CN112553349A - Identification primer, probe, kit and method for homozygote and heterozygote of Hulunbel short-tailed sheep - Google Patents
Identification primer, probe, kit and method for homozygote and heterozygote of Hulunbel short-tailed sheep Download PDFInfo
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Abstract
The invention discloses an identification primer, a probe and a kit for homozygotes and heterozygotes of a Renbell short-tailed sheep, and provides a method for identifying homozygotes and heterozygotes of the Renbell short-tailed sheep based on the primer and the probe. The method comprises the following steps: (1) extracting and diluting DNA of a blood genome of a Hulunbel short-tailed sheep; (2) carrying out taqman SNP fluorescent probe qPCR on the DNA obtained in the step (1); (3) and (5) detecting a product. Through taqman SNP fluorescent probe qPCR, homozygote and heterozygote of the Columbus brevifilis can be rapidly distinguished. The method is used as one of the means for genotyping the Hulunbel short-tailed sheep, and has the advantages of simple and convenient operation, good repeatability, short reaction time and extremely high sensitivity and specificity.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an identification primer, a probe, a kit and a method for homozygotes and heterozygotes of a Hulenbel short-tailed sheep based on a taqman SNP fluorescent probe.
Background
The Plebeier short-tailed sheep is one of meat sheep varieties growing in the Plebeier area in China, and the short and fat tail is a unique mark of the variety. We screened T/Brachyury genes related to spinal development by using a genome re-sequencing technology as candidate genes, confirmed that stable T/Brachyury gene c.G334T mutation exists in a Renbell Brachyury population, and proved that the character of economic value, short tail, is related. In molecular breeding of sheep, homozygous individuals exhibit stable traits relative to those of heterozygous offspring, and thus, in recent years, it is more preferable to select homozygous individuals in a population as a main breeding target.
The traditional sequencing method is mainly based on a Sanger sequencing method and is based on the association of SNP loci with commercial value-related traits, and the Sanger method is a method for obtaining a visible DNA base sequence by starting nucleotides at a fixed point, randomly terminating at a specific base, carrying out fluorescent labeling after each base to generate four groups of nucleotides with different lengths, ending with A, T, C, G, and carrying out electrophoresis detection on a urea denaturation PAGE gel.
Conventional sequencing techniques suffer from the disadvantages of long time, high cost, low throughput, and the composition or secondary structure of the DNA template sometimes causing abnormal termination of the DNA polymerase.
Disclosure of Invention
The invention aims to provide an identification primer, a probe, a kit and a method for a Renbell short-tailed sheep, which overcome the defects that the number of samples for one-time identification is small, the operation is complicated and the use time is long due to the technical limitation of the traditional sanger sequencing method.
The purpose of the invention is realized by the following technical scheme:
an identification primer and a probe for homozygote and heterozygote of a Hulun-Beijing sheep, which are characterized by comprising the following sequences:
a forward primer: 5'-TGCGCCCCTTCCTTTTCAG-3'
Reverse primer: 5'-GGGGGAGTCGGGGTGGATGTAG-3'
taqman probe 1: 5' HEX-TGGCTTGCCCCCCGGCA-BHQ 13
taqman probe 2: 5' FAM-GCTTGCCCCAGGGCACCCA-BHQ 13,
the taqman probes 1 and 2 are fluorescence labeling probes;
a kit for identifying homozygote and heterozygote of a Hulenbel short-tailed sheep comprises the identification primer and the probe, and also comprises the following components: PCR reaction mixed liquor, genome DNA of the Renbell Brachyury, and standard plasmids prepared from homozygote and heterozygote of the T/Brachyury gene of the Renbell Brachyury.
Preferably, the PCR reaction mixture includes SNP Probe Master Mix (2X) 10 ul and RNase-Free ddH2O 4.6 ul。
Preferably, the concentration of the genome DNA of the Renbell short-tailed sheep is 10 ng/ul, and the dosage is 1 ul.
A method for identifying homozygotes and heterozygotes of a Hulenbel short-tailed sheep comprises the following steps:
(a) extracting and diluting DNA of a blood genome of a Hulunbel short-tailed sheep;
(b) the fluorescent quantitative PCR reaction system amplifies the fragment sequence of the Brachyury/T gene of the Renbell Brachyury, and the sequence fragment is from 95879486 sites to 95879323 sites of the homozygous and heterozygous Brachyury/T gene of the Brachyury, such as SEQ ID NO: 5, the PCR reaction system comprises the identification primer and the probe;
(c) and detecting PCR reaction products.
Preferably, the total volume of the PCR reaction system in the step (b) is 20ul, and the PCR reaction system comprises 10 ul of SNP Probe Master Mix (2X), 1.8ul of forward primer, 1.8ul of reverse primer, and the concentration ratio of the forward primer to the reverse primer is 1: 10, 10uM taqman probe 10.4 ul, 10uM taqman probe 20.4 ul, 10 ng/ul DNA template 1 ul, RNase-Free ddH2O 4.6 ul。
Preferably, the PCR reaction is performed according to the following reaction procedure: pre-denaturation at 95 ℃: 30 s, denaturation at 95 ℃: 10 s, 60 ℃ annealing-extension: 30 s, amplifying for 45 cycles, and taking a picture for 30 s at 60 ℃ by performing fluorescence scanning on a fluorescence plate reader after the PCR reaction is finished.
Preferably, the concentration of the forward primer is 1 uM and the concentration of the reverse primer is 10 uM.
Preferably, the SNP Probe Master Mix contains an internal reference fluorescent dye.
The invention has the beneficial effects that:
(1) the genotyping method can replace the traditional sequencing and is one of the means for genotyping the Renbell short-tailed sheep, so the cost is low;
(2) the operation is simple, the qualitative identification speed is high, the taqman probe genotyping operation only needs three steps of obtaining a sample DNA template, configuring a system and reading a result by a machine reaction, the time is consumed for 2 hours, and the traditional sequencing method needs about three days due to transportation and sample quality determination.
(3) The Renbebel short-tailed sheep identification kit based on the taqman probe genotyping can identify 96 samples at most at one time, and compared with the original technology, the flux is greatly improved.
Drawings
FIG. 1 is the genomic DNA of a 1% agarose gel electrophoresis of a Hulenbel short-tailed sheep according to the present invention;
FIG. 2 is a graph showing the typing results of the present invention.
Detailed Description
Example 1
Design of primers and probes for identifying homozygote and heterozygote of Hulunbel short-tailed sheep
The sequences of the identification primers and probes for homozygote and heterozygote of the Hulenbel short-tailed sheep are as follows:
a forward primer: 5'-TGCGCCCCTTCCTTTTCAG-3'
Reverse primer: 5'-GGGGGAGTCGGGGTGGATGTAG-3'
taqman probe 1: 5' HEX-TGGCTTGCCCCCCGGCA-BHQ 13
taqman probe 2: 5' FAM-GCTTGCCCCAGGGCACCCA-BHQ 13,
the taqman probes 1 and 2 are fluorescence labeled probes.
The design idea of identifying the primers and the probes is as follows:
inputting the sequence interval (500 bp before and after the SNP locus) segment of the SNP locus into Primer 5 software, and searching for primers with about 55% of GC value, about 60 ℃ of TM value and Primer dimer and Primer self-complementary sequence not more than 4 as a forward Primer and a reverse Primer. And analyzing the sequences around the SNP sites, finding out sequences with the length of 19-32 bp, the TM value of 5-10 ℃ higher than that of the forward Primer and the reverse Primer and the complementary number of less than 4 generated by the forward Primer and the reverse Primer as probe sequences, inputting the probes and the Primer sequences into Primer Blast in NCBI for sequence comparison, and finding out the primers with the best specificity, the probe sequences as the forward Primer and the reverse Primer and the probes.
Designed probes and primers were input into Primer 5 for validation:
| Tm（℃） | GC(%) | length of | |
| Forward primer | 60.6 | 57.89 | 19 |
| Reverse primer | 65.83 | 68.18 | 22 |
| Probe 1 | 69.9 | 76 | 17 |
| Probe 2 | 69.3 | 74 | 19 |
Example 2
A kit for identifying homozygote and heterozygote of a Renbell Brachyury sheep comprises a forward primer, a reverse primer, fluorescence labeling taqman probes 1 and 2, PCR reaction mixed liquid, genome DNA of the Renbell Brachyury sheep and a standard prepared by identifying homozygote and heterozygote of the T/Brachyury gene of the Renbell Brachyury sheep in example 1The plasmid, wherein the PCR reaction mixture comprises SNP Probe Master Mix (2X) (Novozan) and RNase-Free ddH2O, the dosage is respectively 10 ul and 4.6 ul; the concentration of the standard plasmid is 50 ng/ul, and the dosage is 1 ul; the concentration of the genome DNA of the Hulenbel short-tailed sheep is 10 ng/ul, and the dosage is 1 ul.
Example 3
A method for identifying homozygote and heterozygote of a humenbell brachyury based on the primer and taqman SNP fluorescent-labeled probe in example 1, comprising the following steps:
1. extracting and diluting a sample of DNA of a blood genome of a Hulenbel short-tailed sheep:
according to the column type blood genome DNA extraction kit and the instruction thereof sold on the market, 1% agarose gel electrophoresis of the extracted genome DNA shows that the band is single, bright and has no trailing phenomenon, and as shown in figure 1, a micro ultraviolet spectrophotometer such as Nanodrop is used for measuring the concentration of the genome DNA. When the DNA template is used, the eluent in the genomic DNA extraction kit is used for diluting the DNA concentration, gradient dilution is carried out during dilution, the diluted sample is subjected to concentration measurement, and the DNA concentration is selected to be 5-20 ng/ul, and preferably 10 ng/ul for carrying out the experiment.
2. Amplifying a fragment sequence of the Brachyury/T gene of the Hulenbel Brachyury by using a PCR reaction system, wherein the sequence fragment is from 95879486 sites to 95879323 sites of the Brachyury/T gene of homozygous and heterozygous Brachyury;
a PCR reaction system configuration step:
adding the mixture according to the principle of increasing the volume first and then adding the small volume, adding SNP Probe Master Mix (2X) first, and adding RNase-Free ddH into the system2And O, adding 1.8ul of forward primers and 1.8ul of reverse primers into the system, and finally adding the taqman probes 1 and 2 and the DNA template into the system, wherein a liquid transfer gun is required to blow and uniformly mix the primers in the process of configuration reaction, so that bubbles generated by blowing and uniformly mixing the primers are avoided, and the result is not influenced.
The PCR reaction system was configured as follows, with a total volume of 20ul, consisting of 10 ul of SNP Probe Master Mix (2X) (Novozan), 1.8ul of forward primer, 1.8ul of reverse primer, and a concentrated forward primerThe degree is 1 uM, the concentration of reverse primer is 10uM, 10uM taqman probe is 10.4 ul, 10uM taqman probe is 20.4 ul, 10 ng/ul DNA template is 1 ul, RNase-Free ddH2O4.6 ul, the SNP Probe Master Mix (Novozam) contains an internal reference fluorescent dye. The concentration ratio of the used forward primer to the reverse primer is 1: 10, the asymmetric PCR method reduces the dimer phenomenon caused by the non-labeled probe and the primer, and avoids the wrong interpretation of the machine in the reaction process.
The PCR tube was placed in an ABI 7900HT fluorescent quantitative PCR reaction apparatus, and the reaction program was set as follows:
pre-denaturation at 95 ℃: 30 s;
denaturation at 95 ℃: 10 s;
60 ℃ annealing-extension: 30 s;
amplifying for 45 cycles;
after the PCR reaction is finished, the fluorescence scanning is carried out on a fluorescence plate reader for photographing for 30 s at 60 ℃.
The central position of an amplification fragment of the SNP site of the mark of the Renbell short-tailed sheep is positioned, when the PCR reaction is carried out, a pair of specific probes with different fluorescent marks at two ends are added to identify different alleles, a 5 'end is a report fluorescent group, a 3' end is a quenching fluorescent group, during the PCR process, the two probes can be specifically annealed and combined with a complementary sequence between a forward primer and a reverse primer, and when the probes exist in an integral form, the fluorescent groups only emit weak fluorescence due to energy resonance transfer. After the specific probe is heterozygous with the corresponding allele, the DNA polymerase exerts 5 ' to 3 ' exonuclease activity to cut the reporter fluorophore, and the effect of quenching the fluorophore at the 3 ' end is separated, so that fluorescence is emitted. The 5 'ends of the two probes are labeled with different fluorescent (FAM or HAX) 3' ends are labeled with a BHQ1 quencher binding partner. And judging the SNP allelic type of the corresponding sample according to the detected different fluorescence. As the number of amplification cycles increases, the released fluorescent group accumulates, and the fluorescence intensity is proportional to the amount of amplification product. The probe and the primer designed by the invention can be combined with fluorescent quantitative PCR to quickly and accurately perform qualitative identification on homozygotes and heterozygotes of the Hulen-Berry short-tailed sheep.
The invention provides an identification primer, a probe, a kit and a method for homozygote and heterozygote of a Hulenbel short-tailed sheep, which are specifically described by combining with an embodiment, and persons in related fields can completely change or change and combine the method appropriately according to the method provided by the invention to realize the technology. It is expressly intended that all such modifications or alterations and subcombinations of the disclosed systems are deemed to be within the scope and content of the invention.
Sequence listing
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agtacgtgaa cggggagtgg gtgccggggg gcaagccaga gccgcaggcg cccagctgcg 180
tctacatcca ccccgactcc ccc 203
Claims (9)
1. An identification primer and a probe for homozygote and heterozygote of a Hulun-Beijing sheep, which are characterized by comprising the following sequences:
a forward primer: 5'-TGCGCCCCTTCCTTTTCAG-3'
Reverse primer: 5'-GGGGGAGTCGGGGTGGATGTAG-3'
taqman probe 1: 5' HEX-TGGCTTGCCCCCCGGCA-BHQ 13
taqman probe 2: 5' FAM-GCTTGCCCCAGGGCACCCA-BHQ 13;
the taqman probes 1 and 2 are fluorescence labeled probes.
2. A kit for identifying homozygote and heterozygote of a Hulen-Bei sheep, which comprises the identifying primer and the probe of claim 1, and is characterized by further comprising the following components: PCR reaction mixed liquor, genome DNA of the Renbell Brachyury, and standard plasmids prepared from homozygote and heterozygote of the T/Brachyury gene of the Renbell Brachyury.
3. The kit for identifying homozygote and heterozygote in Hurenbel short-tailed sheep as claimed in claim 2, wherein the PCR reaction mixture comprises SNP Probe Master Mix (2X) 10 ul and RNase-Free ddH2O 4.6 ul。
4. The kit for identifying homozygote and heterozygote in the Hurenberg brachyury sheep as claimed in claim 2, wherein the concentration of the genome DNA of the Hurenberg brachyury sheep is 10 ng/ul and the dosage is 1 ul.
5. A method for identifying homozygote and heterozygote of a Hulenbel short-tailed sheep, which comprises the following steps, and is characterized in that a PCR reaction system comprises the identifying primer and the probe of claim 1:
(a) extracting and diluting DNA of a blood genome of a Hulunbel short-tailed sheep;
(b) the fluorescent quantitative PCR reaction system amplifies the fragment sequence of the Brachyury/T gene of the Renbell Brachyury, and the sequence fragment is from 95879486 sites to 95879323 sites of the homozygous and heterozygous Brachyury/T gene of the Brachyury, such as SEQ ID NO: 5 in sequence table 5;
(c) and detecting PCR reaction products.
6. The method for identifying homozygote and heterozygote in Hurenbel short-tailed sheep according to claim 5, wherein the total volume of the PCR reaction system in step (b) is 20ul, which comprises SNP Probe Master Mix (2X) 10 ul, forward primer 1.8ul, reverse primer 1.8ul, and the concentration ratio of forward primer to reverse primer is 1: 10, 10uM taqman probe 10.4 ul, 10uM taqman probe 20.4 ul, 10 ng/ul DNA template 1 ul, RNase-Free ddH2O 4.6 ul。
7. The method for identifying homozygotes and heterozygotes in the Hulen-Berry brachyury as claimed in claim 5, wherein the PCR reaction is carried out according to the following reaction procedure: pre-denaturation at 95 ℃: 30 s, denaturation at 95 ℃: 10 s, 60 ℃ annealing-extension: 30 s, amplifying for 45 cycles, and taking a picture for 30 s at 60 ℃ by performing fluorescence scanning on a fluorescence plate reader after the PCR reaction is finished.
8. The method for identifying homozygotes and heterozygotes in Hurenberg brachyury as claimed in claim 6, wherein the concentration of the forward primer is 1 uM and the concentration of the reverse primer is 10 uM.
9. The method for identifying homozygotes and heterozygotes in the Hurenbel short-tailed sheep according to claim 6, wherein the SNP Probe Master Mix contains an internal reference fluorescent dye.
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| CN112899375A (en) * | 2021-04-01 | 2021-06-04 | 内蒙古农业大学 | PCR-RFLP method and primer for identifying homozygote heterozygote of Hulenbel short-tailed sheep |
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| CN112899375A (en) * | 2021-04-01 | 2021-06-04 | 内蒙古农业大学 | PCR-RFLP method and primer for identifying homozygote heterozygote of Hulenbel short-tailed sheep |
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Application publication date: 20210326 |