WO2012126153A1 - Method for multiplex detection of genomic dna polymorphism and probes dedicated thereto - Google Patents

Abstract

Provided are a method for multiplex detection of genomic DNA polymorphism and probes dedicated thereto. Also disclosed is a single-stranded DNA molecule comprising successively, from the 5' end to the 3' end, a left-side oligonucleotide fragment complementary to the 5' end of the DNA to be detected, a main fragment and a right-side oligonucleotide fragment complementary to the 3' end of the DNA to be detected. Through ligase reaction, different labels and different allelic sites are distinguished by the single-stranged DNA molecule. Hand-clasping probes of different lengths are successfully ligated by PCR amplification. Through gel or capillary electrophoresis separation and detection systems, simultaneous detection of the polymorphism of a multiple of deoxyribonucleic acids is achieved. High sensitivity and accuracy are attained, and the requirements for high-throughput and low cost are satisfied at the same time.

Description

 Method for multiplex detection of genomic DNA polymorphism and special probe thereof

 The present invention relates to the field of genetic engineering, and in particular to a method for multiplex detection of genomic DNA polymorphisms and a specific probe thereof.

Background technique

 Deoxyribonucleic acid (DNA) polymorphism refers to the difference in nucleotide arrangement among chromosomal DNA alleles. That is, there are two or more genotypes in the DNA region alleles (or fragments), which can be divided into single nucleus. Single nucleotide polymorphi sm (SNP) and insertion/deletion polymorphism (insert/deletion, In/Del).

 (1) Single Nucleotide Polymorphism (SNP)

 A single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by a single nucleotide variation at the genomic level of a chromosome, with at least one allele occurring at least 1% in the population. The polymorphisms exhibited by SNPs involve only a single base variation, which can be caused by a single base transition or transversion, or by the insertion or deletion of a base. But the so-called SNP does not include the latter two cases. SNPs can be divided into two forms in the genome: one is a large number of single base variations throughout the genome; the other is a functional mutation in the coding region of the gene, which is also called cSNP because it is distributed in the coding region of the gene. In general, the SNP ( cSNP ) located in the coding region is relatively small, because within the exon, the mutation rate is only 1/5 of the surrounding sequence. However, it is of great significance in the study of hereditary diseases, so the research of cSNP is more concerned. Methods for distinguishing SNPs include: 1 hybridization based method, 2 enzyme based method, 3 conformation based method, 4 direct sequencing method, and the like. Detection and analysis techniques include: 1 gel analysis technology, 2 fluorescence detection technology, 3 DNA chip detection technology, 4 mass spectrometry detection technology. The ideal method for detecting SNPs must meet the following characteristics: 1. High sensitivity and accuracy (rigid reaction principle), 2 fast, simple, high throughput, 3 relatively inexpensive. However, the existing testing methods cannot meet the above requirements.

 (2) Insertion/deletion polymorphism (insert / deletion, InDel)

Insertion/deletion polymorphism (InDel) refers to the difference in the whole genome between two organisms. One individual has a certain number of nucleotide insertions or deletions in the genome of one individual ( Jander, G., Norri s, SR, Rounsley, SD, Bush, DF, Levin, IM and Last, RL (2002) Arabi dopsi s map-based cloning in the post-genome era. Plant Physiol. 129, 440 - 450 ). Short insertions and deletions (20-30 nucleotides) are mainly caused by mismatches in the DNA replication process, such as mismatches in the sliding strand, while long insertions and deletions occur mainly by unequal length interchange or DNA misalignment. Caused. Based on the insertion of deletion sites in the genome, some PCR primers that amplify these insertion deletion sites are designed, which is the Insert/Delete (InDel) marker. The traditional method for detecting insertion/deletion markers is to design a pair of PCR primers for an insertion/deletion site, which can be detected by PCR amplification, gel or capillary electrophoresis. Generally, only one insertion/deletion marker can be detected at a time, and the flux is very high. low. Invention disclosure

 It is an object of the invention to provide a single stranded DNA molecule.

 The single-stranded DNA molecule provided by the present invention consists of the following components from the 5' end to the 3' end: a left-side oligonucleotide fragment complementary to the 5' end of the DNA to be tested, a host fragment and 3' to the DNA to be tested. a complementary complementary right oligonucleotide fragment;

 The subject fragment is composed of: a length variable region for determining the size of the single-stranded DNA molecule; and a primer pair region and a common fragment respectively located on both sides of the variable length region;

 The primer pair region is obtained by connecting two oligonucleotide chains as follows:

 1) a primer capable of amplifying a pair of primers of said single-stranded DNA molecule;

 2) an oligonucleotide capable of amplifying a reverse complement of the other primer of the pair of primers of the single-stranded DNA molecule;

 The variable length region is an oligonucleotide having a size of 45-1000 nt;

 The common fragment is an oligonucleotide of 13-18 nt in size;

 The subject fragment is not complementary to the gene to be tested.

The size of the one primer and the other primer are both 18-22 nt, and the size of the one primer and the other primer are each specifically 20 nt _;

 The common fragment is an oligonucleotide of a size of specifically 15 nt.

 Each of the following fragments is a fragment corresponding to a DNA molecule in a single nucleotide polymorphism:

 The nucleotide sequence of the variable length region is any one of the following 1) -8):

 Bits 68-112 of sequence 1 in the sequence listing;

 Bits 68-129 of sequence 2 in the sequence listing;

 Bits 62-136 of sequence 3 in the sequence listing;

 62-153 of sequence 4 in the sequence listing;

 Bits 64-125 of sequence 8 in the sequence listing;

 Bits 64-155 of sequence 9 in the sequence listing;

 67-278 of sequence 10 in the sequence listing;

 67-299 of sequence 11 in the sequence listing;

 The nucleotide sequence of the common fragment is at positions 113-127 of sequence 1 in the sequence listing or at positions 130-144 of sequence 2 in the sequence listing or at positions 137-151 of sequence 3 in the sequence listing or in the sequence listing 154-168 of 4 or 126-140 of SEQ ID NO: 8 or 156-170 of SEQ ID NO: 9 or 279-293 of SEQ ID NO: 10 or SEQ ID NO: 11 of the Sequence Listing Positions 300-314; the nucleotide sequence of the primer pair region is position 28-67 of sequence 1 in the sequence listing or position 28-67 of sequence 2 in the sequence listing or the 22nd of sequence 3 in the sequence listing - Positions 22-61 of Sequence 4 in Sequence Listing 61 or Sequences 24-63 of Sequence 8 in the Sequence Listing or Position 24-63 of Sequence 9 in the Sequence Listing or Positions 27-66 of Sequence 10 in the Sequence Listing Or the 27th to 66th of the sequence 11 in the sequence listing.

A second object of the invention is to provide a dedicated clasp probe for detecting DNA single nucleotide polymorphisms. The invention provides a special buckle probe for detecting DNA single nucleotide polymorphism, and the special buckle The hand probe is composed of m sets of probes, and each set of probes corresponds to a SNP site to be tested, and the single nucleotide polymorphism is n polymorphisms of a SNP site to be tested.

 Each set of probes consists of n single-stranded DNA molecules as described.

 n is a natural number greater than or equal to 2;

 m is a natural number greater than or equal to 1;

 The 5'-end left oligonucleotide fragment of each single-stranded DNA molecule specifically binds to an upstream fragment of the SNP site to be tested, and the 3'-end right oligonucleotide of each single-stranded DNA molecule The downstream fragment of the SNP site to be tested specifically binds, and the 3' terminal base of each single-stranded DNA molecule is complementary to the base of the SNP site to be tested.

 The number of the probe sets is the same as the number of the SNP sites to be tested;

 The number of SNP sites to be tested is at least two;

 The size of each of the single-stranded DNA molecules in each of the sets of probes is different, and the size of the single-stranded DNA molecule is determined by the variable length region thereof;

 Phosphorylation of the 5' end of each of the single-stranded DNA molecules, specifically the 5th carbon of the first nucleotide of the left oligonucleotide of each of the single-stranded DNA molecules from the 5' end A phosphate group is attached to the atom.

 The dedicated buckle probe consists of the following two probe sets:

 Probe set 1 consists of two single-stranded DNA molecules as described below:

 1) an oligonucleotide as shown in SEQ ID NO:1 in the Sequence Listing;

 2) an oligonucleotide as shown in SEQ ID NO: 2 in the Sequence Listing;

 Probe set 2 consists of two single-stranded DNA molecules as described below:

 3) an oligonucleotide as shown in SEQ ID NO: 3 in the Sequence Listing;

 4) The oligonucleotide shown in SEQ ID NO: 4 in the Sequence Listing.

 A third object of the present invention is to provide a special buckle probe for detecting DNA insertion or deletion polymorphism.

 The invention provides a special button probe for detecting DNA insertion or deletion polymorphism, wherein the special button probe is composed of a group of probes, and each group of probes corresponds to one DNA insertion or deletion to be tested. a single nucleotide polymorphism, wherein there is a b polymorphism in a DNA insertion or deletion site to be tested,

 Each set of probes consists of single-stranded DNA molecules as described in b.

 b is a natural number greater than or equal to 2;

 a is a natural number greater than or equal to 1;

 The 5'-end left oligonucleotide fragment of each single-stranded DNA molecule specifically binds to an upstream fragment of the insertion or deletion site to be detected, and the 3'-end right oligonucleoside of each single-stranded DNA molecule The acid specifically binds to a downstream fragment of the insertion or deletion site to be tested.

 The number of the single-stranded DNA molecules is the same as the number of insertion or deletion sites of the DNA; the insertion or deletion site of the DNA is at least two;

The size of each single-stranded DNA molecule is different, and the size of the single-stranded DNA molecule is determined by the variable length region thereof; Phosphorylation of the 5' end of each of the single-stranded DNA molecules, specifically the 5th carbon of the first nucleotide of the left oligonucleotide of each of the single-stranded DNA molecules from the 5' end A phosphate group is attached to the atom; the dedicated button probe consists of two probe sets:

 Probe set 1 consists of two single-stranded DNA molecules as described below:

 1) an oligonucleotide as shown in SEQ ID NO:8 in the Sequence Listing;

 2) an oligonucleotide as shown in SEQ ID NO:9 in the sequence listing;

 Probe set 2 consists of two single-stranded DNA molecules as described below:

 3) an oligonucleotide as shown in SEQ ID NO: 10 in the Sequence Listing;

 4) The oligonucleotide shown in SEQ ID NO: 1 in the sequence listing.

 Kits containing the single-stranded DNA molecules or the specialized hand-held probes are also within the scope of the present invention.

 The use of the single-stranded DNA molecule or the specific hand-held probe in the identification of DNA molecular polymorphisms is also within the scope of the present invention.

 The polymorphism is a single nucleotide polymorphism or an insertion or deletion polymorphism;

 The single nucleotide polymorphism is a single nucleotide polymorphism specifically caused by at least two SNP sites; the insertion or deletion polymorphism is specifically caused by at least two insertion or deletion sites. Polymorphism.

 The organism is a plant, and the plant is specifically rough goat grass or barley;

 The single nucleotide polymorphism caused by the at least two SNP sites is specifically the polymorphism of the 62nd nucleotide of the DNA molecule shown in the sequence 5 in the sequence table and the DNA molecule shown in the sequence 6 of the sequence table. Polymorphism of nucleotides;

 The polymorphism of the 62nd nucleotide of the DNA molecule shown in SEQ ID NO: 5 in the sequence listing is the nucleotide 62 of the DNA molecule represented by the sequence 5 in the sequence listing is A or G;

 The 174th nucleotide polymorphism of the DNA molecule represented by the sequence 6 in the sequence listing is the nucleotide number 174 of the DNA molecule represented by the sequence 6 in the sequence listing is T or C;

 The DNA molecule shown in SEQ ID NO: 5 in the sequence listing and the DNA molecule shown in SEQ ID NO: 6 in the sequence listing are the sequences of two SNPs of Aegllops tauschii. The crude goat grass 2280 and 2282 varieties have polymorphisms in the 62nd nucleotide of sequence 5, which are AA and GG, respectively; the crude ramie 2280 and 2282 varieties in sequence 6 are polymorphic at nucleotides 175. Sex, TT and CC respectively.

 The insertion or deletion polymorphisms caused by the at least two insertion or deletion sites are specifically 1) and 2):

1) The DNA molecule shown in SEQ ID NO: 12 in the sequence listing is inserted into CCGTC from positions 120-121 at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 13 in the sequence listing; or the DNA molecule shown in SEQ ID NO: 13 in the sequence listing. Deletion of CCGTC at positions 121-125 at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 12 in the Sequence Listing;

2) The DNA molecule shown in SEQ ID NO: 15 in the sequence table is inserted into GCCCAT from the 1st 16-1 position at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 14 in the sequence listing; or the DNA molecule shown in SEQ ID NO: 14 in the sequence listing. Deletion of GCCCAT from positions 1 17-122 at the 5' end yields the DNA molecule shown in SEQ ID NO: 15 in the Sequence Listing. The DNA sequence shown by the DNA molecule shown in SEQ ID NO: 12 in sequence 12 or the DNA molecule shown in SEQ ID NO: 14 in the sequence listing or the DNA molecule shown in SEQ ID NO: 15 is the DNA sequence of two insertion/deletion markers of barley. .

 A fourth object of the present invention is to provide a method for detecting a single nucleotide polymorphism of a biological genome. The invention provides a method for detecting a single genomic polymorphism of a biological genome, comprising the following steps:

1) ligating and ligating all single-stranded DNA molecules in the special hand-clamping probe for detecting DNA single nucleotide polymorphism with the genomic fragment to obtain a cyclized product of the clasp probe;

 2) PCR is carried out using the cyclized product obtained in the step 1) as a template, and the polymorphism of the single nucleotide of the biological genome is determined according to the size of the obtained PCR product.

 In step 1), before the cyclization of the linkage, further comprising breaking the genomic fragment into a DNA molecule of 80-250 bp size;

 The U.S. </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ br> The concentration of each of the buckled probes in the ligation system is 0. 04 ng / ul, the concentration of the 80-250 bp DNA molecule in the ligation system is 24 ng / ul;

 In step 2), the primer of the PCR is the primer corresponding to the primer pair region in the single-stranded DNA molecule, and the one primer and the other primer in a pair of primers capable of amplifying the single-stranded DNA molecule;

 The nucleotide sequence of the one primer is the 48th-67th position of the sequence 1 in the sequence listing or the 48th-67th position of the sequence 2 in the sequence listing or the 42th-61th position of the sequence 3 in the sequence listing or the sequence in the sequence listing Bits 42-61 of 4;

 The nucleotide sequence of the reverse complementary fragment of the other primer is position 28-47 of sequence 2 in sequence 28-47 of sequence 1 of the sequence listing or positions 22-41 of sequence 3 in the sequence listing. Or the 22-41 of sequence 4 in the sequence listing.

 Determining, by the size of the obtained PCR product, a polymorphism of the single genome of the biological genome by determining the size of the PCR product, and determining a single-stranded DNA molecule in the special-handed probe having the same size as the single-stranded DNA molecule The base at the 3' end of the DNA molecule determines the corresponding SNP site to determine the polymorphism of the single genomic DNA of the organism.

 A fifth object of the present invention is to provide a method for detecting a biological genome insertion or deletion polymorphism.

 The method for detecting biological genome insertion or deletion polymorphism provided by the invention comprises the following steps:

1) ligating and ligating all single-stranded DNA molecules of the special hand-clamping probe for detecting DNA insertion or deletion polymorphism with a genomic fragment to obtain a looper probe cyclization product;

 2) performing PCR using the cyclized product obtained in the step 1) as a template, and determining the polymorphism of the insertion or deletion of the biological genome according to the size of the obtained PCR product;

 In step 1), before the cyclization of the linkage, further comprising breaking the genomic fragment into a DNA molecule of 80-250 bp size;

The linked cyclization system comprises: a ligation buffer, a ligase, a clasp probe, and a large 80-250 bp 01 ng/ul , The concentration of each of the clasp probes in the ligation system is 0. 04 ng / ul, The concentration of the 80-250 bp DNA molecule in the ligation system is 24 ng/ul;

 In step 2), the primer of the PCR is the primer corresponding to the primer pair region in the single-stranded DNA molecule, and the one primer and the other primer in a pair of primers capable of amplifying the single-stranded DNA molecule;

 The nucleotide sequence of the one primer is at positions 44-63 of SEQ ID NO: 8 in the sequence listing or at positions 44-63 of SEQ ID NO: 9 in the sequence listing or at positions 47-66 of SEQ ID NO: 10 in the sequence listing or in the sequence listing Bits 47-66 of 11;

 The nucleotide sequence of the reverse complementary fragment of the other primer is position 24-43 of sequence 8 in the sequence listing or position 24-43 of sequence 9 in the sequence listing or the 27-46 of sequence 10 in the sequence listing. Bits 27-46 of sequence 11 in the sequence or sequence listing.

 Determining the polymorphism of the insertion or deletion of the biological genome according to the obtained PCR product is: if there is a PCR product, determining the corresponding single-stranded DNA molecule in the special buckle probe of the same size by the size of the PCR product, Determining no polymorphisms between the upstream and downstream fragments of the complementary biological genome based on the left oligonucleotide fragment and the right oligonucleotide fragment of the corresponding single-stranded DNA molecule; if there is no PCR product, Insertion or deletion polymorphism between the upstream and downstream fragments of the biological genome complementary to the left and right oligonucleotide fragments of the remaining single-stranded DNA molecules of the dedicated hand-held probe .

 Unless otherwise indicated or individually defined, the scientific and technical terms used herein have the same meaning of the meaning of the meaning of the invention. In addition, the materials, methods, and examples described herein are intended to be illustrative and illustrative, and not limiting or limiting.

DRAWINGS

 Figure 1 is a schematic diagram showing the detection of two alleles of a single nucleotide polymorphism (SNP) marker by two different lengths of hand-held probes.

 Figure 2 is a schematic diagram showing the detection of two alleles of an insertion/deletion polymorphism (InDel) marker by two different lengths of hand-held probes.

 Figure 3 is a miniature column electrophoresis instrument design

 Figure 4 shows the SNP classification of the clasp probe (5% denaturing polyacrylamide gel electrophoresis)

 Figure 5 shows the InDel marker typing of the clasp probe (3% agarose gel electrophoresis)

 Figure 6 shows the InDel labeling of the clasp probe (5% denaturing polyacrylamide gel electrophoresis)

The best way to implement the invention

 The experimental methods used in the following examples are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The present invention is directed to different deoxyribonucleic acid labeling sites and their allelic sites, and the design of different hand-held probes for different lengths and different alleles by ligase reaction, PCR amplification and successful connection The different lengths of the hand-held probes are separated by a gel or capillary electrophoresis system to detect the polymorphism of multiple deoxyribonucleic acid markers simultaneously. The design scheme is shown in Figure 1 and Figure 2. Figure 1 is a schematic diagram showing two alleles of a single nucleotide polymorphism (SNP) marker detected by two different lengths of hand-held probes, wherein SP: a variable length interval, the length can be adjusted in the range of 45-1000 nt PI: a reverse primer for a common PCR primer, 20 nt in length _; P2: a forward primer for a common PCR primer, 20 nt in length _; CS: a common sequence, 15 nt in length _; LF: a left-wing complementary sequence The length is generally between 20-30 nt, which can be adjusted according to the degree of matching with the target DNA and the annealing temperature; RF: is the right-wing complementary sequence, and the length is generally between 20-30 nt, according to the degree of matching with the target DNA and the annealing temperature. Adjustment; Pdp: buckle probe. The total length of the clasp probe Pdpl is 155 nt, and the total length of Pdp2 is 170 nt. They differ by 15 nt. The PCR products of these two clasp probes can be detected on a 3% agarose gel. A: The 3' terminal base of the clasp probe Pdpl is completely matched with the target DNA, that is, C and G are complementary. Under the action of ligase, Pdpl is cyclized, and PCR is amplified by two common primers P1 and P2. The 155 nt PCR product can be separated by 5% polyacrylamide gel or 3% agarose gel or capillary electrophoresis. B: The 3' terminal base of the clasp probe Pdpl does not match the target DNA, that is, C and T are not complementary. Under the action of ligase, Pdpl cannot be cyclized, and PCR is amplified by two common primers P1 and P2. , no PCR products. C: The 3' terminal base of the clasp probe Pdp2 is completely matched with the target DNA, that is, A and T are complementary. Under the action of ligase, Pdp2 is circularized, and PCR is amplified by two common primers P1 and P2. The 170 nt PCR product can be detected in 5% polyacrylamide gel or 3% agarose. D: The 3' terminal base of the clasp probe Pdp2 does not match the target DNA, ie, A and G are not complementary. Under the action of ligase, the clasp probe 2 cannot be cyclized, and two common primers P1 and P2 are used. After PCR amplification, there is no PCR product.

Figure 2 is a schematic diagram showing two alleles of an insert/deletion polymorphism (InDel) marker by two different lengths of hand-held probes, SP: variable length interval, length adjustable range of 45-1000 nt; PI : reverse primer for public PCR primers, 20 nt in length _; P2: forward primer for public PCR primers, 20 nt in length _; CS: common sequence, 15 nt in length _; LF: left-wing complementary sequence, length Generally, it is between 20-30 nt, and can be adjusted according to the degree of matching with the target DNA and the annealing temperature; RF: is a right-wing complementary sequence, and the length is generally between 20-30 nt, which can be adjusted according to the degree of matching with the target DNA and the annealing temperature; Pdp: Buckle probe. The total length of the clasp probe Pdp3 is 185 nt, and the total length of Pdp4 is 200 nt. They differ by 15 nt. The PCR products of these two clasp probes can be detected on a 3% agarose gel. A: The left-wing complementary sequence and the right-wing complementary sequence of the clasp probe Pdp3 are completely matched with the target DNA. Under the action of ligase, the clasp probe 3 is circularized, and PCR is amplified by two common primers P1 and P2. The 185 nt PCR product can be detected by separation on a 5% polyacrylamide gel or 3% agarose gel or capillary electrophoresis. B: After inserting a DNA fragment between the target DNA matched with the Pdp3 left-wing complementary sequence and the right-wing complementary sequence, the clasp probe 3 cannot be circularized by ligase, and PCR amplification is performed using two common primers, P1 and P2. After that, there was no PCR product. C: The left-wing complementary sequence and the right-wing complementary sequence of Pdp4 are completely matched with the target DNA. Under the action of ligase, the clasp probe 4 is circularized, and PCR is amplified by two common primers P1 and P2 to generate a PCR of 200 nt. The product can be isolated and assayed on a 5% polyacrylamide gel or a 3% agarose gel or capillary electrophoresis. D: Pdp4 cannot bind to the target DNA after deleting a DNA fragment in the middle of the target DNA that matches the Pdp4 left-wing complement and the right-wing complement Completely matched, under the action of ligase, the clasp probe 4 could not be cyclized, and after PCR amplification with two common primers P1 and P2, there was no PCR product.

 Example 1. Deduction probe for single nucleotide polymorphism (SNP)

 I. Two SNPs in the genome of Aged Aegilops (tailops tauschii)

 1. Design of the clasp probes PdPCJ819266A, PdPCJ819266G, PdPBJ262615T, PdPBJ262615C

 The genome of Aegilops tauschii is a donor of Triticum aes ii ra^ D. PdPCJ819266 and PdPBJ262615 are two SNPs of Aegilops tauschii (e^io/^s tauschii), of which AA And TT is the genotype of the crude goat grass variety 2280, and GG and CC are the genotypes of the crude goat grass variety 2282, that is, the 62nd nucleotide of the crude goat grass 2280 and 2282 varieties in the sequence 5 is polymorphic. They are ΑΑ and GG respectively; the 175th nucleotide of the Agrocybe aegerita 2280 and 2282 varieties in sequence 6 are polymorphic, namely ΤΤ and CC.

 The button probe PdPCJ819266A specifically recognizes the allele A, and has a length of 147 nt. The clasp probe PdPCJ819266G specifically recognizes the allele G, which is 164 nt in length. The clasp probe PdPBJ262615T specifically recognizes the allele T, and has a length of 176. Nt, the clasp probe PdPBJ262615C specifically recognizes the allele C and has a length of 193 nt. The 5' ends of the four button probes were phosphorylated. The sequence design and structure are shown in Table 1.

 Table 1. Sequence design and structure description of the clasp probe

 Name sequence (5, ― V )

 Design sequence

 PdPCJ819266A P-TCTCCATTAAAGTTACCTTTTGTTTTGCATTGCCTGACCACCTCTACATTGCGGAGT

 AAGATCCTGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGACATTCTAC CGACGCATCATTTGTCAAGCATTGTTT

 Structure description

 Left-wing complementary sequence (LF) TCTCCATTAAAGTTACCTTTTGTTTTG

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

 Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGA

 Public sequence (CS ) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TCATTTGTCAAGCATTGTTT

 PdPCJ819266G P-TCTCCATTAAAGTTACCTTTTGTTTTGCATTGCCTGACCACCTCTACATTGCGGAGT

 AAGATCCTGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCCATTCTACCGACGCATCATTTGTCAAGCATTGTTC

 Structure description

 Left-wing complementary sequence (LF) TCTCCATTAAAGTTACCTTTTGTTTTG

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 ' Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

AGC

 Public sequence (cs) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TCATTTGTCAAGCATTGTTC

 PdPBJ262615T P-CGGGACAAAAGATGGTCAGTCCATTGCCTGACCACCTCTACATTGCGGAGTAAGATC

 CTGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG AGCTGAATGAAGCCATCATTCTACCGACGCATTAACTATATTTGAATGTGCAACAA

 Structure description

 Left-wing complementary sequence (LF) CGGGACAAAAGATGGTCAGTC

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

 Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCAT

 Public sequence (CS ) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TTAACTATATTTGAATGTGCAACAA

 PdPBJ262615C P-CGGGACAAAAGATGGTCAGTCCATTGCCTGACCACCTCTACATTGCGGAGTAAGATC

 CTGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG AGCTGAATGAAGCCATACCAAACGACGAGCGTGCATTCTACCGACGCATTAACTATATTTGAA TGTGCAACAG

 Structure description

 Left-wing complementary sequence (LF) CGGGACAAAAGATGGTCAGTC

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

 Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGAGCGTG

 Public sequence (CS ) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TTAACTATATTTGAATGTGCAACAG

 2. Biosynthesis of clasp probes PdPCJ819266A, PdPCJ819266G, PdPBJ262615T, PdPBJ262615C

 The variable length interval of the clasp probe is a small stretch of ampicillin resistance (AMP) sequence. The EZ-T vector (Cat. No. T 168-10 ) is used as a template to obtain the main sequence of probes of different lengths by two-step PCR. PdPCJ819266A_P_SP_CS PdPCJ819266G_P_SP_CS, PdPBJ262615T_P_SP_CS and PdPBJ262615C_P_SP_CS, which include public PCR primers (PI, P2), variable length interval (SP), common sequence (CS), and the sequence is shown in Table 3.

 1 Structure and design of biosynthetic clasp probe precursor

Design clasp probe precursors PdPCJ819266A_ds, PdPCJ819266G_ds, PdPBJ262615T_ds, PdPBJ262615C_ds, these four sequences with PdPCJ819266A, PdPCJ819266G, PdPBJ262615T, PdPBJ262615C differs in that it has an enzyme cleavage site added to its left and right ends.

GGAACCGGAGCTGAATGAAGCCATCATTCTACCGACGCATTAACTATATTTGAATGTGCAACA ACACTGC

GGAACCGGAGCTGAATGAAGCCATCATTCTACCGACGCATTAACTATATTTGAATGTGCAACA ACACTGC

 Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence

 Left-wing complementary sequence (LF) CGGGACAAAAGATGGTCAGTC

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

 Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCAT

 Public sequence (CS ) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TTAACTATATTTGAATGTGCAACAA

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 PdPBJ262615C_ds GAGTCTTCCTCGGGACAAAAGATGGTCAGTCCATTGCCTGACCACCTCTACATTGCGGA

 GTAAGATCCTGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGCATTCTACCGACGCATTAACTA TATTTGAATGTGCAACAGCACTGC

 Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence

 Left-wing complementary sequence (LF) CGGGACAAAAGATGGTCAGTC

 Public PCR Primer ( P1 ) 5 ' -GTAGAGGTGGTCAGGCAATG-3 '

 Public PCR Primer ( P2 ) 5 ' -ATTGCGGAGTAAGATCCTGC-3 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGAGCGTG

 Public sequence (CS ) CATTCTACCGACGCA

 Right-wing complementary sequence (RF) TTAACTATATTTGAATGTGCAACAG

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 2 PCR synthesis of clasp probe precursors PdPCJ819266A_ds, PdPCJ819266G_ds, PdPBJ262615T_ds and PdPBJ262615C_ds

- buckle hand probe precursor PdPCJ819266A_ds and PdPCJ819266G_ds is synthesized by PCR using four primers PdPCJ819266_Fl, PdPCJ819266_F2, PdPCJ819266_Rl, PdPCJ819266A_R2 or PdPCJ819266G_R2, PCR synthesized hand clasping probe precursor PdPBJ262615T_ds and PdPBJ262615C_ds also using four primers PdPBJ262615_Fl, PdPBJ262615_F2, PdPBJ262615_Rl, PdPBJ262615T_R2 Or PdPBJ262615C_R2 (see Table 3 for the specific sequence). Obtained by the above PCR method Four probes of the host sequence (see Table 3 for specific sequences) were diluted 10 000 times and used as a template for two rounds of PCR. The details are as follows: in the first round of PCR, PdPCJ819266A_P_SP_CS and PdPCJ819266G_P_SP_CS diluted as 10,000 times as above were used as the DNA template of the first round of PCR, and PCR was carried out with PdPCJ819266_Fl and PdPCJ819266_Rl primers, and PdPBJ262615T_P_SP_CS and PdPBJ262615C_P_SP_CS diluted by 10,000 times were also used. For the first round of PCR, PCR was performed with PdPBJ262615_Fl and PdPBJ262615_Rl primers. The reaction system was: 2ul 10x PCR buffer, 1. 6ul 2. 5mM dNTP, 0. 5ul lOuM forward primer, 0. 5ul lOuM reverse primer, Ll DNA template, 0. lul rTaq (5U/ul), plus sterile double distilled water to 20ul. The product of the first round of PCR was diluted 10 000 times and used as a DNA template for the next round of PCR. The second round of the PCR, before PdPCJ819266A_ds body synthesis PdPCJ819266_F2 Wo P PdPCJ819266A_R2 primer before PdPCJ819266G_ds body synthesis PdPCJ819266_F2 Wo P PdPCJ819266G_R2 primer, PdPBJ262615T_ds synthesis using PdPBJ262615_F2 and PdPBJ262615T_R2 primers, synthesized using PdPBJ262615_F2 PdPBJ262615C_ds and PdPBJ262615C_R2 primers. The reaction conditions of the two rounds of PCR were identical, and the reaction conditions were as follows, and the precursor product of the hand-held probe was obtained (see Table 2 for the specific sequence).

 The cycle parameters of the PCR reaction are as follows: (1) 94. C, 3 minutes (2) 94 ° C, 30 seconds; 50 ° C,

30 seconds; 72 ° C, 15 seconds, 5 cycles. (3) Extension at 72 ° C for 5 minutes (4) 94 ° C, 30 seconds; 65 ° C, 30 seconds; 72 ° C, 15 seconds, 28 cycles.

 ( 5 ) Extension at 72 ° C for 5 minutes

 Table 3. PCR primers

 Name sequence (5, ― V )

 PdPCJ819266A—P—SP—CS (is the subject CATTGCCTGACCACCTCTACATTGCGGAGTAAGATCCTGCGGCCGCTTTTTTGCAC fragment, including Pl, P2 primer sequence, SP can be AACATGGGGGATCATGTAACTCGCCTTGACATTCTACCGACGCA

Variable region, CS public sequence)

 PdPCJ819266G_P_SP_CS CATTGCCTGACCACCTCTACATTGCGGAGTAAGATCCTGCGGCCGCTTTTTTGCAC

 AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCCATTCTACCGACGC A

 PdPBJ262615T_P_SP_CS CATTGCCTGACCACCTCTACATTGCGGAGTAAGATCCTGCGGCCGCTTTTTTGCAC

 AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATC ATTCTACCGACGCA

 PdPBJ262615C_P_SP_CS CATTGCCTGACCACCTCTACATTGCGGAGTAAGATCCTGCGGCCGCTTTTTTGCAC

 AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATA CCAAACGACGAGCGTGCATTCTACCGACGCA

 PdPCJ819266_Fl GTTACCTTTTGTTTTGCATTGCCTGACCACC

 PdPCJ819266_Rl GCTTGACAAATGATGCGTCGGTAGAATG

 PdPCJ819266_F2 GAGTCTTCCTTCTCCATTAAAGTTACCTTTTGTTTT

 PdPCJ819266A_R2 GCAGTGAAACAATGCTTGACAAATGATG

PdPCJ819266G_R2 GCAGTGGAACAATGCTTGACAAATGATG PdPBJ262615_Fl AAAAGATGGTCAGTCCATTGCCTGACCACC

 PdPBJ262615_Rl TTCAAATATAGTTAATGCGTCGGTAGAATG

 PdPBJ262615_F2 GAGTCTTCCTCGGGACAAAAGATGGTCAGTC

 PdPBJ262615T_R2 GCAGTGTTGTTGCACATTCAAATATAGTTAATG

 PdPBJ262615C_R2 GCAGTGCTGTTGCACATTCAAATATAGTTAATG

 3 Construction of plasmid P0CP-PdPCJ819266A, P0CP_PdPCJ819266G, P0CP_PdPBJ262615T and POCP- PdPBJ262615C

 The precursor products of the above four hand-held probes were purified and ligated to the P0CP-T vector (sequence 7), respectively. The ligation system was: 1 ul 10 X NEB T4 l igase buffer 1 ul POCP-T (50 ng/ul) , 0. 6 ul second round PCR product (20 ng/ul), 0.4 ul T4 DNA Ligase (400 u/ul), sterilized double distilled water to 10 ul, 16 °C for one night (12h) . 5 ul of the ligated product was separately transformed into Escherichia coli DH5 a strain to obtain a transformant. Monoclonal to 1 ml of LB medium containing kana resistance, cultured at 37 V 200 rpm for 4 h, and 1 ul of bacterial solution was used for PCR identification. The positive transformant P0CP-PdPCJ819266A was identified by PCR with primers PdPCJ819266_F2 and PdPCJ819266A_R2, and the plasmid of 163 bp fragment was obtained and sequenced. The result was the sequence PdPCJ819266A_ds in Table 2. The positive transformants P0CP were identified by PCR with primers PdPCJ819266_F2 and PdPCJ819266G_R2. PdPCJ819266G, a plasmid with a size of 180 bp was sent for sequencing, and the result was the sequence PdPCJ819266G_ds in Table 2. The positive transformant P0CP-PdPBJ262615T was identified by PCR with primers PdPBJ262615_F2 and PdPBJ262615T_R2, and a plasmid of 192 bp fragment was obtained and sent for sequencing. The result was the sequence PdPBJ262615T_ds in Table 2; the positive transformant P0CP_PdPBJ262615C was identified with the primers PdPBJ262615_F2 and P PdPBJ262615C_R2, and a plasmid having a PCR product size of 209 bp was obtained and sequenced, and the result was the sequence PdPBJ262615C_ds in Table 2. The plasmids of the above positive clones were separately extracted: P0CP_PdPCJ819266A, P0CP_PdPCJ819266G, POCP-PdPBJ262615T and P0CP_PdPBJ262615C.

 The above method for transforming the DH5 α strain is as follows: The stored competent DH5 α bacterial solution is taken out from a refrigerator at 80 ° C, placed on ice for about 5 minutes to naturally melt, and 80 ul of the bacterial liquid and 5 ul of the ligation product are placed. Mix the tube on ice, gently rotate the tube, mix it on ice for 20 minutes, heat at 42 °C for 45-50 seconds, then place on ice for 2 minutes, then add 950 ul SOC medium at 37 ° The C shaker was shaken at about 150 rpm for 1 hour and 30 minutes. Finally, 50-100 ul of the transformed SOC medium was applied to the LB/Kana resistant plate, and the kanamycin content was 50 ug/ml.

The above method for extracting the positive clone plasmid is as follows: 500 ul of the above positive clone liquid is taken in 25 ml LB medium at 37 ° C for 230 rpm overnight, and the plasmid is extracted according to the medium amount of plasmid extraction method, and finally 200 ul of TE and RNase are added, and the temperature is maintained at 37 ° C. One hour, plasmids P0CP-PdPCJ819266A, P0CP-PdPCJ819266G, P0CP-PdPBJ262615T and P0CP_PdPBJ262615C were obtained. 25 ug of each of the four plasmids extracted (100 ug each) were double-digested, and the enzymes digested were fyl (NEW ENGLAND BioLabs) and ts Λ 3⁄4. Bts l (NEW ENGLAND BioLabs ), double digestion The system is: 50 ul 10x NEB4 buffer 5 ul 100 x BSA, 12 ul Mly l (10 U/ul), 14 ul Nb. Bts l (lOU/ul), 200 ul Plasmid DNA (500 ng/ul), sterilized double distilled water to 500 ul, incubated at 37 °C for 5 hours, and heated at 80 V for 20 minutes to inactivate the enzyme. 500 ul of the digested product was freeze-dried to 150 ul, and then separated and purified.

 3. Isolation and purification of clasp probes PdPCJ819266A, PdPCJ819266G, PdPBJ262615T, PdPBJ262615C

 1) Separation of the clasp probe

 The polyacrylamide electrophoresis instrument used for the separation of the clasp probe is a mini-column electrophoresis instrument modified by the Liuyi Instrument Factory (see Figure 3). The glass column is 90 mm long, the inner diameter is 5-7 mm, and the nozzle is made of diamond grinding. level. Before preparing the gel, put the bottom of the washed and dried glass tube on the latex tube, firstly use the 0.8% agarose gel to pour into the lower electrophoresis column about 2 cm, and then 5% polypropylene after the agarose gel is solidified. The amide gel, after the polyacrylamide gel was solidified, the agarose gel of the lower electrophoresis column was picked out with a syringe needle, leaving a space of 2 cm under the electrophoresis column. Before electrophoresis, add the electrophoresis solution to the 2 cm space, and insert the electrophoresis column into the well of the holder. The electrophoresis voltage is 300 V and the current is 7 mA. When the xylene hydrazine is electrophoresed under the gel column, the lower electrophoresis column is sealed with a dialysis bag with a molecular weight cut off of 3.5 KDa, and then electrophoresed for about 25 min, then the electrophoresis is removed. The column was placed in a 1.5 ml EP tube. The column was inverted and the dialysis bag was removed.

 2) Purification of the buckle probe

 Add the above obtained solution to 1/10 volume of 3 M NaOAC (pH=5.2), add 3 volumes of cold absolute ethanol, freeze at -20 °C for 30 min, and centrifuge to separate the precipitate. Wash 7 times of 75% ethanol in ml, dry at room temperature for 5 min, and dissolve the precipitate with 30 ul of sterile double distilled water. The DNA content was determined by taking 1 ul of the solution, and determined by ND-1000 spectrophotometer (Thermo), 0D260/280=1.89, 0D260/230=1.90, the concentration was about 35 ng/ul, and about 1050 ng was recovered. Dilute the hand-held probe to 2 ng/ul with sterile distilled water. The quality of the recovered probe is high (see Figure 4, Lane 2). The four probe fragments separated are from small to large: PdPCJ819266A, 147 nt PdPCJ819266G, 164 nt; PdPBJ262615T, 176 nt; PdPBJ262615C, 193 nt. Since the clasp probe only accounts for about 2.77% of the total plasmid DNA and 100 ug of the plasmid, the recovery rate of the clasp probe can reach 37.9% of the theoretical yield.

 The isolated PdPCJ819266A, PdPCJ819266G, PdPBJ262615T and PdPBJ262615C were sent for sequencing, and the result was that PdPCJ819266A had the oligonucleotide shown in SEQ ID NO: 1 in the sequence listing, wherein the nucleotides 1 to 27 at the 5' end of the sequence 1 in the sequence listing The acid is complementary to the DNA 1 to be tested (nucleotides 35-61 of SEQ ID NO: 5 in the sequence listing), the public PCR primer fragment at positions 28-47, and the public PCR primer fragment at positions 48-67. Bits 68-112 are variable length regions, positions 113-127 are common sequences, and positions 128-147 are complementary to the DNA to be tested (nucleotides 62-81 of sequence 5 in the sequence listing). The base at position 147 is Τ, and the 5' end is phosphorylated;

PdPCJ819266G has the oligonucleotide shown in SEQ ID NO: 2 in the sequence listing, wherein the nucleotides 1 to 27 of the sequence 2 from the 5' end of the sequence are the DNA 1 ' to be tested (DNA 1 to be tested and DNA 1 to be tested) The only difference is that the base of position 62 of sequence 5 in the sequence listing is replaced by A for G; sequence 5 of the sequence listing In the sequence listing, nucleotides 35-61 of sequence 5 are reverse-complementary, positions 28-47 are common PCR primer fragments, positions 48-67 are common PCR primer fragments 2, and positions 68-129 are The variable length region, the 130th-144th position is a common sequence, and the 145th to 164th are complementary to the DNA 1' to be tested (the nucleotides 62-81 of the sequence 5 in the sequence listing), and the 164th base Phosphorylation at the C, 5'end;

 PdPBJ262615T has the oligonucleotide shown in SEQ ID NO: 3 in the sequence listing, wherein nucleotides 1 to 1 of the sequence 3 from the 5' end are the same as the DNA 2 to be tested (SEQ ID NO: 154-174 of Sequence 6 in the Sequence Listing) The nucleotides are reverse-complementary, the common PCR primer fragment is located in positions 22-41, the common PCR primer fragment is in positions 42-61, the variable length region is in positions 62-136, and the 137-151 is The public sequence, 152-176 is a reverse complement to the DNA 2 to be tested (nucleotides 175-199 of SEQ ID NO: 6 in the sequence listing), the base at position 176 is Α, and the 5' end is phosphorylated;

 PdPBJ262615C has the oligonucleotide shown in SEQ ID NO: 4 in the sequence listing, wherein nucleotides 1 to 1 of the sequence 4 from the 5' end are the same as the DNA 2' to be tested (SEQ ID NO: 154 in Sequence Listing 6) 174 nucleotides) (The only difference between DNA 2' to be tested and DNA to be tested is that the base at position 175 of sequence 6 in the sequence listing is replaced by C for reverse complementation, and the 22nd to 41st positions are public PCR. Primer fragment 1, positions 42-61 are common PCR primer fragments 2, positions 62-153 are variable length regions, positions 154-168 are common sequences, and 169-193 are for DNA 2' to be tested (in the sequence listing) The nucleotides 175-199 of SEQ ID NO:6 are reverse-complementary, the base at position 193 is G, and the 5'-end is phosphorylated;

The above probes PdPCJ819266A, PdPCJ819266G, PdPBJ262615T and PdPBJ262615C ₀ can also be synthesized manually.

 Second, the SNP classification of the clasp probe

 1. Genomic DNA preparation

 The crude goat grass variety 2280 and the crude goat grass variety 2282 were extracted by CTAB method (both described in Neal SC, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR appl ications. BioTechniques 14 : 748 - 751, The public can obtain the genomic DNA from the Institute of Botany of the Chinese Academy of Sciences, using large ultrasound, wavelength 700 W, 40 KhZ, breaking for 35 min, breaking the genome to 80-250 bp, and finally adjusting the DNA concentration to 150 ng/ul. .

 2, the connection of the clasp probe

 The above-mentioned 4 ng/ul dilutions of 4 hand-held probe mixtures (average of 0.5 ng/ul per hand-hand probe) were subjected to ligation and cyclization using the template for the above-described broken genomic DNA.

 The connection reaction system of the clasp probe:

 10 ul lO X Ampl igase® buffer; 2 ul clasp probe (2 ng/ul) (final concentration of each clasp probe in the ligation system is approximately 0.04 ng/ul); 16 ul genomic DNA (150 ng/ul) (final concentration of genomic DNA molecule in the final ligation system of 24 ng/ul); 0. 8 ul Ampl igas® thermostable DNA ligase (5 U/ul) (ligase in the final ligation system) The final concentration in the medium is 0. 04 U / ul ); plus sterile double distilled water to 100 ul

The reaction conditions of the clasp probe are: 95 ° C for 3 minutes; then 94 ° C for 30 seconds, 55 ° C for 10 minutes, 10 cycles.

 3. Public PCR primer amplification

 The above-mentioned ligation product was 0.5 ul, and the two common primers (Pl, P2) of the clasp probe were used for PCR amplification, and the successful cyclization probe was amplified.

 PCR amplification system: 2 ul 10 X PCR buffer; 0.4 ul dNTP (10 mM); 0.5 ul PI

(10 uM) ; 0.5 ul P2 (10 uM); 0.5 ul of ligation product; 0· 1 ul rTag (5 U/ul); plus sterile double distilled water to 20 ul

 PCR reaction conditions: first 94 ° C for 3 minutes; then 94 ° C for 30 seconds, 57 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles; then 72 ° C for 5 minutes.

 After electroporation by 5% urea polyacrylamide, the results were observed after silver staining. The results are shown in Figure 4. Lane 1 is the result of two SNPs of a DNA molecule of S. serrata 2280 (sequence 5 in the sequence listing), Lane 2 For the results of isolation and purification of the clasp probe, Lane 3 is the result of two SNPs typing of the 2282-segment DNA molecule of the crude Aegilops (sequence 6 in the Sequence Listing). Because PdPCJ819266 and PdPBJ262615 are two SNPs of the crude goat grass iAegilops tauschii), AA and TT are the genotypes of the crude goat grass variety 2280, while GG and CC are the genotypes of the crude goat grass variety 2282. Lane 1 showed that the clasp probe PdPCJ819266A specifically recognized the allele A, which was 147 nt in length, and the clasp probe PdPBJ262615T specifically recognized the allele T with a length of 176 nt. Lane 3 results showed that the clasp probe PdPCJ819266G specifically recognized the allele G, which was 164 nt in length, and the clasp probe PdPBJ262615C specifically recognized the allele C with a length of 193 nt. SNP typing was performed by designing hand-held probes of different lengths and finally separating them by 5% polyacrylamide gel or 3% agarose gel or capillary electrophoresis.

 Example 2. Insertion/deletion polymorphism (InDel) marker clasp probe

 1. Two insertion/deletion (InDel) labeling detection of barley genome

 1. Handle probe PdPInDell-l, PdPInDell- 2, PdPInDel2_l, PdPInDel2 - 2 design

 PdPInDell and PdPInDel2 are two InDel markers on chromosome 5 (5H) of barley iHordeum vulgare L., InDell-1 and InDel2_l are genotypes specific to barley variety Vada, and InDell-2 and InDel2_2 are specific for barley variety L94. genotype. The clasp probe PdPInDell-1 specifically recognizes the InDell-1 genotype with a length of 160 nt. The clasp probe PdPInDell-2 specifically recognizes the InDell-2 genotype and has a length of 190 nt. The clasp probe PdPInDel2_l specifically recognizes the InDel2_l genotype. With a length of 318nt, the clasp probe PdPInDel2-2 specifically recognizes the InDel2-2 genotype and has a length of 342 nt.

 The 5' ends of the four clasp probes were phosphorylated. The sequence design and structure are shown in Table 4.

 Table 4 Sequence design and structure description of the clasp probe

 Name sequence (5, ― V )

 Design sequence

PdPInDell-1 p--A(l;AAC("A(7r('AACAAT(> : CICICATTi-CCTG CCACCTCTAC. T(. T (.GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC

GGAGC^; : iA -^::A;;;( _: ;;AA(; AGCAGCAAi^^

Structure description

Left-wing complementary sequence (LF) ACGAA (; CACl^CAACAATCCCTCT

Public PCR Primer (P1) b ⁷ -GTAGAGGTGGT(^AGGCAATG-3 '

Public PCR Primer (P2)

Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGC

Common sequence (CS)

Right-wing complementary sequence (RF) ACAGCAGCAACATGCGGTAG

 PdPInDell-2 p--ACGAACi; Ai;T(/UCAATC(:(rr(rriATTGC( TGACA^

 TC GCGGCCGCTTTTTTGCACAACATGGGGGATCATGTMCTCGCCTTGATCGTTGGGAACC

 一一 , '

Structure description

Left-wing complementary sequence (LF)

Public PCR Primer (P1) 5 " --GIAGAGGTGGTCA GCAAIG--3 ·

Public PCR Primer (P2) --AT:{:Ci:GA^T A AT( = .--:

Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGAGCGTG

Common sequence (CS)

Right-wing complementary sequence (RF)

 PdPInDel2-l P- A(]A(]GTCCTT/ AACATCA'rGCTTCATT(,OT

 , 'C G' GCCGCTTTTTTGCACMCATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC AACAACGTTGCGCAAACTATTAACTGGCGAACTGCTTACTCTAGCTTCCCGGCAACAATTAAT AGACTGGATGGAGGCGGATAAAGTTGCAGGAC A 1 Γ::: ΓΑ( ^:(. - (: ΛΤί^ΑΤΤΤΑΤΤΑΑ (; AGTG Structure Description

Left-wing complementary sequence (LF) (l; Ci; A("AAAAGAT(;GTCA(? TC)

Public PCR Primer (P1) 5, --GTAGAGGTGGTCAGGCAATG--3 '

Public PCR Primer (P2)

Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTGCTTACTCTAGCTTCCCGGCAACAATTAATAGACT GGATGGAGGCGGATAAAGTTGCAGGAC Common sequence (cs)

 Right-wing complementary sequence (RF) TCA']TTATTAACAGTGATAATG(,GC ( 3⁄4 λ,.; g

 PdPInDel2- 2 P--^CACGTCirrTATAGAACATCATGCTTi;ATTGC( GACCAiCT^

 AG.V CiV^CGGCCGCTTTTTTGCACMCATGGGGGATCATGTAACTCGCCTTGATCGTTGGGA ACCGGAGCTGAATGAAGCCATACCAAACGACGGGCGTGACACCACGATGCCTGTAGCAATGGC AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACCCTAGCTTCCCGGCAACAATTAAT AGACTGGATGGAGGCGGATA GTTGCAGGACCACTTCTGCGCTCGGCCCTTC^. A'i

Structure description

 Left-wing complementary sequence (LF) CGG ACAA AGA GG'i('AGT('

 Public PCR Primer (P1) 5 ' -GTAGAGGTGGT(AG( ;AATG-3' '

 Public PCR Primer (P2) ' --A'; TGi'Gi: (: TAAGAT(X.TG('-- '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGGGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACCCTAGCTTCCCGGCAACAATTAATAGACT GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC

 Common sequence (CS)

 Right-wing complementary sequence (RF) ACA'i'T'i'ATGTC TTT TTAACAGTGATA

 2. Biosynthesis of clasp probes PdPInDell-l, PdPInDell_2, PdPInDel2_l, PdPInDel2_2

 The variable length interval of the clasp probe is a small stretch of ampicillin resistance (AMP) sequence. The EZ-T vector (catalogue number T168-10) is used as a template to obtain the main sequence PdPInDell of probes of different lengths by two-step PCR amplification. l_P_SP_CS, PdPInDel 1-2_P_SP_CS, PdPInDel2-l_P_SP_CS and PdPInDel2-2_P_SP_CS, which include common PCR primers (PI, P2), variable length interval (SP), common sequence (CS), and the sequence details are shown in Table 4 and Table 6.

 1 Structure and design of biosynthetic clasp probe precursor

 The clasp probe precursors PdPInDell-l_ds, PdPInDel l_2_ds, PdPInDel2_l_ds, PdPInDel2-2_ds are designed. The difference between these four sequences and PdPInDell-1, PdPInDell_2, PdPInDel2_l and PdPInDel2-2 is that an enzyme is added to the left and right ends. Cut site, its structure is shown in Table 5o

 Table 5 Sequence design and structure description of the clasp probe precursor

 Name sequence (5, ― V )

 Design sequence

 PdPInDell-1 - ds GAGTCTTCCTACG CCACTCAA (: AATCCCTCTCATTGCCTGACCACCTCTACA

 GAGTAAGA^;; TG-.GGCCGCTTTTTTGCAm

TGGGAACCGGAGC 1 Γ::: ΓΛΓ3⁄4 0'; (: ACAGCAGCAACATGCGGTAGCACTGC Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence

 Left-wing complementary sequence (LF) AC'GAAC'CACl'CAAC ATCiXTCT

 Public PCR Primer (P1)

 Public PCR Primer (P2) ; ' --ΑΤ Οί'ΟίτΑί ί Α (:; ;'C(" G(>-3⁄4 '

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGC

 Common sequence (CS)

 Right-wing complementary sequence (RF) ACAGCAGCAACATGCGGTAG

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 PdPInDell- 2 - ds GAGTCTTCCTACG/UC(;A(;T(;AA(^AATi;Ci;T(T(ATTG(X;TGACi;Ai;CTCT)

 G "A " : TGCGGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGT TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTG:; V; (; = ,:\:X: :A G AAGCA CATCCCi. A G(;ACG(: CACTGC

 Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence

 Left-wing complementary sequence (LF) ACGAACCACTCAAi'AAT('C('TCT

 Public PCR Primer (P1) 5 " --GIAGAGGTGGTCA GCAAIG--3 ·

Public PCR Primer (P2) AT i:Ci:GA- TAA AT(.(^ ( .--:;. ^>>

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGAGCGTG

 Common sequence (CS) G,' r ί; 'A

 Right-wing complementary sequence (RF) AGCAACATGCGGTAGGACGO

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 PdPInDel2- 1 - ds GAGTCTTCCTA ( A ( GTC(:TTATAGAAi;ATCATGCTT(;ATTGiX;TGACi;A ( Cl^

 ( AAG U , TG ( GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGAT

CGT ^r rGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTA

GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTGCTTACTCTAGCTTCCCGGCAA CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACGAT :" (: GA::: (:: TAACAOTGATAATGGGCCACTGC

 Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence Left-wing complementary sequence (LF) ("Ci;CA('AAAAi;ATG(;T(^ACT(^)

Public PCR Primer (PI) 5 ^? --GTAGAGGTGGTCAGGCAATG--:V

 Public PCR Primer (P2)

 Variable length interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTGCTTACTCTAGCTTCCCGGCAACAATTAATAGACT GGATGGAGGCGGATAAAGTTGCAGGAC

 Public sequence (CS) ·. ': \ 'Γ ''Γ, '-, '人 , ■, :, '' . ':-::.',

 Right-wing complementary sequence (RF) 'i'CAT'i'TATTAAi'AG'i GATAATGGGC

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 PdPInDel2- 2 - ds GAGTCTTCCTACACGT (:CTTATAGAACATi;/VrG(TT(ATTGCi i

 GCGGAGTAA: Ti ' GGCCGCTTTTTTGCACAACATGGGGGATCATGTMCTCGCCTTGAT CGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGGGCGTGACACCACGATGCCTGTA GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACCCTAGCTTCCCGGCAA CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC( T ^::i' (^: A ^H:,AAiAT rATOTCATrm

 Structure description

 Double-stranded DNA restriction endosystem GAGTCTTCCT

Enzyme recognition sequence

 Left-wing complementary sequence (LF) CGGGACAAAAGATGGTCAGTC

 Public PCR Primer (P1) 5 ' --GTAGAGGTGGTCAGGCAATG--3'

Public PCR Primer (P2) -- Α Ϊί-;;. ί-0Α0ΤΑΑ ^; 'Α (·0';'^ - Variable Length Interval (SP) GGCCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

 AGCTGAATGAAGCCATACCAAACGACGGGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACCCTAGCTTCCCGGCAACAATTAATAGACT GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC

 Public sequence (CS) ; ', ΑΤ ; ', Λ [ , :::Λ::.;:::.Λ

 Right-wing complementary sequence (RF) ACATTTATGTCATTTATTAACAGTGATA

 Double-stranded DNA single-strand nicking enzyme CACTGC

Recognition sequence

 2 PCR method to synthesize clasp probe precursor PdPInDell-l_ds, PdPInDell_2_ds, PdPInDel2-l_ds and PdPInDel2_2_ds

The PCR synthesis of the design of the clasp probe precursors PdPInDell-l_ds and PdPInDel l_2_ds was performed using four primers PdPInDell_Fl, PdPInDel 1_F2, PdPInDel 1_1_R1, PdPInDel 1_1_R2 (PdPInDel 1-2_R1 and PdPInDel 1-2_R2 ), and the hand probe precursor PdPInDel2_l_ds And the PCR synthesis of PdPInDel2-2_ds also used four primers PdPInDel2_Fl, PdPInDel2_F2, respectively. PdPInDel2-l_Rl, PdPInDel2_l_R2 (PdPInDel2_2_Rl and PdPInDel2_2_R2) (see Table 6 for the specific sequence). The precursor sequence of the clasp probe was synthesized by two rounds of PCR using four primers.

 The details are as follows: The first round of PCR, using the above diluted 10,000 times PdPInDel l-l_P_SP_CS and PdPInDel l-2_P_SP_CS as the first round of PCR DNA template, using PdPInDel l_Fl and PdPInDel l-l_Rl (PdPInDel l_2_Rl) primers for PCR PdPInDel2-l_P_SP_CS and PdPInDel2_2_P_SP_CS diluted as 10,000 times as above were used as the DNA template of the first round of PCR, and PCR was carried out using PdPInDel2_Fl and P PdPInDel2-l_Rl (PdPInDel2_2_Rl) primers. The reaction system was: 2ul 10x PCR buffer, 1 6ul 2. 5mM dNTP, 0.5 ul lOuM forward primer, 0.5 ul lOuM reverse primer, lul DNA template, 0. lul rTaq (5 U/ul), plus sterile double distilled water to 20 ul. The product of the first round of PCR was diluted 10,000 times and used as a DNA template for the next round of PCR. For the second round of PCR, the PdPInDel l-l_ds precursor was synthesized using PdPInDel l_F2 and PdPInDel 1_1_R2 primers, the PdPInDel l-2_ds precursor was synthesized using PdPInDel 1_F2 and PdPInDel 1_2_R2 primers, and the PdPInDel2-l_ds precursor was synthesized using PdPInDel2_F2 and PdPInDel2_l_R2 primers. The PdPInDel2-2_ds precursor was synthesized using PdPInDel2_F2 and PdPInDel2_2_R2 primers. The reaction conditions of the two rounds of PCR were the same, and the reaction conditions were as shown in the PCR system, and the precursor product of the clasp probe was obtained (see Table 5 for the specific sequence).

 The cycle parameters of the PCR reaction are as follows: (1) 94 ° C, 3 minutes; (2) 94 ° C, 30 seconds; 50 ° C, 30 seconds; 72 ° C, 15 seconds, 5 cycles; (3) 72 ° C extension for 5 minutes; (4) 94 ° C, 30 seconds; 65 ° C, 30 seconds; 72 ° C, 15 seconds, 28 cycles; (5) 72 ° C extension for 5 minutes

 Table 6 PCR primers and probe fragments

PdPInDell— Fl Ci:Ai :AA¾Vi::: ::TCr(〕AT'i'(，CC'i'GACCA(X

PdPInDell—Fl Ci:Ai :AA3⁄4Vi::: ::TCr(]AT'i'(,CC'i'GACCA(X

PdPInDell-1 - Rl O : 1; C ? ^;- ί.,·';'ί;-: d: 't;; - 0' A 5 ^; --

PdPInDell— F2 GAGTCTTCCTA^. AAr. (; (; Ί (; ΛΑ^ΑΛ'; ::Χ.: ί

 PdPInDell - 1 - R2 GCAGTGCTACCGCAT (, TTG (: TG () TGT' G

PdPInDell- 2— Rl 0ί_. ί; ί_. ϊ i; ϊ G .' ί ^: ί. \>: d::: Α. ^{: :}

 PdPInDell - 2 - R2 GCAGTGCCGTCCTACCGCAT(rTG(;'^':

 PdPInDel2_Fl

 PdPInDel2-l_Rl ATTATCACTGTTA/V]Vl\ATGA G ( ;:;.i-:;; A--

PdPInDel2_F2 GAGTCTTCCTACACGT (XTTA[AGAACATCATGCTT

 PdPInDel2-l_R2 GCAGTGGCCCATi Λ'ί i^CTGTTAATAAATGA'i G

 PdPInDel2-2_Rl TGTTAATAAATGACATAAATGT'i^C:^! Γ ^ AOAA

 PdPInDel2-2_R2 GCAGTG!AE CACKrrTAATAAATCACA'E ΑΑΑΚΓΓΤ^

 3 Construction of plasmid POCP-PdPInDell-U POCP - PdPInDel 1 - 2, POCP - PdP_InDel2 - 1 and POCP - PdPInDel2 - 2

The precursor products of the above four hand-held probes were purified and ligated into the P0CP-T vector (sequence 7), respectively, and the ligation system was: 1 ul 10 X NEB T4 ligase buffer 1 ul POCP-T (50 ng/ul), 0.6 ul second round of PCR product (20 ng/ul), 0.4 ul T4 DNA Ligase (400 u/ul), sterilized double distilled water to 10 ul, connected at 16 °C for one night (12h). 5 ul of the ligation products were separately transformed into E. coli DH5a strain to obtain transformants. Select monoclonal to > 1 ml LB medium containing kanamycin, culture at 37 V 200 rpm for 4 h, and take 1 ul of bacterial solution for PCR identification. The positive transformant POCP-PdPInDell-1 was identified by PCR using primers PdPInDel 1_F2 and PdPInDel 1_1_R2, and a plasmid of 176 bp fragment was obtained and sequenced. The result was the sequence PdPInDell-l_ds in Table 5; using primers PdPInDell_F2 and P PdPInDel 1_2_R2 The positive transformant POCP-PdPInDell-2 was identified by PCR, and the plasmid with the size of 206 bp was obtained and sequenced. The result was the sequence PdPInDell-2_ds in Table 5. The positive transformants P0CP-PdPInDel2-l were identified by PCR with primers PdPInDel2_F2 and PdPInDel2_l_R2. The plasmid of 334 bp fragment was obtained and sequenced, and the result was the sequence PdPInDel2-l_ds in Table 5. The positive transformant P0CP-PdPInDel2-2 was identified by the primers PdPInDel2_F2 and PdPInDel2-2_R2, and the PCR product was 358 bp fragment. The plasmid was sent for sequencing and the result was the sequence PdPInDel2-2_d _{S in} Table 5. The plasmids of the above positive clones were separately extracted: P0CP-PdPInDell-l, POCP-PdPInDel 1-2, P0CP-PdPInDel2_l and POCP-PdPInDel2-2.

The above method for transforming the DH5 α strain is as follows: The stored competent DH5 α bacterial solution is taken out from a refrigerator at 80 ° C, placed on ice for about 5 minutes to naturally melt, and 80 ul of the bacterial liquid and 5 ul of the ligation product are placed. Mix the tube on ice, gently rotate the tube, mix it on ice for 20 minutes, heat at 42 °C for 45-50 seconds, then place on ice for 2 minutes, then add 950 ul SOC medium at 37 ° The C shaker was shaken at about 150 rpm for 1 hour and 30 minutes. Finally, 50-100 ul of the transformed SOC medium was applied to the LB/Kana resistant plate, and the kanamycin content was 50 ug/ml. The above method for extracting the positive clone plasmid is as follows: 500 ul of the above positive clone liquid is taken in 25 ml LB medium at 37 ° C for 230 rpm overnight, and the plasmid is extracted according to the medium amount of plasmid extraction method, and finally 200 ul of TE and RNase are added, and the temperature is maintained at 37 ° C. One hour, plasmids POCP_PdPInDell_l, POCP-PdPInDel 1- 2, P0CP-PdPInDel2-l and P P0CP-PdPInDel2-2 were obtained. 25 ug of each of the four extracted plasmids (80 ug in total) were double-digested, and the double-digested enzymes were fyl (NEW ENGLAND BioLabs) and sputum 3⁄4. Btsl (NEW ENGLAND BioLabs), double digestion The system is: 50 ul 10x NEB4 buffer 5 ul 100 x BSA, 12 ul Mlyl (10 U/ul), 14 ul Nb. Btsl (lOU/ul), 200 ul plasmid DNA (500 ng/ul), plus sterilization Double distilled water to 500 ul, incubated at 37 °C for 5 hours, 80 ° heating for 20 minutes to inactivate the enzyme. 500 ul of the digested product was freeze-dried to 150 ul, and then separated and purified.

 3. Separation and purification of the clasp probes PdPInDell-l, PdPInDell_2, PdPInDel2_l, PdPInDel2_2

 1) Separation of the clasp probe

 The polyacrylamide electrophoresis instrument used for the separation of the clasp probe is a mini-column electrophoresis instrument modified by the Liuyi Instrument Factory (see Figure 3). The glass column is 90 mm long, the inner diameter is 5-7 mm, and the nozzle is made of diamond sand. level. Before preparing the gel, put the bottom of the washed and dried glass tube on the latex tube, and then use the 0.8% agarose gel to pour into the lower electrophoresis column for about 2 cm. After the agarose gel is solidified, 5% polyacrylamide is added. Glue, after the polyacrylamide gel was solidified, the agarose gel of the lower electrophoresis column was picked out with a syringe needle, leaving a space of 2 cm under the electrophoresis column. Before electrophoresis, add the electrophoresis solution to the 2 cm space, and insert the electrophoresis column into the well of the holder. The electrophoresis voltage is 300 V and the current is 7 mA. When the xylene hydrazine is electrophoresed under the gel column, the lower electrophoresis column is sealed with a dialysis bag with a molecular weight cut-off of 3.5 KDa, and then electrophoresed for about 25 min (adjust the time according to the fragment size). Remove the electrophoresis column, invert the electrophoresis column, carefully remove the dialysis bag, and remove the electrophoresis solution from the column space in a 1.5 ml EP tube.

 2) Purification of the clasp probe

 Add the above solution to 1/10 volume of 3 M NaOAC (pH=5.2), add 3 volumes of cold absolute ethanol, freeze at -20 °C for 30 min, centrifuge to separate the precipitate, and use 1 ml of Wash 75% ethanol twice, dry at room temperature for 5 min, and dissolve the precipitate with 30 ul of sterile double distilled water. The DNA content was determined by taking 1 ul of solution, and determined by ND-1000 spectrophotometer (Thermo), 0D260/280=1.90, 0D260/230=1.90, the concentration was about 28 ng/ul, and about 840 ng was recovered. The needle was diluted to 2 ng/ul with sterile distilled water, and the recovery of the hand-held probe reached 38.8% of the theoretical yield.

The isolated clasp probes PdPInDell-1, PdPInDell_2, PdPInDel2_l, and PdPInDel2-2 were sent for sequencing, and the result was that PdPInDell-1 had the oligonucleotide shown in SEQ ID NO: 8 in the sequence listing (160 nt in size), in which the sequence table The nucleotides 1 to 23 of the sequence 8 from the 5' end are complementary to the DNA 3 to be tested (nucleotides 98-120 of SEQ ID NO: 12 in the sequence listing), and the public PCR primer fragment is located at positions 24-43. 1. The 44th-63th position is a common PCR primer fragment 2, the 64th-125th position is a variable length region, the 126th-140th position is a common sequence, and the 14th-160th is a DNA sequence to be tested (the sequence 12 in the sequence listing) Reverse nucleotides 121-140). PdPInDel l-2 has the oligonucleotide (size 190 nt) shown in SEQ ID NO: 9 in the sequence listing, wherein the nucleotides 1 to 23 of the sequence 9 from the 5' end of the sequence are the DNAs to be tested 4 (in the sequence listing) The nucleotides 98-120 of SEQ ID NO:13 are reverse-complementary, the 24-48th position is the common PCR primer fragment 1, the 44th-63th position is the common PCR primer fragment 2, and the 64th-155th position is the variable length region. 156-170 is a common sequence, and 171-190 is the DNA to be tested 4 (nucleotides 121-140 of SEQ ID NO: 13 in the sequence listing, the only difference between DNA4 and DNA3 is shown in SEQ ID NO: 12 in the sequence listing. Insert 5 bases between positions 120 and 121 of DNA3 to obtain DNA4, which is inversely complementary to positions 186-190 of the oligonucleotide shown in SEQ ID NO: 9 in the sequence listing, 186-190 The 5 bases of the position are the complementary sequences of the insert, and the 5 'end is phosphorylated;

 PdPInDel2-l has the oligonucleotide (size 318 nt) shown in SEQ ID NO: 10 in the sequence listing, wherein nucleotides 1-26 of the sequence 10 from the 5' end of the sequence are the same as the DNA to be tested 5 (in the sequence listing) The nucleotides 91-116 of SEQ ID NO: 14 are reverse-complementary, the common PCR primer fragment at positions 27-46, the common PCR primer fragment at positions 47-66, and the variable length region at positions 67-278. , positions 279-293 are common sequences, positions 294-318 and DNA to be tested 5 (nucleotides 117-141 of sequence 14 in the sequence listing, the only difference between DNA6 and DNA5 is shown in sequence 15 in the sequence listing Insert 6 bases between DNAs 116-117 to obtain DNA5, which is complementary to the third to third positions of the oligonucleotides shown in sequence 10 in the sequence listing, positions 313-318 6 bases are complementary sequences of the insert, phosphorylation at the 5' end; PdPInDel2-2 has the oligonucleotide shown in SEQ ID NO: 11 in the sequence listing (size 342 nt), wherein the sequence of the sequence 11 is from the 5' end The nucleotides 1-26 are complementary to the DNA to be tested 6 (nucleotides 91-116 of SEQ ID NO: 15 in the sequence listing), 27- The 46th position is the common PCR primer fragment 1, the 47th to the 66th is the common PCR primer fragment 2, the 67th to the 299th is the variable length region, the 300th to the 314th is the common sequence, and the 315th to the 342th is the DNA to be tested. (nucleotides 117-144 of SEQ ID NO: 15 in the sequence listing) reverse complementation, phosphorylation at the 5' end;

 The above probes PdPInDel l-1, PdPInDel l_2, PdPInDel2_l, PdPInDel2- 2 can also be artificially synthesized.

 Second, the SNP classification of the clasp probe

 1. Genomic DNA preparation

 The barley varieties Vada and L94 were extracted by CTAB method (both described in Neal SC, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR appl ications. BioTechniques 14 : 748-751, publicly available from the Chinese Academy of Sciences The genomic DNA obtained by the Institute was broken with a large ultrasonic wave at a wavelength of 700 W, 40 KhZ for 35 min, the genome was broken to 80-250 bp, and the final DNA concentration was adjusted to 150 ng/ul.

 The two InDel markers of the two-segment DNA molecule on the fifth chromosome of the barley variety Vada are DNA3 shown in SEQ ID NO: 12 in the sequence listing and DNA5 shown in SEQ ID NO: 14 on the fifth chromosome of the barley variety L94. The two InDel markers of the segment DNA molecule are DNA4 shown in SEQ ID NO: 13 in the sequence listing and DNA6 shown in SEQ ID NO: 15 respectively.

2, the connection of the clasp probe The above-mentioned 4 ng/ul dilutions of 4 hand-held probe mixtures (average 0.5 ng/ul per hand-hand probe) were subjected to ligation and cyclization using the template for the above-described shattered genomic DNA.

 Linking probe reaction system: 10 ul lOXAmpligase® buffer; 2 ul button probe (2 ng/ul) (the final concentration of each button probe in the ligation system is approximately 0.04 ng/ul); 16 ul genomic DNA (150 ng/ul) (final concentration of genomic DNA molecule in the final ligation system of 24 ng/ul); 0.8 ul Amp li gas® thermostable DNA ligase (5 U/ul) (ligase in The final concentration in the final connection system is 0.04 U/ul); plus sterilized double distilled water to 100 ul

 The reaction conditions of the clasp probe are: 95V for 3 minutes; then for 94°C for 30 seconds, for 55 minutes for 10 minutes, for 10 cycles.

 3. Public PCR primer amplification

 The above-mentioned ligation product was 0.5 ul, and the two common primers (Pl, P2) of the clasp probe were used for PCR amplification, and the successful cyclization probe was amplified.

 PCR amplification system: 2 ul 10 X PCR buffer; 0.4 ul dNTP (10 mM); 0.5 ul PI (10 uM); 0.5 ul P2 (10 uM); 0.5 ul ligation product; 0· 1 ul rTag (5 U /ul); Add sterilized double distilled water to 20 ul

 PCR reaction conditions: first 94 ° C for 3 minutes; then 94 ° C for 30 seconds, 57 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles; then 72 ° C for 5 minutes.

 The results are shown by 3% agarose gel electrophoresis. The results are shown in Fig. 5. V represents the barley variety Vada, and L represents the barley variety L94. It can be seen from the figure that the lane V result proves that the button probe PdPInDell-Ι can The InDel marker represented by DNA3 shown in SEQ ID NO: 12 in the sequence of specific recognition sequence has a length of 160 nt, and the clasp probe PdPInDel2-l can specifically recognize the InDel marker represented by DNA5 shown in SEQ ID NO: 14 in the sequence listing, and has a length of 318 nt. The results of lane L demonstrated that the hand probe PdPInDell-2 can specifically recognize the InDel marker represented by DNA4 in sequence 13 in the sequence listing, and the length is 190 nt. The button probe PdPInDel2-2 can specifically recognize the sequence 15 in the sequence listing. The InDel marker represented by DNA6 is 342 nt in length.

 After electrophoresis by 5% urea polyacrylamide, the results were observed after silver staining. The results are shown in Fig. 6. V represents the barley variety Vada, L represents the barley variety L94, and P is a mixture of 4 kinds of hand-held probes. The results of lane V demonstrated that the button probe PdPInDell-Ι can specifically recognize the InDel marker represented by DNA3 in sequence 12 in the sequence listing, and the length is 160 nt, and the button probe PdPInDel2_l can specifically recognize the sequence 14 in the sequence listing. The InDel marker represented by DNA5 is 318 nt in length. The results of lane L demonstrated that the hand probe PdPInDell-2 can specifically recognize the InDel marker represented by DNA4 in sequence 13 in the sequence listing, and the length is 190 nt. The button probe PdPInDel2-2 can specifically recognize the sequence 15 in the sequence listing. The InDel marker represented by DNA6 is 342 nt in length.

The above results demonstrate that the clasp probe PdPInDell-Ι can specifically recognize Vada's InDell genotype with a length of 160 nt. The clasp probe PdPInDell-2 can specifically recognize the L94 InDell genotype with a length of 190 nt. PdPInDel2-l can specifically recognize the InDel2 genotype of Vada, which is 318 nt in length. The clasp probe PdPInDel2-2 can specifically recognize the InDel2 genotype of L94. The degree is 342 nt. The difference in length of the four probes can be distinguished in a 3% agarose gel.

Industrial application

 The experiments of the present invention prove that the single-stranded DNA molecules provided by the present invention are designed for different deoxyribonucleic acid labeling sites and their allelic sites, and the hand-held probes of different lengths are designed to be distinguished by the ligase reaction. Different markers and different alleles, PCR amplification of successfully connected different lengths of hand-held probes, gel or capillary electrophoresis separation and detection system, to achieve simultaneous detection of multiple deoxyribonucleic acid-labeled polymorphisms, can achieve High sensitivity and accuracy while meeting high throughput and low cost requirements.

Claims

Rights request 1. A single-stranded DNA molecule consisting of the following components from the 5' end to the 3' end: complementary to the 5' end of the DNA to be tested, the left oligonucleotide fragment, the host fragment and the 3' to the DNA to be tested a complementary complementary right oligonucleotide fragment;  The subject fragment is composed of: a length variable region for determining the size of the single-stranded DNA molecule; and a primer pair region and a common fragment respectively located on both sides of the variable length region;  The primer pair region is obtained by connecting two oligonucleotide chains as follows:  1) a primer capable of amplifying a pair of primers of said single-stranded DNA molecule;  2) an oligonucleotide capable of amplifying a reverse complement of the other primer of the pair of primers of the single-stranded DNA molecule;  The variable length region is an oligonucleotide having a size of 45-1000 nt;  The common fragment is an oligonucleotide of 13-18 nt in size;  The subject fragment is not complementary to the gene to be tested.  2. A single-stranded DNA molecule according to claim 1 wherein: The size of the one primer and the other primer are both 18-22 nt, and the size of the one primer and the other primer are each specifically 20 nt _;  The common fragment is an oligonucleotide of 15 nt in size;  The nucleotide sequence of the variable length region is any one of the following 1) -4):  Bits 68-112 of sequence 1 in the sequence listing;  Bits 68-129 of sequence 2 in the sequence listing;  Bits 62-136 of sequence 3 in the sequence listing;  62-153 of sequence 4 in the sequence listing;  Bits 64-125 of sequence 8 in the sequence listing;  Bits 64-155 of sequence 9 in the sequence listing;  67-278 of sequence 10 in the sequence listing;  67-299 of sequence 11 in the sequence listing;  The nucleotide sequence of the common fragment is at positions 113-127 of sequence 1 in the sequence listing or at positions 130-144 of sequence 2 in the sequence listing or at positions 137-151 of sequence 3 in the sequence listing or in the sequence listing 154-168 of 4 or 126-140 of SEQ ID NO: 8 or 156-170 of SEQ ID NO: 9 or 279-293 of SEQ ID NO: 10 or SEQ ID NO: 11 of the Sequence Listing Positions 300-314; the nucleotide sequence of the primer pair region is position 28-67 of sequence 1 in the sequence listing or position 28-67 of sequence 2 in the sequence listing or the 22nd of sequence 3 in the sequence listing - Positions 22-61 of Sequence 4 in Sequence Listing 61 or Sequences 24-63 of Sequence 8 in the Sequence Listing or Position 24-63 of Sequence 9 in the Sequence Listing or Positions 27-66 of Sequence 10 in the Sequence Listing Or the 27th to 66th of the sequence 11 in the sequence listing. 3. A special button probe for detecting DNA single nucleotide polymorphism, wherein the special button probe is composed of m groups of probes, and each set of probes corresponds to a SNP site to be tested. Single nucleotide polymorphism There are n polymorphisms in the SNP locus to be tested.  Each set of probes consists of n single-stranded DNA molecules according to claim 1 or 2, n being a natural number greater than or equal to 2;  m is a natural number greater than or equal to 1;  The 5'-end left oligonucleotide fragment of each single-stranded DNA molecule specifically binds to an upstream fragment of the SNP site to be tested, and the 3'-end right oligonucleotide of each single-stranded DNA molecule The downstream fragment of the SNP site to be tested specifically binds, and the 3' terminal base of each single-stranded DNA molecule is complementary to the base of the SNP site to be tested.  4. The dedicated buckle probe of claim 3, wherein:  The number of the probe sets is the same as the number of the SNP sites to be tested;  The number of SNP sites to be tested is at least two;  The size of each of the single-stranded DNA molecules in each of the sets of probes is different, and the size of the single-stranded DNA molecule is determined by the variable length region thereof;  Phosphorylation of the 5' end of each of the single-stranded DNA molecules, specifically the 5th carbon of the first nucleotide of the left oligonucleotide of each of the single-stranded DNA molecules from the 5' end A phosphate group is attached to the atom; the dedicated button probe consists of two probe sets:  Probe set 1 consists of two single-stranded DNA molecules as described below:  1) an oligonucleotide as shown in SEQ ID NO:1 in the Sequence Listing;  2) an oligonucleotide as shown in SEQ ID NO: 2 in the Sequence Listing;  Probe set 2 consists of two single-stranded DNA molecules as described below:  3) an oligonucleotide as shown in SEQ ID NO: 3 in the Sequence Listing;  4) The oligonucleotide shown in SEQ ID NO: 4 in the Sequence Listing.  5. A special button probe for detecting DNA insertion or deletion polymorphism, wherein the special button probe is composed of a group of probes, and each group of probes corresponds to a DNA insertion or deletion site to be tested. The single nucleotide polymorphism is a polymorphism of b in a DNA insertion or deletion site to be tested,  Each set of probes is composed of b single-stranded DNA molecules according to claim 1 or 2, and b is a natural number greater than or equal to 2;  a is a natural number greater than or equal to 1;  The 5'-end left oligonucleotide fragment of each single-stranded DNA molecule specifically binds to an upstream fragment of the insertion or deletion site to be detected, and the 3'-end right oligonucleoside of each single-stranded DNA molecule The acid specifically binds to a downstream fragment of the insertion or deletion site to be tested.  6. The special buckle probe of claim 5, wherein:  The DNA has at least 2 insertion or deletion sites;  The size of each single-stranded DNA molecule is different, and the size of the single-stranded DNA molecule is determined by the variable length region thereof; Phosphorylation of the 5' end of each of the single-stranded DNA molecules, specifically the 5th carbon of the first nucleotide of the left oligonucleotide of each of the single-stranded DNA molecules from the 5' end Connecting a phosphate group to an atom; The dedicated buckle probe consists of the following two probe sets:  Probe set 1 consists of two single-stranded DNA molecules as described below:  1) an oligonucleotide as shown in SEQ ID NO:8 in the Sequence Listing;  2) an oligonucleotide as shown in SEQ ID NO:9 in the sequence listing;  Probe set 2 consists of two single-stranded DNA molecules as described below:  3) an oligonucleotide as shown in SEQ ID NO: 10 in the Sequence Listing;  4) The oligonucleotide shown in SEQ ID NO: 11 in the Sequence Listing.  7. A kit comprising the single-stranded DNA molecule of claim 1 or 2 or the special hand-held probe of any of claims 3-6.  The use of the single-stranded DNA molecule according to claim 1 or 2 or the special hand-held probe according to any one of claims 3 to 6 for the identification of DNA molecular polymorphism;  The polymorphism is a single nucleotide polymorphism or an insertion or deletion polymorphism;  The single nucleotide polymorphism is a single nucleotide polymorphism specifically caused by at least two SNP sites; the insertion or deletion polymorphism is specifically caused by at least two insertion or deletion sites. Polymorphism  The single nucleotide polymorphism caused by the at least two SNP sites is specifically the polymorphism of the 62nd nucleotide of the DNA molecule shown in the sequence 5 in the sequence table and the DNA molecule shown in the sequence 6 of the sequence table. Polymorphism of nucleotides;  The polymorphism of the 62nd nucleotide of the DNA molecule shown in SEQ ID NO: 5 in the sequence listing is the nucleotide 62 of the DNA molecule represented by the sequence 5 in the sequence listing is A or G;  The 174th nucleotide polymorphism of the DNA molecule represented by the sequence 6 in the sequence listing is the nucleotide number 174 of the DNA molecule represented by the sequence 6 in the sequence listing is T or C;  The insertion or deletion polymorphisms caused by the at least two insertion or deletion sites are specifically 1) and 2): 1) The DNA molecule shown in SEQ ID NO: 12 in the sequence listing is inserted into CCGTC from positions 120-121 at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 13 in the sequence listing; or the DNA molecule shown in SEQ ID NO: 13 in the sequence listing. Deletion of CCGTC at positions 121-125 at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 12 in the Sequence Listing;  2) The DNA molecule shown in SEQ ID NO: 15 in the sequence table is inserted into GCCCAT from positions 56-117 at the 5' end to obtain the DNA molecule shown in SEQ ID NO: 14 in the sequence listing; or the DNA molecule shown in SEQ ID NO: 14 in the sequence listing. The deletion of GCCCAT at positions 117-122 of the terminus results in the DNA molecule shown in SEQ ID NO: 15 in the Sequence Listing.  The DNA sequence shown by the DNA molecule shown in SEQ ID NO: 12 in sequence 12 or the DNA molecule shown in SEQ ID NO: 14 in the sequence listing or the DNA molecule shown in SEQ ID NO: 15 is the DNA sequence of two insertion/deletion markers of barley. .  9. A method for detecting a single nucleotide polymorphism of a biological genome, comprising the steps of: 1) cyclizing all single-stranded DNA molecules in the special-handle probe according to claim 3 or 4 with a genomic fragment to obtain a cyclized product of a clasp probe; 2) performing PCR using the cyclized product obtained in the step 1) as a template, and determining the polymorphism of the single genome of the biological genome according to the size of the obtained PCR product;  In step 1), before the cyclization of the linkage, further comprising breaking the genomic fragment into a DNA molecule of 80-250 bp size;  01 U/ul, The concentration of the ligase in the ligation system is 0. 04 U / ul, the connection cyclization system comprises: a ligation buffer, a ligase, a ligating probe, a DNA fragment of 80-250 bp in size. The concentration of each of the buckled probes in the ligation system is 0. 04 ng / ul, and the concentration of the 80-250 bp DNA molecule in the ligation system is 24 ng / ul;  In step 2), the primer of the PCR is the primer corresponding to the primer pair region in the single-stranded DNA molecule, and the one primer and the other primer in a pair of primers capable of amplifying the single-stranded DNA molecule;  The nucleotide sequence of the one primer is the 48th-67th position of the sequence 1 in the sequence listing or the 48th-67th position of the sequence 2 in the sequence listing or the 42th-61th position of the sequence 3 in the sequence listing or the sequence in the sequence listing Bits 42-61 of 4;  The nucleotide sequence of the reverse complementary fragment of the other primer is position 28-47 of sequence 2 in sequence 28-47 of sequence 1 of the sequence listing or positions 22-41 of sequence 3 in the sequence listing. Or the 22-41 of sequence 4 in the sequence listing.  10. A method of detecting a biological genome insertion or deletion polymorphism, comprising the steps of: 1) cyclizing all single-stranded DNA molecules of a dedicated button probe of claim 5 or 6 to a genomic fragment, Obtaining a looper probe cyclization product;  2) performing PCR by using the cyclized product obtained in the step 1) as a template, and determining the polymorphism of the insertion or deletion of the biological genome according to the obtained PCR product;  In step 1), before the cyclization of the linkage, further comprising breaking the genomic fragment into a DNA molecule of 80-250 bp size;  01 U/ul, The concentration of the ligase in the ligation system is 0. 04 U / ul, the connection cyclization system comprises: a ligation buffer, a ligase, a ligating probe, a DNA fragment of 80-250 bp in size. The concentration of each of the buckled probes in the ligation system is 0. 04 ng / ul, and the concentration of the 80-250 bp DNA molecule in the ligation system is 24 ng / ul;  In step 2), the primer of the PCR is the primer corresponding to the primer pair region in the single-stranded DNA molecule, and the one primer and the other primer in a pair of primers capable of amplifying the single-stranded DNA molecule;  The nucleotide sequence of the one primer is at positions 44-63 of SEQ ID NO: 8 in the sequence listing or at positions 44-63 of SEQ ID NO: 9 in the sequence listing or at positions 47-66 of SEQ ID NO: 10 in the sequence listing or in the sequence listing Bits 47-66 of 11;  The nucleotide sequence of the reverse complementary fragment of the other primer is 24-24th of sequence 8 in the sequence listing or 24-24th of sequence 9 in the sequence listing or 27-46 of sequence 10 in the sequence listing. Bits 27-46 of sequence 11 in the sequence or sequence listing.