CN116987798A - Primer combination, kit and method for detecting short tandem repeat sequence - Google Patents
Primer combination, kit and method for detecting short tandem repeat sequence Download PDFInfo
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Abstract
The invention provides a primer combination for detecting short tandem repeat sequences, which comprises 4 groups of primers, wherein the 1 st group consists of primers with sequences of 1-8 and is used for detecting loci THO1, D21S11, D2S1338 and Penta E; group 2 consisted of primers of sequences 9-22 for detection against loci D5S818, D13S317, D7S820, D16S539, CSF1P0, D18S51, D19S 433. The invention redesigns the primer and the detection method suitable for detecting the short tandem repeat sequence of the Chinese crowd aiming at the locus with high genetic polymorphism of the Chinese crowd, and the detection method is quick, simple, convenient and visual and has low experiment cost.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a primer, a kit and a method for detecting a short tandem repeat sequence.
Background
The short tandem repeat (short tandem repeats, STR) is a class of DNA tandem repeats that are widely found in the human genome and have a core sequence of 2 to 6 bases. The short tandem repeat sequence has extremely high recognition rate among different individuals, and according to statistics, 12 STR loci among irrelevant individuals have 10-14 probability of complete identity, 10-4 probability of complete identity among siblings, and the difference conforms to Mendelian genetic rules in the genetic process. Therefore, STR amplification detection techniques are widely used in individual identification, paternity test, population genetics research, and chimeric rate detection.
With the high degree of automation of capillary electrophoresis equipment, a large number of STR sites are defined by standards for identification of various large humans (european STR standard ESS, DNA standard locus ISSOL adopted by international criminal police organization, joint DNA indexing system CODIS of federal survey agency, etc.), and more researchers have been devoted to the development of this field.
At present, domestic STR detection is mostly dependent on imported STR kits. Numerous studies have reported that genetic polymorphisms at the loci of short tandem repeats differ somewhat between ethnicities.
In view of this, there is an urgent need to develop STR detection methods and kits that are more suitable for the chinese population and that can be adapted to the detection instruments commonly used in modern times.
Disclosure of Invention
The invention provides a primer combination, a kit and a method for detecting a short tandem repeat sequence, which are used for solving at least one of the technical problems.
To solve the above problems, as one aspect of the present invention, there is provided a primer set for detecting short tandem repeats, comprising 4 sets of primers, wherein set 1 consists of primers of sequences 1 to 8 for detection against loci THO1, D21S11, D2S1338, penta E; group 2 consists of primers of sequences 9-22 for detection against loci D5S818, D13S317, D7S820, D16S539, CSF1P0, D18S51, D19S 433; group 3 consists of primers of sequences 23-32 for detection against loci Amelogenin, vWA, D S1179, TPOX, FGA; group 4 consists of primers of sequences 33-42 for detection against genes D6S1043, D12S391, D3S1358, D10S1248, penta D.
Preferably, one primer in the primer pair for detecting each locus is labeled with a fluorescent dye, and the 4 sets of primers are labeled with 4 different fluorescent dyes, respectively, at the 5' ends of the primers.
Preferably, the 1 st group of primers is marked by blue fluorescent dye, the 2 nd group of primers is marked by green fluorescent dye, the 3 rd group of primers is marked by yellow fluorescent dye, and the 4 th group of primers is marked by red fluorescent dye.
Preferably, the blue fluorescent label is 6-FAM, the green fluorescent label is HEX, the yellow fluorescent label is TAMRA, and the red fluorescent label is ROX.
Preferably, the concentration of each primer is 0.05 to 0.4. Mu. Mol/L, preferably 0.06 to 0.2. Mu. Mol 1/L.
The invention also provides a kit for detecting the short tandem repeat sequence, which comprises the primer combination.
The invention also provides application of the primer combination in preparation of a kit for detecting the short tandem repeat sequence.
The invention also provides an application of the primer combination or the kit in detecting the short tandem repeat sequence.
The invention also provides a method for detecting the short tandem repeat sequence, which comprises the following steps:
step 1, obtaining DNA of a sample to be detected;
step 2, adopting the primer combination or the kit, and carrying out PCR amplification by taking the DNA obtained in the step 1 as a template, preferably adopting a fluorescent label composite amplification system for amplification;
and 3, detecting and typing analysis is carried out on the PCR amplification product obtained in the step 2, so as to obtain the types of all loci of the sample to be detected, and preferably, capillary electrophoresis detection is adopted.
Preferably, the PCR amplification system comprises the following components: 50mmol/L KC1, 20mmol/L Tris-HC1, 2.5mmol/L MgC1 2 0.3mmol/L dNTP (concentration of each deoxyribonucleotide is 0.3)mmol/L), 3.2% glycerol and 0.07% Tween 20, 100units/mL Taq DNA polymerase, 0.05. Mu. Mol/L-0.4. Mu. Mol/L primer;
the amplification volume is 10. Mu.L to 50. Mu.L, preferably 10. Mu.L to 25. Mu.L;
the PCR amplification procedure was: 95 ℃ for 2-5 min;95 ℃ and 20s; 58-60 ℃ and 20-2 min30s; 68-72 ℃ for 30s-2min;25-30 cycles; 68-72 ℃ and 25-45 min;
the PCR amplification procedure is preferably: 95 ℃ for 5min;95 ℃ and 20s;60 ℃ for 2min;68 ℃, lmin;25 cycles; 68 ℃ for 25min.
By adopting the technical scheme, the primers and the detection method suitable for detecting the short tandem repeat sequences of the Chinese population are redesigned according to the loci with high genetic polymorphism of the Chinese population, and the detection method is quick, simple, convenient and visual and has low experiment cost.
Drawings
FIG. 1 is a graph showing the amplification detection results of the first sample.
FIG. 2 is a graph showing the amplification detection results of the second sample.
Detailed Description
The following describes embodiments of the invention in detail, but the invention may be practiced in a variety of different ways, as defined and covered by the claims.
In one aspect, the invention provides a primer combination for detecting short tandem repeats, which consists of a sequence shown in 001-042 in a sequence table, and is used for detecting loci TH01, D21S11, D2S1338, penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, D18S51, D19S433, amelogenin, vWA, D S1179, TPOX, FGA, D S1043, D12S391, D10S1248, D3S1358 and Penta D respectively.
The short tandem repeat sequence detected by the invention comprises loci D2S1338, D12S391, D6S1043, D10S1248, D18S51, D19S433 and D3S1358 with high genetic polymorphism of Chinese people besides loci with high genetic polymorphism of human beings. These loci all belong to loci with high discrimination, high heterozygosity and high information content in Chinese population. Therefore, the detection method designed by the invention is more suitable for Chinese people, and is beneficial to popularization and application of the technology. In addition, the serial repeated units of the STR gene locus selected by the invention are four, five or six, so that the slip peak proportion can be effectively reduced, and the obtained detection result has higher accuracy.
In a preferred embodiment of the invention, the primer combination further consists of a 1 st to 4 TH set of primers, wherein the 1 st set consists of primers shown in sequences 1 to 8, the detection is performed for loci TH01, D21S11, D2S1338, pentaE, the 2 nd set consists of primers shown in sequences 9 to 22, the detection is performed for loci D5S818, D13S317, D7S820, D16S539, CSF1PO, D18S51, D19S433, the 3 rd set consists of primers shown in sequences 23 to 32, the detection is performed for loci Amelogenin, vWA, D S1179, TPOX, FGA, the 4 TH set consists of primers shown in sequences 33 to 42, the detection is performed for loci D6S1043, D12S391, D3S1358, D10S1248, pentaD.
In a further preferred embodiment of the invention, one primer of the primer pair for each locus is labeled with a fluorescent dye for capillary electrophoresis detection to determine its allelic identity and size. Meanwhile, the 4 groups of primers are respectively marked by adopting 4 fluorescent dyes with different colors and are respectively marked at the 5' ends of the primers. In a more preferred embodiment of the invention, the 1 st primer set is labeled with a blue fluorescent dye, the fluorescent label is 6-FAM, the 2 nd primer set is labeled with a green fluorescent dye, the fluorescent label is HEX, the 3 rd primer set is labeled with a yellow fluorescent dye, the fluorescent label is TAMRA, the 4 th primer set is labeled with a red fluorescent dye, the fluorescent label is ROX, and meanwhile an orange fluorescent label is selected as an internal standard, and the fluorescent label is CY5. Therefore, after the amplified products of each group are mixed with the orange fluorescent marked internal standard, electrophoresis can be carried out in the same capillary, and the simultaneous detection and analysis of 21 STR loci are realized.
Five fluorescent markers of 6-FAM, HEX, TAMRA, ROX and CY5 are adopted for fluorescent marking, and the fluorescein marking technology is mature and has low cost. However, the fluorescent marker used in the present invention is not limited thereto, and those skilled in the art may select other alternative fluorescent markers according to the actual circumstances.
In the detection method provided by the invention, the sequence information of the specific amplification primers adopted by the 21 STR loci is shown in table 1.
TABLE 1 primer Condition Table for detecting short tandem repeats according to the invention
In the detection method provided by the invention, all primers of 21 loci can be mixed together, and the composite amplification of alleles of the 21 loci can be completed in one PCR reaction. Therefore, the composite amplification system has the advantages of simple operation, high efficiency and low cost, and is more beneficial to popularization and application of the method.
Furthermore, in a preferred embodiment of the present invention, the concentration of each primer is 0.05 to 0.4. Mu. Mol 1/L, more preferably 0.06 to 0.2. Mu. Mol/L. In some specific embodiments, the primer concentrations for the 21 loci are shown in table 2.
TABLE 2 primer concentration table for detecting short tandem repeats of the invention
In one aspect, the invention provides a kit for detecting short tandem repeats comprising a primer combination according to the invention.
In another aspect, the invention provides the use of a primer combination according to the invention for the preparation of a kit for the detection of short tandem repeats.
In a further aspect, the invention provides a primer combination according to the invention or the use of a kit according to the invention for the detection of short tandem repeat sequences.
In a final aspect the invention provides a method of detecting a short tandem repeat sequence comprising the steps of:
1. and obtaining the DNA of the sample to be detected.
2. The primer combination or the kit is adopted, the DNA obtained in the step 1 is used as a template for PCR amplification, and a fluorescent labeling multiplex amplification system is preferably adopted for amplification.
3. And (3) detecting and typing the PCR amplification products obtained in the step (2) to obtain the types of the loci of the sample to be detected, wherein capillary electrophoresis detection is preferably adopted.
In the detection method provided by the invention, a PCR amplification system adopted for amplification comprises a buffer system. The buffer system comprises: KCl, tris-HCl, mgC1 2 Dntps, glycerol and tween 20, wherein dntps are equimolar mixtures of four deoxyribonucleotides (dATP, dTTP, dCTP and dGTP). The DNA polymerase used in the amplification method is Taq DNA polymerase, and can be selected from hot start DNA polymerase, antibody blocking modification and/or chemical modification.
In a further provided embodiment of the invention, the PCR amplification system used in the amplification of the invention comprises the following components in the amounts: 50mmol/L KCl,20mmol/L Tris-HCl,2.5mmol/L MgCl2, 0.3mmol/L dNTP (the concentration of each deoxyribonucleotide is 0.3 mmol/L), 3.2% glycerol and 0.07% Tween 20, 100units/mL Taq DNA polymerase, 0.05 μmol 1/L-0.4 μmol/L primer. The amplification volume is 10. Mu.L to 50. Mu.L, preferably 10. Mu.L to 25. Mu.L.
In the detection method provided by the invention, the procedure adopted for PCR amplification is divided into three steps, namely, the first step: 95 ℃ for 2-5 min; and a second step of: 95 ℃ for 20s; 58-60 ℃,20s-2min30s; 68-72 ℃ for 30s-2min;25-30 cycles; and a third step of: 68-72 deg.c for 25-45 min. In a further preferred embodiment of the invention, the procedure used for PCR amplification according to the invention is in particular: the first step: 95 ℃ for 5min; and a second step of: 95 ℃ for 20s;60 ℃ for 2min;68 ℃ for 1min;25 cycles; and a third step of: 68 ℃ for 25min.
In the detection method provided by the invention, when the primer with fluorescent label is adopted for amplification, the amplified product also carries the fluorescent label. In the capillary electrophoresis process, the carried fluorescent marker can emit a fluorescent signal under the excitation of laser, and the fluorescent signal can be identified and collected by a sequencer (such as 3730, 3730XL and the like) or a genetic analyzer (such as CLASSIC 116 and the like), and can be applied to a detection instrument commonly used in modern times.
Compared with the prior art, the invention has the following advantages.
(1) The short tandem repeat sequence aimed at by the detection method provided by the invention comprises 21 loci, and the loci with high genetic polymorphism of Chinese people are selected.
(2) The serial repeated units of the STR gene locus selected by the invention are four, five or six, so that the slip peak proportion can be effectively reduced, and the obtained detection result has higher accuracy.
(3) The detection method provided by the invention can be used for amplifying by adopting a fluorescent-labeled composite amplification system, and the obtained amplification product can be electrophoresed in the same capillary, so that the simultaneous detection and analysis of 21 STR loci are realized.
(4) The detection method provided by the invention can adopt FAM, HEX, TAMRA, ROX and CY5 fluorescent markers for fluorescent marking, and has mature fluorescein marking technology and low cost.
(5) Experimental results show that the detection method provided by the invention has good specificity, can be used for qualitative analysis such as genetic research of individuals identification and genetic identification population, and can also be used for quantitative analysis such as detection of the chimeric rate after hematopoietic stem cell transplantation.
In summary, the invention determines the primers corresponding to the 21 STR loci employed in the detection method through a great deal of creative work, and groups and combines them to satisfy simultaneous detection of 21 STR locus information in one sample. Meanwhile, the invention determines the optimal primer proportion, amplification system and amplification condition through a large amount of exploration, and the obtained detection method has good specificity and accurate parting result and can be used for qualitative and quantitative analysis.
The detection method of the short tandem repeat sequence and the primer and the raw materials used in the kit can be purchased from the market. In order that those skilled in the art can better understand the technical solution of the present invention, the following description of the present invention will be further described with reference to examples.
Examples: 21 STR loci detection of 2 cases of human genome DNA samples
In the embodiment, a magnetic bead method is adopted to extract DNA, so as to obtain a human genome DNA sample; performing PCR amplification on the obtained DNA sample, wherein the adopted primer fluorescent labels are 6-FAM, HEX, TAMRA and ROX respectively; performing capillary electrophoresis detection on the PCR amplified product; and (5) parting analysis. Meanwhile, the types of 21 STR loci in the obtained human genomic DNA sample are determined by combining the STR detection kit of Life company and Sanger sequencing method.
The method comprises the following steps:
1. configuration of primer composition stock solution
A5X primer stock was prepared according to the formulation shown in Table 3.
TABLE 3 formulation of 5X primer stock
2. PCR amplification
In PCR amplification, 21 STR sites of a DNA sample were amplified in an amplification system as shown in Table 4.
TABLE 4 concrete composition of PCR amplification reaction System
Wherein the 5X buffer comprises the following components in percentage by weight: 250mmol/L KC1, 100mmol/L TrisHC1, 12.5mmol/L MgC12, 1.5mmol/L dNTP (1.5 mmol/L concentration of each deoxyribonucleotide), 16% glycerol and 0.35% Tween 20.
The PCR amplification procedure is divided into three steps: the first step: 95 ℃ for 5min; and a second step of: 95 ℃ for 20s;60 ℃ for 2min;68 ℃ for 1min;25 cycles; and a third step of: preserving at 68deg.C, 25min, and 4deg.C.
3. Capillary electrophoresis
The amplification product obtained in the step 2 is subjected to internal molecular weight labeling with mechlorethamine and CY5 according to the following 1 mu L: 8.8. Mu.L: 0.2 μl was mixed and loaded into 96-well plates, denatured at 95deg.C for 3min, ice-bath for 3min, and subjected to capillary electrophoresis using a remote CLASSIC 116 sequencer to collect data.
4. Typing analysis
The experimental data collected in step 3 were analyzed by fragment analysis software GeneMapper to obtain typing data of the test samples.
The results are shown in figures 1-2 of the specification, wherein figure 1 is the first sample detection, peak 1 and peak 2 are the allele amplification peak of the THO1 locus, peak 3 and peak 4 are the allele amplification peak of the D21S11 locus, peak 5 and peak 6 are the allele amplification peak of the D2S1338 locus, and peak 7 and peak 8 are the allele amplification peak of the PentaE locus, peak 9 and peak 10 are the allele amplification peak of the D5S818 locus, peak 11 is the allele amplification peak of the D13S317 locus, peak 12 and peak 13 are the allele amplification peak of the D7S820 locus, peak 14 and peak 15 are the allele amplification peak of the D16S539 locus, peak 16 and peak 17 are the allele amplification peak of the CSF1PO locus, peak 18 and peak 19 are the allele amplification peak of the D18S51 locus, peak 20 and peak 21 are the allele amplification peak for the D19S433 locus, peak 22 and peak 23 are the allele amplification peak for the Amelogenin locus, peak 24 and peak 25 are the allele amplification peak for the vWA locus, peak 26 and peak 27 are the allele amplification peak for the D8S1179 locus, peak 28 and peak 29 are the allele amplification peak for the TPOX locus, peak 30 and peak 31 are the allele amplification peak for the FGA locus, peak 32 and peak 33 are the allele amplification peak for the D6S1043 locus, peak 34 and peak 35 are the allele amplification peak for the D12S391 locus, peak 36 and peak 37 are the allele amplification peak for the D3S1358 locus, peak 38 and peak 39 are the allele amplification peak for the D10S1248 locus, and peak 40 and peak 41 are the allele amplification peak for the PentaD locus. FIG. 2 shows the second sample amplification test, wherein peak 1 and peak 2 show the THO1 locus, peak 3 and peak 4 show the D21S11 locus, peak 5 shows the D2S1338 locus, peak 6 shows the PentaE locus, peak 7 and peak 8 shows the D5S818 locus, peak 9 shows the D13S317, peak 10 shows the D7S820 locus, peak 11 shows the D16S539, peak 12 and peak 13 shows the D21S11, peak 14 shows the D18S51, peak 16 and peak 17 shows the D19S433, peak 18 and peak 19 shows the Amelogenin locus, peak 9 shows the D13, peak 10 shows the D15 shows the D22, and peak 23 shows the D22 and the D25 shows the D22, and the D23 shows the D22 and the D23 shows the D24, the D22 shows the D25, the D22 shows the D22 and the D25 shows the D23.
According to the detection result, the amplified segment sizes of the alleles of each STR locus of the DNA sample are obtained, so that the allele types of each STR locus can be obtained, the obtained allele types are compared with the actual allele types, the obtained allele types are consistent with the actual allele types, the detection result accuracy is good, and the detection method provided by the embodiment realizes the compound amplification of 21 loci, is simple and convenient to operate, has low cost and high efficiency, and is suitable for application and popularization.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A primer set for detecting short tandem repeats, comprising 4 sets of primers, wherein set 1 consists of primers of sequences 1-8 for detection against loci THO1, D21S11, D2S1338, penta E; group 2 consists of primers of sequences 9-22 for detection against loci D5S818, D13S317, D7S820, D16S539, CSF1P0, D18S51, D19S 433; group 3 consists of primers of sequences 23-32 for detection against loci Amelogenin, vWA, D S1179, TPOX, FGA; group 4 consists of primers of sequences 33-42 for detection against genes D6S1043, D12S391, D3S1358, D10S1248, penta D.
2. The primer combination for detecting short tandem repeat according to claim 1, wherein one primer in the primer pair for detecting each locus is labeled with a fluorescent dye, and the 4 sets of primers are labeled with 4 different colors of fluorescent dyes, respectively, at the 5' ends of the primers.
3. The primer combination for detecting short tandem repeats according to claim 2, wherein the 1 st primer set is labeled with a blue fluorescent dye, the 2 nd primer set is labeled with a green fluorescent dye, the 3 rd primer set is labeled with a yellow fluorescent dye, and the 4 th primer set is labeled with a red fluorescent dye.
4. The primer combination for detecting short tandem repeat according to claim 3, wherein the blue fluorescent label is 6-FAM, the green fluorescent label is HEX, the yellow fluorescent label is TAMRA, and the red fluorescent label is ROX.
5. The primer combination for detecting short tandem repeat according to claim 4, wherein the concentration of each primer is 0.05 to 0.4. Mu. Mol/L.
6. A kit for detecting short tandem repeats, comprising a primer combination according to any one of claims 1-5.
7. Use of a primer combination according to any one of claims 1-5 for the preparation of a kit for detecting short tandem repeats.
8. Use of a primer combination according to any one of claims 1-5, or a kit according to claim 6, for detecting short tandem repeats.
9. A method for detecting short tandem repeats, comprising the steps of:
step 1, obtaining DNA of a sample to be detected;
step 2, performing PCR amplification by using the primer combination according to any one of claims 1 to 5 or the kit according to claim 6, and using the DNA obtained in step 1 as a template;
and 3, detecting and typing analysis is carried out on the PCR amplification product obtained in the step 2, and the types of all loci of the sample to be detected are obtained.
10. The method of claim 9, wherein the PCR amplification system comprises the following components: 50mmol/L KC1, 20mmol/L Tris-HC1, 2.5mmol/L MgC1 2 0.3mmol/L dNTP, 3.2% glycerol and 0.07% Tween 20, 100units/mL Taq DNA polymerase, 0.05. Mu. Mol/L-0.4. Mu. Mol/L primer;
the amplification volume is 10 mu L-50 mu L;
the PCR amplification procedure was: 95 ℃ for 2-5 min;95 ℃ and 20s; 58-60 ℃ and 20-2 min30s; 68-72 ℃ for 30s-2min;25-30 cycles; 68-72 deg.c, 25-45 min.
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