A method and kit for DNA length melting determination

Abstract

The application discloses a DNA length melting determination method and a kit, which overcomes the problem that multiple target DNAs cannot be distinguished according to melting temperatures when the melting temperatures of the target DNAs are similar in the prior art. The determination method is simpler and faster, and can be widely used in fields such as food authenticity identification based on nucleic acid analysis and medical diagnosis, and has strong practical application value. The application adds a small-molecule quaternary ammonium salt to eliminate the influence of GC content on the melting temperature, and establishes two methods of fluorescence labeling and non-labeling fluorescence for determining the DNA length. The length of unknown DNA is determined by using the melting temperature after adding the three additives and calculating the GC%. The application realizes the application of DNA hydrolysis cutting reaction monitoring, and combines multiple PCR and the length melting determination method to detect three animal-derived components of bovine, goat and sheep in milk and dairy products.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of analytical biology detection technology based on nucleic acid analysis, and relates to a DNA length melting determination method and kit. BACKGROUND

[0002] Nucleic acid analysis is an important food detection method, and PCR technology is most widely used because of its high specificity, high sensitivity, high repeatability and reliable detection results. PCR (polymerase chain reaction) is widely used for in vitro amplification of DNA, and the amplification product usually has a certain length, so that the length of the PCR product can be determined to identify the specificity of the PCR reaction and the target source of the multiplex PCR reaction. Plate gel electrophoresis and capillary gel electrophoresis are the most commonly used methods for determining the length of PCR products, but they usually require a transfer solution and the process is complex. Real-time PCR technology mainly monitors the production of PCR amplification products through the combination of polymerase chain reaction and fluorescent reporter molecules. Real-time PCR is mainly divided into probe method and dye method. The fluorescently labeled probe method can be divided into three types: one is a primer probe for PCR amplification, commonly known as Scorpion, Amplifluor, LUX TM , etc.; another is a TaqMan hydrolysis probe, a molecular beacon hybridization probe combined with a template DNA; and the third is a nucleic acid analogue. The probe method has high specificity, but usually requires a labeled reporter group and a quencher group, which is difficult to synthesize and design. LUX TM The primer probe only needs to be labeled with one fluorescent group, and it is a hairpin structure itself. When in a free state, the specific sequence can quench the fluorescent signal, and when combined with DNA, the hairpin structure is opened, and the fluorescent signal rises exponentially. However, this hairpin primer requires special software for design, and the process is relatively complex. In order to reduce the difficulty of primer design, the fluorescent group is labeled on the linear primer, and the number of C bases is increased at the 5' end of the primer. When DNA replicates, the G base complementary to the C base can quench the fluorescent signal, which can also monitor the formation of amplification products. Real-time PCR can also use dye binding method to detect fluorescent signal, and common DNA double-stranded binding dyes include SYTO 9, SYBR Green I, EvaGreen, ResoLight, LC Green, etc.

[0003] High resolution melting curve analysis is a kind of efficient, fast and reliable PCR product analysis technology. Melting analysis can realize rapid and simple identification of PCR products, but cannot obtain length information because the DNA melting temperature (Tm) value depends not only on length but also on GC content. The present application eliminates the influence of GC content on melting temperature by adding small molecule quaternary ammonium salt (tetramethylammonium chloride or tetraethylammonium chloride or betaine), and establishes two methods of fluorescence labeling and non-labeling fluorescence melting for determining DNA length.

[0004] Multiplex PCR can reduce detection cost, save detection time, realize synchronous detection of multiple components of the same sample, and is applied to dairy authenticity identification. Multiplex PCR combined with high resolution melting technology can distinguish multiple target DNAs according to melting temperature. However, when the melting temperatures of target DNAs are similar, they cannot be distinguished by melting, and can only be improved by adjusting the size and GC% of primers. In order to overcome the above limitations, when the melting temperatures of target DNAs are similar but the lengths of PCR products are different, additives are added to change the melting temperature, so as to realize the purpose of distinguishing different target DNAs. SUMMARY

[0005] The present application aims to provide a melting determination method and kit for DNA length, which overcomes the problem in the prior art that multiple target DNAs cannot be distinguished according to melting temperature when the melting temperatures of target DNAs are similar. The determination method of the present application is simpler and faster, and can be widely used in fields such as food authenticity identification based on nucleic acid analysis and medical diagnosis, and has strong practical application value.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A melting determination method for DNA length, characterized in that it comprises the following steps:

[0008] 1) The influence of GC content on melting temperature is eliminated by adding tetramethylammonium chloride or tetraethylammonium chloride or betaine small molecule quaternary ammonium salt to the DNA sample;

[0009] 2) Standard curves of melting temperature and length corresponding to different additives and standard curves of GC content and melting temperature difference with and without additives are established;

[0010] 3) Tetramethylammonium chloride or tetraethylammonium chloride or betaine additive is added to the DNA of unknown length for melting, and the corresponding melting temperature is read and entered into the two standard curves to calculate the DNA length;

[0011] 4) For the determination of the length of DNAase hydrolysis reaction product, add tetramethylammonium chloride or tetraethylammonium chloride or betaine additive to the DNAase hydrolysis reaction product, read the corresponding melting temperature, and bring in two standard curves to calculate the length of DNA hydrolysis product;

[0012] 5) For the determination of the length of PCR product, design amplicon by corresponding PCR primer, add tetramethylammonium chloride or tetraethylammonium chloride or betaine additive to the PCR product, read the corresponding melting temperature, and bring in two standard curves to calculate the length of PCR product;

[0013] 6) For the differentiation of multiplex PCR product and the detection of actual sample, design multiple pairs of specific PCR primers by comparing nucleotide sequences and specific tests, and add tetramethylammonium chloride or tetraethylammonium chloride or betaine additive to the single, double and triple PCR amplification products for melting differentiation.

[0014] In step 1), the formation of the melting curve is monitored by fluorescence labeling and non-labeled fluorescence, and the corresponding melting temperature is read.

[0015] In step 2), the length of DNA is calculated by using either melting temperature or GC content.

[0016] In step 3), the length of DNA is determined by adding any one of tetramethylammonium chloride, tetraethylammonium chloride, and betaine additive.

[0017] In step 5), the specific primer pair for amplifying goat DNA is Primer 1, the specific primer pair for amplifying bovine DNA is Primer 2, and the specific primer pair for amplifying sheep DNA is Primer 3.

[0018] The sequence of the primer pair Primer 1 is:

[0019] Upstream primer: 5'-GGGGGGGGGGGCCATAATTACAACAA-3'

[0020] Downstream primer: 5'-CCCTATCAGCTGCAGTAGGGTT-3'

[0021] The sequence of the primer pair Primer 2 is:

[0022] Upstream primer: 5'-AGTAAGCGTAATTATGATAC-3'

[0023] Downstream primer: 5'-CCCCCTTGATTCTCTTGGTGTAGAG-3'

[0024] The sequence of the primer pair Primer 3 is:

[0025] Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'

[0026] Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'.

[0027] In step 1), step 3), step 4), and step 5), the fluorescently labeled melting system is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ , 10 mM HEPES, and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, and the non-labeled fluorescently labeled melting system is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ , 10 mM HEPES (pH 7.9), 0.5x or 2x DNA binding dye, and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, and the non-labeled fluorescently labeled melting system is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg

[0028] In step 4), the PCR reaction system is composed of: 50 nM of forward primer and reverse primer, 1x Taq PCR Master Mix, 2 ng / μL of PCR purified product, and finally 10 μL of reaction system is supplemented with double distilled water, and double distilled water is used as a negative control; the PCR amplification reaction program is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 57℃ annealing for 20 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min.

[0029] In step 5), the PCR reaction system is composed of: primer concentration ratio of primer pair Primer 1:Primer 2:Primer 3 is 9:3:5, 1x QIAGEN Multiplex PCR Master Mix, 0.5x DNA binding dye, 20 ng / μL of DNA, and finally 10 μL of reaction system is supplemented with double distilled water, and double distilled water is used as a negative control; the PCR amplification program is: 94℃ pre-denaturation for 10 min; 94℃, 15 s denaturation, 57℃, 60 s annealing and extension, 25 cycles; 72℃, 5 min re-extension, and fluorescence signal is collected at each cycle of extension stage.

[0030] In step 5), the actual sample is collected from fresh milk, dairy products or other genetic samples, and the position of the melting peak of the reference sample and the sample to be tested is compared to identify and detect.

[0031] A DNA length melting assay kit, characterized in that the kit contains tetramethylammonium chloride or tetraethylammonium chloride or betaine additive for establishing an additive melting system, a DNA binding dye, a 1x reaction buffer: 6mM Mg 2+ and 10mM HEPES, and a standard curve of melting temperature and length corresponding to different additive length determination, and a standard curve of GC content and melting temperature difference with and without additive.

[0032] Compared with the prior art, the present application has the advantages and effects as follows:

[0033] 1、 The present application uses the addition of tetramethylammonium chloride or tetraethylammonium chloride or betaine and other small molecule quaternary ammonium salt to eliminate the influence of GC content on melting temperature, and establishes two methods of fluorescent labeling and non-labeled fluorescent melting for determining DNA length. The rapid determination of DNA length can be realized without special electrophoresis equipment, avoiding the cumbersome steps of gel preparation and sample transfer separation. In addition, for the target DNA that cannot be distinguished due to similar melting temperature values in multiplex melting analysis, the small molecule quaternary ammonium salt can be added to effectively distinguish and distinguish. The method for determining the length of DNA established by the present application reduces the cost of DNA analysis and detection, and improves the detection efficiency.

[0034] 2、 The present application is directed to Cas12a catalytic DNA hydrolysis enzyme reaction, and the melting temperature difference of the corresponding hydrolysis fragment is very small. By adding an additive, the melting temperature difference is increased to realize the monitoring of the hydrolysis reaction.

[0035] 3、 The present application designs specific primers for goats, cows and sheep, and the melting temperature difference of the corresponding amplicon is very small. By adding an additive, the melting temperature difference is increased to realize the differential detection of goats, cows and sheep. The DNA length melting method of the present application is a simple, rapid, efficient and practical nucleic acid analysis technology. DETAILED DESCRIPTION:

[0036] Figure 1 The influence of the number of C bases at the 5' end of the primer on fluorescent labeling melting, wherein (A) is the sequence information of the 171bp primer; (B) is the capillary electrophoresis of 171bp; (C) is the amplification curve of 171bp; (D) is the melting curve of 171bp;

[0037] Figure 2 The influence of the heating rate on fluorescent labeling melting, the heating rate changes from 0.05℃ / s to 1℃ / s;

[0038] Figure 3Fluorescently labeled melting for three groups of DNA length and Tm value negative correlation typical representative, - represents no additives, 1, 2, 3 respectively represent Additive 1, Additive 2, Additive 3;

[0039] Figure 4 Two standard curves for fluorescently labeled melting to determine DNA length. Among them, (A) the relationship between melting temperature and length negative reciprocal when adding and not adding additives; (B) the relationship between GC content and the difference in melting temperature when adding and not adding additives, mean ± standard deviation, 3 repeated melting;

[0040] Figure 5 Application of fluorescently labeled melting to determine the length of PCR products. Among them, (A) polyacrylamide gel electrophoresis; (B) melting curve;

[0041] Figure 6 Melting curves of different length DNA fragments and different types of dyes in the additive system. Among them, (A) Dye 1; (B) Dye 2; (C) Dye 3; (D) Dye 4;

[0042] Figure 7 Effect of dye concentration on melting curve in different additive systems. Among them, (A) Additive 1; (B) Additive 2; (C) No Additive; (D) Additive 3;

[0043] Figure 8 Non-labeled fluorescent melting for three groups of DNA length and Tm value negative correlation typical representative, - represents no additives;

[0044] Figure 9 Two standard curves for non-labeled fluorescent melting to determine DNA length. Among them, (A) the relationship between melting temperature and length negative reciprocal when adding and not adding additives; (B) the relationship between GC content and the difference in melting temperature when adding and not adding additives, mean ± standard deviation, 3 repeated melting;

[0045] Figure 10 Application of non-labeled fluorescent melting to determine the length of PCR products. (A) Polyacrylamide gel electrophoresis; (B) Melting curve;

[0046] Figure 11 Application of non-labeled fluorescent melting to detect CRISPR-Cas12a cleavage PCR products. (A) Polyacrylamide gel electrophoresis; (B) Melting curve;

[0047] Figure 12Melting and electrophoresis detection of multiplex PCR products. (A) Melting curves with and without additives; (B) Capillary gel electrophoresis. - represents without additives, lanes 1-7 represent goat, cow, sheep, cow + sheep, goat + cow, goat + sheep, goat + cow + sheep, respectively;

[0048] Figure 13 Melting and electrophoresis detection of commercially available dairy products with additives. (A) Melting curves; (B) Capillary gel electrophoresis. 1-4 represent goat, cow, sheep, goat + cow + sheep control samples, respectively, and 5-12 are eight commercially available dairy products. DETAILED DESCRIPTION:

[0049] In order to make the purposes, technical solutions and advantages of the present application clearer, further detailed description will be given below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0050] The present application is a DNA length melting determination method and kit. The present application eliminates the influence of GC content on melting temperature by adding small molecule quaternary ammonium salt, and establishes two methods of fluorescence labeling and non-labeling fluorescence for determining DNA length. The length of unknown DNA is determined by adding the above three additives and calculating the GC%. The combination of multiplex PCR and melting determination length method is used to detect bovine, goat and sheep three animal-derived ingredients in milk and dairy products.

[0051] The melting detection method of the present application comprises the following steps:

[0052] 1) The influence of GC content on melting temperature is eliminated by adding tetramethylammonium chloride or tetraethylammonium chloride or betaine and other small molecule quaternary ammonium salt to the DNA sample; the formation of melting curve can be monitored by fluorescence labeling and non-labeling fluorescence, and the corresponding melting temperature is read.

[0053] 2) The standard curves of different additives corresponding to melting temperature and length, and the standard curves of GC content and the difference between melting temperature with and without additives are established; DNA length can be calculated by using either melting temperature or GC content.

[0054] 3) For unknown length of DNA, add tetramethylammonium chloride or tetraethylammonium chloride or betaine additive for melting, read the corresponding melting temperature and enter the two standard curves to calculate the DNA length; DNA length can be determined by adding any one of tetramethylammonium chloride, tetraethylammonium chloride and betaine additives for melting.

[0055] 4) For the determination of the length of DNAse hydrolysis reaction product, add tetramethylammonium chloride or tetraethylammonium chloride or betaine additive to the DNAse hydrolysis reaction product, read the corresponding melting temperature and bring in two standard curves to calculate the length of DNA hydrolysis product;

[0056] 5) For the determination of the length of PCR product, the amplicon can be obtained by designing corresponding PCR primers, and tetramethylammonium chloride or tetraethylammonium chloride or betaine additive is added to the PCR product, the corresponding melting temperature is read and two standard curves are brought in to calculate the length of PCR product; the composition of the PCR reaction system is: 50 nM of forward primer and reverse primer, 1 × Taq PCR MasterMix, 2 ng / μL of PCR purified product, finally 10 μL of reaction system is made up with double distilled water, and double distilled water is used as negative control. The reaction program of PCR amplification is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 57℃ annealing for 20 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min.

[0057] 6) For the differentiation of multiplex PCR products and the detection of actual samples, a plurality of specific PCR primers are designed by comparing nucleotide sequences and specific tests, and tetramethylammonium chloride or tetraethylammonium chloride or betaine additive is added to the single, double and triple PCR amplification products for melting differentiation.

[0058] The specific primer pair for amplifying goat DNA is Primer 1, the specific primer pair for amplifying bovine DNA is Primer 2, and the specific primer pair for amplifying sheep DNA is Primer 3.

[0059] The sequence of the primer pair Primer 1 is:

[0060] Upstream primer: 5'-GGGGGGGGGGGCCATAATTACAACAA-3'

[0061] Downstream primer: 5'-CCCTATCAGCTGCAGTAGGGTT-3'

[0062] The sequence of the primer pair Primer 2 is:

[0063] Upstream primer: 5'-AGTAAGCGTAATTATGATAC-3'

[0064] Downstream primer: 5'-CCCCCTTGATTCTCTTGGTGTAGAG-3'

[0065] The sequence of the primer pair Primer 3 is:

[0066] Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'

[0067] Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'

[0068] The PCR reaction system is composed of primer pair Primer1:Primer2:Primer3 with a primer concentration ratio of 9:3:5, 1×QIAGEN Multiplex PCR Master Mix, 0.5×DNA binding dye, 20 ng / μL of DNA, and finally 10 μL of reaction system is supplemented with double distilled water, and double distilled water is used as a negative control. The PCR amplification procedure is as follows: 94℃ pre-denaturation for 10 min; 94℃, 15 s denaturation, 57℃, 60 s annealing and extension, 25 cycles are set; 72℃, 5 min re-extension, and the fluorescence signal is collected at each cycle of the extension stage.

[0069] The actual sample is collected from fresh milk, dairy products or other genetic samples, and the position of the melting peak of the reference sample and the sample to be detected is identified and detected.

[0070] The fluorescently labeled melting system in the step 1), step 3), step 4), step 5) is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ , 10 mM HEPES and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, and ultrapure water is used instead of the additive group. The non-labeled fluorescently labeled melting system is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ , 10 mM HEPES (pH 7.9), 0.5× or 2× DNA binding dye and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, and ultrapure water is used instead of the additive group. The high-resolution melting procedure is 37-99℃, and the melting rate is 0.05℃ / s.

[0071] The present application also includes a melting detection kit for DNA length, which contains tetramethylammonium chloride or tetraethylammonium chloride or betaine additives for establishing additive melting systems, DNA binding dyes, 1× reaction buffer: 6 mM Mg 2+ and 10 mM HEPES, and standard curves of melting temperature and length corresponding to different additives for length determination, and standard curve graphs of GC content and melting temperature difference with and without additives.

[0072] Example 1: Optimization of fluorescently labeled melting conditions

[0073] 1.1 PCR and melting reaction system and procedure

[0074] PCR reaction system composition: 50 nM of forward primer and reverse primer, 1 x Taq PCR Master Mix, 2 ng / μL of PCR purified product, finally with double distilled water to make up 10 μL reaction system, and double distilled water as negative control. Fluorescently labeled melting system: 1 μL of synthetic DNA or PCR amplified product, 6 mM Mg 2+ , 10 mM HEPES and additives, and no additives group with ultrapure water instead. The reaction program of PCR amplification is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 57℃ annealing for 20 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min. The melting program is 37-99℃, and the melting rate is 0.05℃ / s.

[0075] 1.2 The effect of the number of G bases on the fluorescence labeled melting

[0076] 1, 2, 3, 5, and 10 different numbers of C bases are added to the 5' end of the forward primer of the PCR product with a length of 171 bp, and the FAM group is labeled. After the completion of DNA replication, the complementary strand produces a corresponding number of G bases Figure 1 A) Capillary gel electrophoresis Figure 1 B) shows that the electrophoretic band of the PCR product is normal, indicating that the PCR amplification can proceed normally after adding C bases. The primer with different numbers of C bases has obvious amplification signal Figure 1 C), and as the number of C bases added to the primer end increases to 1, 2, 3, 5, and 10, the quenching effect of G bases on the fluorescence signal also gradually increases, and the corresponding amplification signal also rises. The melting results show that the melting peak signal is very low when 1-2 C bases are added, and there is no obvious melting peak. When the number of C bases is 3, 5, and 10, a typical melting peak is formed, and the melting peak signal is continuously enhanced Figure 1 D). Therefore, the number of C bases will affect the quenching degree of the FAM fluorescence group signal, and further affect the formation of the melting peak. The present application adds different numbers of C bases to the 5' end of the primer of different fragment lengths, to ensure that each DNA fragment can produce effective melting information.

[0077] 1.3 The effect of heating rate on fluorescence labeling

[0078] The heating rate can affect the fitting of the melting curve. In order to select the most suitable melting rate, the heating rates of 0.05, 0.1, 0.3, 0.5, 0.7, and 1℃ / s are set for the melting reaction of three DNA double strands with lengths of 29 bp, 36 bp, and 50 bp in the additive melting system. From Figure 2It can be seen that as the heating rate gradually increases, the melting temperature gradually increases, the corresponding melting peak becomes wider, and the melting signal continuously decreases. Moreover, the higher the heating rate, the greater the overlap of the melting peaks of the three DNA fragments. When the heating rate is 0.05℃ / s, the melting peak shape is more complete, and the melting temperature reading is more accurate. To obtain better resolution and more accurate melting temperature, 0.05℃ / s is selected as the optimal heating rate.

[0079] Example 2: Establishment of a method for determining DNA length by fluorescent labeling and melting

[0080] 2.1 Typical examples of fluorescently labeled melting in three groups of DNA lengths and Tm values ​​that are negatively correlated

[0081] The addition of tetramethylammonium chloride, tetraethylammonium chloride, or betaine can eliminate the influence of GC content on melting temperature. To verify the feasibility of using these three additives to determine DNA length via melting temperature, three groups of DNA fragments were selected for melting analysis. The selected DNA fragments all showed an inverse relationship between length and melting temperature; that is, shorter DNA fragments had higher melting temperatures, and longer DNA fragments had lower melting temperatures. The melting results for DNA fragments of 50 bp and 70 bp in different systems are shown below. Figure 3 The results showed that in the system without additives, the melting temperature of 50bp was higher than that of 70bp because the GC content of 50bp was higher than that of 70bp, and the GC content has a greater impact on the melting temperature. When Additive 1 was added, the effect of GC content on the melting temperature was eliminated, and the melting temperatures of both 50bp and 70bp decreased, but the decrease in melting temperature was different for the two, resulting in the melting temperature of 50bp ultimately being lower than that of 70bp. When Additive 2 was added, the melting temperatures of both decreased, and the melting temperature of 50bp was lower than that of 70bp. When Additive 3 was added, the melting temperatures of all fragments increased, but the melting temperature of 50bp was still lower than that of 70bp. DNA lengths of 93bp, 108bp, 175bp, and 236bp also showed high GC content. Without additives, the melting temperature of these fragments was higher than that of the longer fragments, and the melting temperature was negatively correlated with the fragment length. However, after adding the three additives, the melting temperature and fragment length became positively correlated.

[0082] 2.2 Establishment of a fluorescently labeled melting method for determining DNA length

[0083] For 12 identified DNA fragments, melting was performed under optimized experimental conditions in additive systems of Additive 1, Additive 2, or Additive 3, with a no-additive control included. The melting temperatures of fragments of different lengths were recorded. A standard curve was fitted using the negative reciprocal of fragment length as the x-axis and melting temperature as the y-axis. Figure 4A). From the point graph without additive, it can be seen that the melting temperature and length have no correlation. In the standard curve graph with Additive 1, the DNA length negative reciprocal and melting temperature are positively correlated, the larger the length of the DNA fragment, the higher the corresponding melting temperature, and the linear correlation coefficient R 2 = 0.959. In the standard curve graph with Additive 2 and Additive 3, the DNA length and melting temperature are also positively correlated, and the linear correlation coefficients are R 2 = 0.975, R 2 = 0.966. If the melting temperature of a certain fragment is known, the fragment size can be calculated by bringing it into the standard curve equation of Additive 1 or Additive 2 or Additive 3.

[0084] Because the effect of GC% on melting temperature can be eliminated by adding various additives, theoretically, the change value of melting temperature after adding additives has a correlation with GC%. The melting temperature without additive minus 0.41 x GC% and length negative reciprocal has a good linear relationship, and the linear correlation coefficient R 2 = 0.991 Figure 4 B). The standard curve is made with the GC% of the DNA fragment as the horizontal coordinate and the melting temperature difference with and without additive as the vertical coordinate. After adding Additive 1, the GC% and the melting temperature difference with and without Additive 1 are positively correlated, and the linear correlation coefficient R 2 = 0.952, the larger the GC%, the more the melting temperature decreases after adding Additive 1. GC% and the melting temperature difference with and without Additive 2 are positively correlated, and the correlation coefficient R 2 = 0.964. GC% and the melting temperature difference with and without Additive 3 are negatively correlated, and the correlation coefficient R 2 = 0.964. Therefore, the GC% size can also be calculated according to the melting temperature difference with and without additive, and then brought into the standard equation of the melting temperature without additive minus 0.41 x GC% and length negative reciprocal in B to calculate the size of the DNA fragment. Figure 4

[0085] To test the practicability of the established method, three DNA fragments with amplicon lengths of 73 bp, 106 bp and 209 bp were subjected to fluorescence-labeled melting determination. Figure 5 A is a polyacrylamide gel electrophoresis graph, and the amplification products have obvious electrophoresis bands and lengths that meet the expectations. The amplification products were reacted in the additive melting system and the control system without additive, and the melting results are shown in Figure 5 ​B. When no additive was added, the melting curves of the three DNA fragments were difficult to distinguish from each other. After the addition of additive, the melting temperature increased with the increase of DNA length, and the three melting peaks could be distinguished from each other.

[0086] The melting temperature after the addition of additive was read and brought into the standard equation of DNA length and melting temperature obtained by fitting (see Figure 4 A), and the length of 81.89 bp, 111.53 bp and 144.08 bp was calculated. The difference between the melting temperature without additive and the melting temperature after the addition of additive was brought into the standard equation obtained by fitting (see Figure 4 B), and the GC% was calculated and brought into the standard equation of -1 / Length and Tm (No Additive)-0.41 GC% (see Figure 4 B), and the length of 88.08 bp, 117.58 bp and 154.06 bp was calculated. Therefore, both methods can be used to determine the length of PCR product. The specific results of the determination of the three fragments by the two methods are shown in Table 1.

[0087] Table 1 Results of fluorescence-labeled melting determination of DNA length

[0088]

[0089] Note: a is the DNA length calculated according to the melting temperature, and b is the DNA length calculated according to the GC%. The average value ± standard deviation, 3 repeated experiments.

[0090] Example 3: Optimization of non-labeled fluorescence melting conditions

[0091] 3.1 Reaction system and procedure of PCR and melting

[0092] The PCR amplification system and procedure and the melting procedure were the same as those in 1.1 of the embodiment. The non-labeled fluorescence-labeled melting system was: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ , 10 mM HEPES (pH 7.9), 0.5x DNA binding dye and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, and ultrapure water instead of the additive group.

[0093] 3.2 Effect of dye type on non-labeled fluorescence melting

[0094] In order to establish a non-labeled fluorescence melting system with the most suitable dye, the melting effects of four dyes in the additive system were compared. Figure 6The melting results of the four dyes showed that the six different length DNA fragments had similar rules in the additive system. With the increase of DNA length, the melting curve was constantly right-shifted and the melting temperature gradually increased, indicating that the four dyes could all indicate the melting behavior of DNA in the additive system. The melting results of Dye 1 (A) showed that the signal on the left side of the melting peak of each DNA fragment was higher than that on the right side, and the melting results of Dye 2 (B) were similar. In addition, there was an interference peak on the right side of the main melting peak of 107 bp, which further affected the identification of the melting temperature. In comparison, the melting curves of Dye 3 (C) and Dye 4 (D) were more complete, and the interference in accurately reading the melting temperature was smaller. The melting curve of Dye 4 had less overlap than that of Dye 3 (C, D), and was more suitable for analysis of multiple fragments. Finally, Dye 4 was selected. Figure 6 Figure 6 Figure 6 Figure 6 Figure 6

[0095] 3.3 Effect of dye concentration on non-labeled fluorescent melting

[0096] Different concentrations of dyes were added to the melting system of Additive 1 or Additive 2 or Additive 3 for reaction. DNA dyes are detected by non-specific binding to the small groove of double-stranded DNA. Since Additive 1 or Additive 3 can also bind to AT bases and embed in the DNA groove, Additive 1 or Additive 3 may compete with the dye for binding to the DNA double strand. This results in that when high concentration of Additive 1 or Additive 3 is present, the DNA has low melting signal in low concentration of dye, and even no melting peak. When the dye concentration gradually increases, the fluorescence signal gradually rises, and the shape of the melting peak becomes obvious and complete. For Additive 1 or Additive 3, when the dye concentration is 2x, the DNA produces a relatively obvious melting peak, so 2x dye concentration is used for the experiment (A, D). For Additive 2 and the additive-free system, increasing the dye concentration makes the melting peak right-shifted. Since the DNA has a complete melting peak when the dye concentration is 0.5x, 0.5x dye concentration is used for the experiment (B, C). Figure 7 Figure 7

[0097] Example 4: Establishment of a method for non-labeled fluorescent melting determination of DNA length

[0098] 4.1 Non-labeled fluorescent melting of three groups of DNA length and typical representative of Tm value negative correlation

[0099] ​​​​​​​To verify that the three additives can also eliminate the influence of GC% on melting temperature in DNA binding dye, three groups of DNA fragments with typical length and negative correlation between length and melting temperature were selected for melting. Figure 8 In Fig. 2A, the GC% of 50 bp is 56%, and the GC% of 68 bp is 43%. The shaded part is the control without additive. Because the influence of GC% on melting temperature is relatively large, the melting temperature of 50 bp is higher, and the melting peak is located to the right of 68 bp. When Additive 1 or Additive 2 is added, the melting temperature of the two DNA fragments is simultaneously reduced, and the size of the melting temperature is no longer dependent on the GC%, but mainly related to the length of the DNA, so the melting temperature of 50 bp is lower than that of 60 bp. After adding Additive 3, the melting temperature of the two DNA fragments is simultaneously increased, and the melting temperature of 50 bp is lower than that of 68 bp. In addition, a group of DNA fragments with 92 bp and GC% of 43% and 107 bp and GC% of 36% are melted. Without additive, the melting temperature is 92 bp > 107 bp, while in the three additive melting systems, the melting temperature is 92 bp < 107 bp. The sequence information of the third group is 172 bp and GC% is 47% and 231 bp and GC% is 37%. Without additive, the melting temperature is 172 bp > 231 bp, and in the three additive melting systems, the melting temperature is 172 bp < 231 bp. The above results show that by adding different additives to the DNA binding dye, the melting temperature of long DNA fragments is higher than that of short fragments, which proves that the addition of additives can eliminate the influence of GC ratio on melting, so that the length of DNA and the melting temperature are positively correlated.

[0100] After optimizing the reaction conditions and verifying different lengths of DNA fragments, in order to establish a standard curve for melting to determine the length of DNA fragments, 16 different DNA fragments were reacted in Additive 1, Additive 2, No Additive or Additive 3 melting system and no additive melting system. The melting temperature of different length fragments was read, and the length and the negative reciprocal of the length were taken as the abscissa, and the melting temperature was taken as the ordinate to make a point graph. Figure 9 In Fig. 2A, the melting temperature of the group without additive and the negative reciprocal of the length of DNA has no obvious linear relationship. After adding Additive 1, Additive 2 and Additive 3, there is an obvious linear relationship between the two, and after linear fitting, the linear correlation coefficients R 2 are 0.979, 0.991 and 0.983, respectively. Therefore, for unknown length of DNA fragments, after melting by adding different additives, the melting temperature is read and brought into the fitting equation to calculate the length.

[0101] In label-free fluorescent melting systems, the role of additives is also to eliminate the influence of GC% on melting temperature; therefore, GC% and the melting temperature difference should also be correlated. A standard curve was fitted by plotting DNA length on the x-axis and the melting temperature without additives minus 0.41% GC on the y-axis, and the correlation coefficient R0.41% was used. 2 =0.976 ( Figure 9 A). Plotting GC% on the x-axis and the melting temperature difference between additive-free and additive-free samples on the y-axis, a positive correlation was found between GC% and the melting temperature difference between Additive 1, Additive 2, and No Additive. After linear fitting, the correlation coefficient R0 was found to be... 2 The values ​​were 0.957 and 0.964, respectively. GC% and Additive 3 were negatively correlated with the melting temperature difference without additives, with a correlation coefficient R0. 2 =0.938. Since the GC% and the melting temperature difference between adding and not adding the three additives show a good linear relationship, the GC% can also be calculated for DNA of unknown length using the melting temperature difference between adding and not adding the additives. Then, the GC% can be substituted into... Figure 9 The length of DNA can be calculated from the standard equation obtained by B-fit. In summary, by adding different additives to DNA-binding dyes and then melting them, the length of DNA can be calculated using both melting temperature and GC percentage.

[0102] To verify the practicality of the established label-free fluorescence melting method for determining DNA length, DNA fragments from different sources were detected. To detect common PCR amplification products, three pairs of specific primers were designed, amplifying three DNA fragments of 73 bp, 106 bp, and 209 bp. The amplification products were subjected to polyacrylamide gel electrophoresis, and the results are shown below. Figure 10 A. The electrophoresis image shows three complete electrophoretic bands, and the band size matches the actual length. Additionally, 1 μL of the amplification product was added to both the additive and non-additive systems for reaction. Figure 10 As shown in B, without the additive, the melting curves of the three PCR products overlapped and were not clearly distinguishable. After adding the additive, the melting temperature increased with increasing DNA length, and the melting curves became distinguishable. Read the corresponding melting temperatures and substitute them into... Figure 9 In the fitted equation, the length is calculated. The difference in melting temperature with and without additives is calculated and substituted into the equation. Figure 10 The fitting equation in B is used to calculate GC%, and then substituted into... Figure 10 The size of the DNA fragment was calculated using the standard equation in B, which is the melting temperature without additives minus 0.41 × GC% and the negative reciprocal of the length. The specific calculation results are shown in Table 2. Both calculation methods can obtain the DNA length, and the measured value is close to the actual length.

[0103] Table 2 DNA length results of non-labeled fluorescent melting assay

[0104]

[0105] Note: a DNA length calculated according to melting temperature, b DNA length calculated according to GC%. Mean ± standard deviation, 3 repeated experiments.

[0106] Example 5: Application of melting detection in monitoring DNA hydrolysis cleavage reaction

[0107] CRISPR-Cas12a has a cis cleavage activity, which can cut a 210 bp DNA fragment into 89 bp and 121 bp, and the corresponding GC% is 55% and 48% respectively. The polyacrylamide gel electrophoresis diagram of CRISPR-Cas12a system cleaving 210 bp PCR product shows that Figure 11 A), the system without Cas12a has only a 210 bp band, while the system with Cas12a has 89 bp, 121 bp and 210 bp three electrophoresis bands. Taking the CRISPR-Cas12a system cleaving 210 bp PCR product in the system with and without additives, the melting result shows that when there is no additive, the melting temperature of the two DNA fragments of 89 bp and 121 bp produced by Cas12a cleavage is close, so they are fused into one melting peak, and there is also a 210 bp that is not completely cleaved, a total of 2 melting peaks exist. When a certain concentration of additive is added, the influence of GC% on melting temperature is eliminated, and the melting temperature is mainly related to the length, so the melting of 89 bp and 121 bp can be distinguished, so there are three melting peaks Figure 11 B). It is proved that this melting detection method can be used for the analysis of CRISPR-Cas12a cleaving PCR product.

[0108] Example 6: Application of DNA length melting assay in multiplex PCR detection

[0109] For the multiplex identification and detection of goat, cow and sheep animal-derived ingredients in dairy products, the sample DNA was extracted for multiplex detection and authenticity identification. Among them, the sample sources: fresh milk samples were purchased from Caotan Dairy Farm in Xi'an Weiyang District; Fresh goat milk was purchased from Xi'an Weiyang District market in Shaanxi Province; Sheep milk and various brands of commercial milk powder samples were purchased from online stores. DNA extraction: magnetic bead method blood genomic DNA extraction kit.

[0110] 5.1 Multiplex PCR melting detection

[0111] (1) PCR primer design

[0112] The corresponding nucleotide sequences for each species were found using NCBI. Primers were designed and screened using Primer 5 software, and primer specificity was evaluated using Primer-blast software. The specific primer pair for goat DNA was Primer1, the specific primer pair for amplifying bovine DNA was Primer2, and the specific primer pair for amplifying sheep DNA was Primer3. The specific primer sequences are as follows:

[0113] The sequence of primer pair Primer1 is as follows:

[0114] Upstream primer: 5'-GGGGGGGGGGGCCATAATTACAACAA-3'

[0115] Downstream primer: 5'-CCCTATCAGCTGCAGTAGGGTT-3'

[0116] The sequence of primer pair Primer2 is as follows:

[0117] Upstream primer: 5'-AGTAAGCGTAATTATGATAC-3'

[0118] Downstream primer: 5'-CCCCCTTGATTCTCTTGGTGTAGAG-3'

[0119] The sequence of primer pair Primer3 is as follows:

[0120] Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'

[0121] Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'

[0122] (2) Multiplex PCR reaction system and procedure

[0123] The primer concentration ratio of Primer 1:Primer 2:Primer 3 was 9:3:5. 1×QIAGEN MultiplexPCR Master Mix, 0.5×dye, and 20 ng / μL DNA were used. Double-distilled water was added to make up to 10 μL of the reaction mixture, with double-distilled water used as a negative control. The PCR amplification program was as follows: 94℃ pre-denaturation for 10 min; 94℃ for 15 s denaturation, 57℃ for 60 s annealing and extension, for 25 cycles; and 72℃ for a final extension of 5 min. Fluorescence signals were collected during the extension phase of each cycle. The melting system and program were the same as in 3.1 of Example 3.

[0124] (3) Multiplex PCR melting detection

[0125] The additives were added to the single, double and triple PCR amplification products for melting discrimination. Without the additive, the multi-component samples did not form multiple melting peaks, and multiple detection could not be performed. After adding two kinds of additives respectively, the melting temperatures of the single-component samples were different, and they could be distinguished from each other according to the positions of the melting peaks; the double-component samples formed two melting peaks; the triple-component samples produced three melting peaks, and seven different component samples could be discriminated by melting Figure 12 A). Figure 12 B is a capillary gel electrophoresis diagram of PCR amplification products, and the amplification products with different components produce corresponding electrophoresis bands. The electrophoresis results are consistent with the melting results, proving that the method can realize multiple detection.

[0126] 5.2 Actual sample melting detection

[0127] DNA extracted from eight kinds of commercially available samples was detected by DNA length melting determination combined with multiple PCR method to test its practical application. Figure 13 A is the melting peak curve of goat, cow, sheep, goat + cow + sheep reference controls and eight dairy products after adding additives. By comparing and analyzing the melting peaks of the reference controls, the components of the eight samples were confirmed, and the capillary electrophoresis method was used for verification (13B). The detection results of the two methods are consistent, proving that the method can be used for species-specific dairy ingredient identification, detection and identification.

[0128] Example 7: A kit for determining DNA length and a method of use

[0129] (1) Kit composition

[0130] The kit contains tetramethylammonium chloride or tetraethylammonium chloride or betaine additive for establishing an additive melting system, DNA binding dye, 1x reaction buffer: 6mM Mg 2+ and 10mM HEPES, and fluorescence-labeled, non-labeled fluorescence under different additives corresponding to the melting temperature and length of the standard curve, the standard curve of GC content and the melting temperature difference with and without additive.

[0131] (2) Kit use method

[0132] For fluorescently labeled DNA samples, take 1 μL of the DNA to be tested, add 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride, 1x reaction buffer for high-resolution melting. For non-labeled fluorescent DNA samples, in addition to the fluorescent labeling melting system, 2x or 0.5x DNA binding dye is added. After melting, the melting temperature is read and the corresponding standard equation is calculated. Based on the melting temperature and GC%, the length information of the DNA to be tested can be obtained.

[0133] The preferred embodiments of the present application are not intended to limit the scope of protection of the present application, and any equivalent structural changes made based on the content of the specification and drawings of the present application shall be included in the patent protection scope of the present application. SEQUENCE LISTING <110> Shaanxi University of Science and Technology <120> A melting assay method and kit for DNA length <130> 2022 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Artificial sequence <400> 1 gggggggggg gccataatta caacaa 26 <210> 2 <211> 22 <212> DNA <213> Artificial sequence <400> 2 ccctatcagc tgcagtaggg tt 22 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 agtaagcgta attatgatac 20 <210> 4 <211> 25 <212> DNA <213> Artificial sequence <400> 4 cccccttgat tctcttggtg tagag 25 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <400> 5 cgtagatgta gtatgacttt tcct 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <400> 6 gtgaagttag ttaggagagt aattata 27

Claims

1. A method for determining the length of DNA by melting, characterized in that: Includes the following steps: 1) The effect of GC content on melting temperature can be eliminated by adding tetramethylammonium chloride, tetraethylammonium chloride, or betaine additive to the DNA sample; 2) Establish standard curves for melting temperature and length corresponding to different additives, as well as standard curves for GC content and melting temperature difference between adding and not adding additives; 3) For DNA of unknown length, add tetramethylammonium chloride, tetraethylammonium chloride, or betaine additive to melt it, and read the corresponding melting temperature and input it into two standard curves to calculate the DNA length; 4) To determine the length of the DNA hydrolysis product, add tetramethylammonium chloride, tetraethylammonium chloride, or betaine additive to the DNA hydrolysis product, read the corresponding melting temperature, and input it into two standard curves to calculate the length of the DNA hydrolysis product. 5) To determine the length of PCR products, the amplicons are obtained by designing corresponding PCR primers. Tetramethylammonium chloride, tetraethylammonium chloride, or betaine additive is added to the PCR products, and the corresponding melting temperature is read and input into two standard curves to calculate the length of the PCR products. 6) For the differentiation of multiplex PCR products and the detection of actual samples, multiple pairs of specific PCR primers are designed by comparing nucleotide sequences and specificity tests. Tetramethylammonium chloride, tetraethylammonium chloride, or betaine additives are added to single, double, and triple PCR amplification products to melt and differentiate them. The actual samples were collected from fresh milk and dairy products, and the positions of the melting peaks of the reference sample and the sample to be tested were compared for identification and detection.

2. The method for determining DNA length by melting according to claim 1, characterized in that: In step 1), the formation of the melting curve is monitored by both fluorescent labeling and unlabeled fluorescence, and the corresponding melting temperature is read.

3. The method for determining DNA length by melting according to claim 1, characterized in that: In step 3), the DNA length is determined by melting and adding any one of tetramethylammonium chloride, tetraethylammonium chloride, or betaine additive.

4. The method for determining DNA length by melting according to claim 1, characterized in that: In step 6), the specific primer pair used to amplify goat DNA is Primer1, the specific primer pair used to amplify bovine DNA is Primer2, and the specific primer pair used to amplify sheep DNA is Primer3. The sequence of the primer pair Primer1 is as follows: Upstream primer: 5'- GGGGGGGGGGGCCATAATTACAACAA -3' Downstream primer: 5'-CCCTATCAGCTGCAGTAGGGTT-3' The sequence of primer pair Primer2 is as follows: Upstream primer: 5'-AGTAAGCGTAATTATGATAC-3' Downstream primer: 5'-CCCCCTTGATTCTCTTGGTGTAGAG-3' The sequence of primer pair Primer3 is as follows: Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3' Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'.

5. The method for determining DNA length by melting according to claim 1, characterized in that: In steps 1), 3), 4), and 5), the melting system for the fluorescently labeled material is: 1 μL of synthetic DNA or PCR amplification product, 6 mM Mg 2+ 10 mM M HEPES and 1–3 M tetraethylammonium chloride or 4–6 M betaine or 2–4 M tetramethylammonium chloride; for the additive-free group, ultrapure water was used instead. The label-free fluorescent melting system consisted of: 1 μL synthetic DNA or PCR amplification product, 6 mM Mg 2+ 10 mM HEPES at pH 7.9, 0.5× or 2× DNA-binding dye, and 1-3 M tetraethylammonium chloride or 4-6 M betaine or 2-4 M tetramethylammonium chloride; for the additive-free group, ultrapure water was used instead; the high-resolution melting program was 37-99℃, and the melting rate was 0.05℃ / s.

6. The method for determining DNA length by melting according to claim 1, characterized in that: In step 4), the PCR reaction system consists of: 50 nM forward and reverse primers, 1×Taq PCR Master Mix, 2 ng / µL of purified PCR product, and finally, double-distilled water to make up to 10 µL of the reaction system, with double-distilled water used as a negative control. The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 57℃ annealing for 20 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min.

7. The method for determining DNA length by melting according to claim 1, characterized in that: In step 5), the PCR reaction system consisted of the following: primers in the Primer 1:Primer 2:Primer 3 ratio of 9:3:5, 1×QIAGEN Multiplex PCR Master Mix, 0.5×DNA binding dye, 20 ng / µL DNA, and finally, double-distilled water was added to make up to 10 µL of the reaction system. Double-distilled water was used as a negative control. The PCR amplification program was as follows: 94℃ pre-denaturation for 10 min; 94℃ for 15 s denaturation, 57℃ for 60 s annealing and extension, for 25 cycles; and 72℃ for 5 min extension. Fluorescence signals were collected during the extension phase of each cycle.

8. A DNA length melting assay kit, characterized in that: This kit contains tetramethylammonium chloride or tetraethylammonium chloride or betaine additive for establishing the additive melting system, DNA binding dye, and a 1× reaction buffer: 6 mg / mL. 2+ The kit contains standard curves of melting temperature and length corresponding to 10 mM HEPES and different additives for length determination, as well as standard curves of GC content and melting temperature difference with and without additives. The method for determining DNA length in the kit is the method described in any one of claims 1-7.

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