrophoresis provided by the invention is shown in FIG. 2:
step 110: extracting genes from a sample, preparing corresponding templates and primers, and carrying out PCR amplification to obtain a gene fragment product to be analyzed;
step 120: placing the amplified product in a corresponding well site of a sample plate, e.g., a 96-well plate, with the same product sample placed in all well sites of each row;
step 130: setting corresponding electrophoresis voltage according to the preset fragment length detected by each channel;
step 140: performing capillary electrophoresis, and acquiring electrophoresis results of all channels by utilizing laser-induced fluorescence and CCD imaging;
step 150: and splicing the results of all channels to obtain complete gene fragment information.
Preferably, the algorithm for splicing nucleic acid fragments to be separated in parallel as described above, wherein when the nucleic acid to be detected is DNA, the method for separating the nucleic acid to be detected into fragment compositions containing a plurality of nucleic acid fragments is a PCR method; when the nucleic acid to be detected is RNA, reverse transcription is carried out, and then the nucleic acid to be detected is divided into fragment compositions containing a plurality of nucleic acid fragments by utilizing a PCR method.
Preferably, the algorithm for splicing nucleic acid fragments separated in parallel as described above, wherein the fluorescent dye is labeled at the locus to be detected in the nucleic acid to be detected.
Preferably, the splicing algorithm for parallel separation of nucleic acid fragments as described above, the method for labeling the fluorescent dye on the locus to be detected in the nucleic acid to be detected is as follows: fluorescent dyes are labeled at the 5' end of one primer in each locus.
Preferably, the algorithm for splicing nucleic acid fragments separated in parallel as described above, the loci where the amplified fragments do not overlap use fluorescent dyes with the same excitation light, and the loci where the amplified fragments overlap use fluorescent dyes with different excitation lights.
The fluorescent dye is marked at the 5' end of one primer in each locus, and the primers of different loci are marked with different fluorescent markers. Amplified allele products carry fluorescence, length alleles are separated through electrophoresis, and the alleles in gel are detected through a fluorescence spectroscopy system. Loci are distinguished by the color of fluorescence, and fragment length alleles are determined by fragment mobility.
Preferably, the fluorescent dye comprises a plurality of FAM, VIC, NED, PET, LIZ, dR110, TAMRA, ROX, JOE, HEX, TET, as described above.
In practice, different colored fluorochromes may be combined, the emission wavelengths of the combined fluorochromes differing as much as possible, typically 20-30 nm. Methods of combining are well known to those skilled in the art.
Preferably, the algorithm for splicing nucleic acid fragments separated in parallel as described above, the number of partitions between the length intervals of said nucleic acid fragments being less than or equal to the maximum number of channels of said multichannel capillary array electrophoresis apparatus.
Preferably, the splicing algorithm for parallel separation of nucleic acid fragments is as described above, and the size of the fragment of the nucleic acid to be detected is 100bp-20 kb; more preferably 100bp-3 kb; more preferably 100bp to 500 bp.
The invention provides a novel capillary electrophoresis thought, and based on the method provided by the invention, the effect of shortening the analysis time is more obvious in theory if the nucleic acid fragment to be analyzed is larger. The multi-channel capillary array electrophoresis apparatus in the prior art can provide up to 96 channels, so that the present invention can be used for the analysis of ultra-large fragments. Therefore, it can be seen that the short panel of analysis in this method lies in the amplification length of PCR, and the range of the existing long PCR is generally 3-20kb, and if it is larger than 3kb, the long taq enzyme can be used for amplification. Most commonly in the art, fragments of sizes ranging from 100bp to 500bp are analyzed.
According to one aspect of the invention, the invention also provides the use of a splicing algorithm for the parallel separation of nucleic acid fragments as described above for detecting the presence or absence of a specific locus and for STR typing.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Examples
In this example, an 8-channel capillary array was used to detect DNA fragments having a total length of 500bp (as shown in FIG. 3).
Molecular weight calibration was performed using LIZ-500 molecular weight internal standards with molecular weights of 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, 500, respectively.
The fluorescent dye is marked at the 5' end of one primer in each locus to be analyzed, and the DNA fragment to be analyzed is divided into the fragment sizes corresponding to the following channels by a PCR method:
design the channel 1 to separate the 160bp fragment of 100-.
According to the designed fragment length of each channel, the corresponding electrophoresis voltage is calculated by utilizing the relational expression of the migration velocity v of electrophoresis and the electric field intensity E of electrophoresis, so that the migration velocity of the initial separation fragments in each channel is kept consistent, namely the migration velocity of the 100bp fragment in the channel 1, the migration velocity of the 139bp fragment in the channel 2, the migration velocity of the 200bp fragment in the channel 3 and the like are consistent. In this embodiment, the electrophoresis voltage of channel 1 is-6.0 kV, the voltage of channel 2 is-6.8 kV, the voltage of channel 3 is-8.3 kV, the voltage of channel 4 is-9.7 kV, the voltage of channel 5 is-11.3 kV, the voltage of channel 6 is-12.9 kV, the voltage of channel 7 is-15.6 kV, the voltage of channel 8 is-18.3 kV, the electrophoresis diagram of each channel is as shown in FIG. 4, and then splicing is performed, so that a complete fragment electrophoresis diagram can be obtained, as shown in FIG. 5.
The operation in splicing is shown in fig. 1.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (11)

1. A splicing algorithm for separating nucleic acid fragments in parallel, comprising:
step 120: selecting a channel i, carrying out peak identification on the internal standard signal, and recording the position of each peak;
step 130: arranging the peak positions of the internal standard signals in the order from small to large;
step 140: selecting corresponding peak positions according to the signal characteristics of each channel, thereby determining the signal extraction range of each channel;
step 150: extracting the rest signals except the internal standard in the range corresponding to the channel i;
step 160: calculating the electrophoresis voltage V of channel iiAnd channel 1 electrophoretic voltage V1Ratio k ofi；
Step 170: the signal extracted in step 150 is according to the ratio kiScaling to make channel i coincide with the signal of channel 1;
step 180: repeating the step 120 and the step 170 to obtain the results of all the channels after expansion and contraction;
step 190: splicing the results stored in each channel according to the sequence of the channels to obtain complete gene fragment information;
the method for parallel separation of nucleic acid fragments comprises: dividing nucleic acid to be detected into fragment compositions containing a plurality of nucleic acid fragments, wherein the fragment compositions are marked by fluorescent dyes, carrying out multichannel simultaneous detection on the fragment compositions by using a multichannel capillary array electrophoresis apparatus, and adjusting the voltage at two ends of each channel according to the preset length interval of the nucleic acid fragment to be detected of each channel so as to enable the migration rate of electrophoresis of the preset nucleic acid fragment to be detected in each channel to be approximately the same; collecting electrophoresis result information of each channel and splicing to obtain complete nucleic acid information to be detected,
during detection, adding a molecular weight internal standard DNA fragment into each channel;
the splicing algorithm for parallelly separating the nucleic acid fragments is applied to detecting the existence of a specific locus and STR typing.
2. The algorithm for splicing nucleic acid fragments to be separated in parallel according to claim 1, further comprising, before step 120, step 110: and denoising and smoothing the internal standard signals in each channel.
3. The splicing algorithm for separating nucleic acid fragments in parallel according to claim 1, wherein when the nucleic acid to be detected is DNA, the method for dividing the nucleic acid to be detected into fragment compositions containing a plurality of nucleic acid fragments is a PCR method;
when the nucleic acid to be detected is RNA, reverse transcription is carried out, and then the nucleic acid to be detected is divided into fragment compositions containing a plurality of nucleic acid fragments by utilizing a PCR method.
4. The algorithm for splicing nucleic acid fragments separated in parallel according to claim 1, wherein the fluorescent dye is labeled at a locus to be detected in the nucleic acid to be detected.
5. The splicing algorithm for parallel separation of nucleic acid fragments according to claim 4,
the fluorochrome is labeled at the 5' end of one primer in each locus.
6. The algorithm for splicing nucleic acid fragments separated in parallel according to claim 5, wherein loci where the amplified fragments do not overlap are labeled with the same fluorescent dye, and loci where the amplified fragments overlap are labeled with different fluorescent dyes.
7. The algorithm for splicing nucleic acid fragments separated in parallel according to claim 5 or 6, wherein the fluorescent dye comprises a plurality of FAM, VIC, NED, PET, LIZ, dR110, TAMRA, ROX, JOE, HEX, TET.
8. The algorithm for splicing nucleic acid fragments for parallel separation according to claim 1, wherein the number of partitions between the length intervals of the nucleic acid fragments is not more than the maximum number of channels of the multichannel capillary array electrophoresis apparatus.
9. The splicing algorithm for parallel separation of nucleic acid fragments according to claim 1, wherein the size of the fragment of the nucleic acid to be detected is 100bp-20 kb.
10. The splicing algorithm for parallel separation of nucleic acid fragments according to claim 1, wherein the size of the fragment of the nucleic acid to be detected is 100bp-3 kb.
11. The splicing algorithm for parallel separation of nucleic acid fragments according to claim 1, wherein the size of the fragment of the nucleic acid to be detected is 100bp to 500 bp.
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