CN113549689A - Kit and method for detecting PROS1 gene exon - Google Patents
Kit and method for detecting PROS1 gene exon Download PDFInfo
- Publication number
- CN113549689A CN113549689A CN202111109673.7A CN202111109673A CN113549689A CN 113549689 A CN113549689 A CN 113549689A CN 202111109673 A CN202111109673 A CN 202111109673A CN 113549689 A CN113549689 A CN 113549689A
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- primer pair
- sequence
- artificial sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kit and a method for detecting PROS1 gene exon. Specifically, the invention provides a primer pair set and a corresponding kit which are rapid, comprehensive and simple to operate and can be used for detecting the exon of the PROS1 gene and the specific SNP, and a method for detecting the exon of the PROS1 gene and the specific SNP by using the kit. The kit comprises a plurality of pairs of primer pairs for specifically amplifying the PROS1 gene exons, and can efficiently and once amplify all the PROS1 gene exons. In addition, part of the primer pairs can also amplify intron sequences containing specific SNPs, which is beneficial to realizing more comprehensive detection of PROS1 gene mutation and has application value and clinical significance for diagnosing hereditary Protein S Deficiency (PSD).
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for detecting PROS1 gene exon.
Background
Protein S (PS) is one of the natural human anticoagulant proteins. Protein S is a vitamin K-dependent plasma protein produced primarily by the liver, vascular endothelium, monocytes and megakaryocytes. Thrombin formation is inhibited by functioning as a cofactor for activated protein c (apc) in the degradation of coagulation factors Va and viii a. Protein S is a single chain protein, consisting of 676 amino acids. Normally, about 60% of PS binds to the regulatory component C4b binding protein of the complement system to form a complex, and the remaining 40% functions as a cofactor for APC. PS has APC-dependent anticoagulant function.
Thromboembolism (VTE) is caused by injury to the vessel wall, slowing or stopping of blood flow, and a hypercoagulable state of blood. Including lower limb Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE). In recent years, pulmonary embolism is more and more emphasized by the characteristics of acute onset, atypical clinical manifestations, high mortality rate and the like. Genetic Protein S Deficiency (PSD) is currently an established risk factor for venous thromboembolism. PSD is an autosomal dominant genetic disease with incomplete penetrance, associated with mutations or polymorphisms in the gene encoding protein S (PROS 1).
The PROS1 gene has 15 exons and 14 introns in total, spans 101kb, and is located on the long arm of chromosome 3, at position 11.1. About 200 or more mutations in PROS1 have been identified at home and abroad to cause changes in PS synthesis or function. Typically, patients with homozygous or compound heterozygous mutations of PROS1 may have life-threatening thrombosis, such as neonatal haemorrhagic purpura. Hereditary PS deficiency is a hereditary thrombotic disease with a low incidence but severe consequences, and its major symptoms are superficial thrombophlebitis, deep vein thrombosis and pulmonary embolism.
The vast majority of mutations in the PROS1 gene can be detected by direct sequencing methods.
Currently, no kit is available on the market that can directly detect all exons of the PROS1 gene. This is probably because protein S is composed of two highly homologous genes, one is an active PROS1 gene and the other is an inactive pseudogene PROS2, and the nucleotide sequences of the pseudogene PROS2 and PROS1 have 97% identity, and thus there is a certain difficulty in designing primers. The PCR efficiency is low because of the secondary structure of the primers or the upstream and downstream primers, or the low binding efficiency of the primers and the DNA template, and the high GC content of the DNA template sequence between the upstream and downstream primers. The conventional commercial PROS1 detection is to detect the gene polymorphism of a certain known locus, and cannot detect the mutation of the unknown locus of the PROS1 gene.
Therefore, the development of a kit and a method which are simple and convenient to operate, good in specificity, high in accuracy and capable of efficiently detecting all exons of the PROS1 gene is urgently needed in the field so as to make up for the defects in the aspect of domestic PROS1 gene detection.
Disclosure of Invention
The invention aims to provide a kit and a method capable of accurately and efficiently detecting all exons of a PROS1 gene.
In a first aspect of the invention, there is provided a set of specific primer pairs for amplifying an exon of a PROS1 gene, the set of primer pairs comprising:
(Z2) primer pair 2 comprising the sequence set forth in SEQ ID NO: 3 and the upstream primer shown as SEQ ID NO: 4, a downstream primer; and
(Z4) primer pair 4 comprising the sequence set forth in SEQ ID NO:7 and the upstream primer shown as SEQ ID NO: 8, a downstream primer;
wherein, a sequence amplified by the primer pair 2 has a polymorphic site SNP which is rs 8178610G/A; and a polymorphic site SNP exists in the sequence amplified by using the primer pair 4, and the SNP is rs 8178623G/A.
In another preferred embodiment, the SNP1 indicates the presence of a G/A at the rs8178610 site.
In another preferred embodiment, the SNP1 indicates the presence of a G/A at position rs 8178623.
In another preferred embodiment, the primer pair set further comprises a primer pair selected from the group consisting of:
(Z1) primer pair 1, comprising the sequence set forth in SEQ ID NO: 1 and the upstream primer shown as SEQ ID NO: 2, or a reverse primer;
(Z3) primer pair 3, comprising the sequence set forth in SEQ ID NO: 5 and the upstream primer shown as SEQ ID NO: 6 as shown in the figure;
(Z5) primer pair 5 comprising the sequence set forth in SEQ ID NO: 9 and the upstream primer shown as SEQ ID NO: 10, a downstream primer;
(Z6) primer pair 6 comprising the sequence set forth in SEQ ID NO: 11 and the upstream primer shown as SEQ ID NO: 12, a downstream primer;
(Z7) primer pair 7, comprising the sequence set forth in SEQ ID NO: 13 and the upstream primer shown as SEQ ID NO: 14, a downstream primer;
(Z8) primer pair 8 comprising the sequence set forth in SEQ ID NO: 15 and the sequence shown in SEQ ID NO: 16, or a reverse primer;
(Z9) primer pair 9 comprising the sequence set forth in SEQ ID NO: 17 and the upstream primer shown as SEQ ID NO: 18, a downstream primer;
(Z10) primer pair 10 comprising the sequence set forth in SEQ ID NO: 19 and the upstream primer shown as SEQ ID NO: 20, a downstream primer;
(Z11) primer pair 11 comprising the sequence set forth in SEQ ID NO: 21 and the upstream primer shown as SEQ ID NO: 22;
(Z12) primer pair 12 comprising the sequence set forth in SEQ ID NO: 23 and the upstream primer shown as SEQ ID NO: 24, a downstream primer;
(Z13) primer pair 13 comprising the sequence set forth in SEQ ID NO: 25 and the sequence shown in SEQ ID NO: 26, a downstream primer;
(Z14) primer pair 14 comprising the sequence set forth in SEQ ID NO: 27 and the upstream primer shown as SEQ ID NO: 28, a downstream primer;
(Z15) primer pair 15 comprising the sequence set forth in SEQ ID NO: 29 and the upstream primer shown in SEQ ID NO: 30, or a reverse primer as shown.
In another preferred embodiment, the primer pair set comprises 15 pairs of primers shown in the primer pairs 1-15.
In another preferred example, the primer pair 1 specifically amplifies exon 1 of the PROS1 gene; the primer pair 2 specifically amplifies PROS1 gene exon 2; the primer pair 3 specifically amplifies PROS1 gene exon 3; the primer pair 4 specifically amplifies PROS1 gene exon 4; the primer pair 5 specifically amplifies PROS1 gene exon 5 and exon 6; the primer pair 6 specifically amplifies PROS1 gene exon 7; the primer pair 7 specifically amplifies PROS1 gene exon 8; the primer pair 8 specifically amplifies PROS1 gene exon 9; the primer pair 9 specifically amplifies PROS1 gene exon 10; the primer pair 10 specifically amplifies PROS1 gene exon 11; the primer pair 11 specifically amplifies PROS1 gene exon 12; the primer pair 12 specifically amplifies PROS1 gene exon 13; the primer pair 13 specifically amplifies PROS1 gene exon 14; the primer pair 14 specifically amplifies PROS1 gene exon 15, and the primer pair 15 specifically amplifies PROS1 gene exon 15.
In another preferred embodiment, the amplification product amplified by the primer pair 2 further comprises sequences of partial intron 1 and intron 2 of the PROS1 gene; wherein, a polymorphic site SNP exists in the amplified intron 1 sequence, the SNP is rs 8178610G/A, a polymorphic site SNP exists in the intron 2 sequence, and the SNP is rs 531436627G/A.
In another preferred embodiment, the amplification product amplified by the primer pair 4 further comprises the sequences of partial intron 3 and intron 4 of the PROS1 gene; wherein, polymorphic site SNP exists in the amplified intron 3 sequence, and the SNP is rs 8178623G/A.
In a second aspect of the invention, there is provided a use of the primer set according to the first aspect of the invention for preparing a diagnostic reagent or kit for:
(a) detecting PROS1 gene mutation and mutation sites thereof; and/or
(b) Diagnosing a genetic protein S deficiency.
In another preferred example, the mutation site of the PROS1 gene is located in an exon and/or an intron of the PROS1 gene.
In a third aspect of the present invention, a kit for detecting an exon of a PROS1 gene is provided, wherein the kit comprises: primer pairs according to the first aspect of the invention and instructions.
In another preferred example, the instructions state that the kit is used to detect the PROS1 gene exon, as well as SNP1 and SNP2, with the PROS1 gene as the reference sequence.
In another preferred embodiment, the SNP1 indicates the presence of a G/A at the rs8178610 site.
In another preferred embodiment, the SNP1 indicates the presence of a G/A at position rs 8178623.
In another preferred embodiment, the specification states that:
(P1) when SNP1, i.e. G → a mutation at rs8178610, is present, it indicates that the susceptibility or prevalence probability of PSD is higher than that of the normal population; and/or
(P2) when SNP2, namely G → A mutation exists at rs8178623, the susceptibility or the prevalence probability of PSD is higher than that of normal population.
In another preferred example, the reference sequence of the PROS1 gene has an NCBI accession number of NG _ 009813.1.
In another preferred embodiment, the primer pairs 1-15 are each placed in separate containers.
In another preferred embodiment, the primer pair is a dry powder or liquid preparation.
In another preferred example, the primer pair 1 specifically amplifies exon 1 of the PROS1 gene; the primer pair 2 specifically amplifies PROS1 gene exon 2; the primer pair 3 specifically amplifies PROS1 gene exon 3; the primer pair 4 specifically amplifies PROS1 gene exon 4; the primer pair 5 specifically amplifies PROS1 gene exon 5 and exon 6; the primer pair 6 specifically amplifies PROS1 gene exon 7; the primer pair 7 specifically amplifies PROS1 gene exon 8; the primer pair 8 specifically amplifies PROS1 gene exon 9; the primer pair 9 specifically amplifies PROS1 gene exon 10; the primer pair 10 specifically amplifies PROS1 gene exon 11; the primer pair 11 specifically amplifies PROS1 gene exon 12; the primer pair 12 specifically amplifies PROS1 gene exon 13; the primer pair 13 specifically amplifies PROS1 gene exon 14; the primer pair 14 specifically amplifies PROS1 gene exon 15, and the primer pair 15 specifically amplifies PROS1 gene exon 15.
In another preferred embodiment, the PCR amplification reagents comprise: 2 × Taq Master Mix, Mg2+DMSO, and ddH2O。
In a fourth aspect of the invention, there is provided a non-diagnostic method for detecting an exon of the PROS1 gene, the method comprising the steps of:
(S1) providing a sample DNA to be tested;
(S2) performing PCR amplification on the test sample DNA using the primer set according to the first aspect of the present invention, thereby obtaining an amplification product containing the exon sequence of the PROS1 gene; and
(S3) sequencing the amplification product containing the pro S1 gene exon sequence, thereby obtaining sequence information of the pro S1 gene exon sequence; and
(S4) comparing the sequence information obtained in the step (S3) with a reference sequence of the PROS1 gene, thereby obtaining a mutation site of the PROS1 gene in the test sample DNA.
In another preferred example, in the step (S2), the PCR amplification includes the steps of:
(i) respectively preparing a class I PCR reaction system R1 and a class II PCR reaction system R2;
wherein R1 contains the following components: 10ng-1 mug sample to be tested, 0.1-0.5 mug primer pair 1, 2 XTaq Master Mix, 0.5-2mM Mg2+1-4% by volume of DMSO and ddH2O；
R2 contains the following components: 10ng-1 ug of sample to be tested, 0.1-0.5 uM of primer pair 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, 2 XTaq Master Mix, 0.5-2mM Mg2+And ddH2O; and
(ii) PCR amplification was performed for R1 and R2 according to the following procedure:
5min at 95 ℃; the following procedure was performed for 12 cycles: 25s at 94 ℃, 40s at 62 ℃ (-0.3 ℃), 45s at 72 ℃; the following procedure was performed for 24 cycles: 25s at 94 ℃, 30s at 58 ℃ and 45s at 72 ℃; 5min at 72 ℃; 25 ℃ for 25 s;
thereby obtaining amplification products of nucleic acid sequences containing exons 1, 2, 3, 4, 5 and 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, respectively, of the PROS1 gene.
In another preferred example, the sample DNA to be tested is of human origin.
In another preferred example, the sample DNA to be tested is derived from a subject having or suspected of having a genetic protein S deficiency.
In another preferred example, the sample DNA to be tested is derived from a sample selected from the group consisting of: a blood sample, a saliva sample, a buccal swab, or a combination thereof.
In another preferred example, the test sample DNA is obtained by extracting DNA from a blood sample, a saliva sample, or a buccal swab.
In another preferred embodiment, in step (ii), 62 ℃ (-0.3 ℃) represents a 0.3 ℃ reduction in annealing temperature per cycle, i.e., the annealing temperature during PCR is 62 ℃ for the first cycle, followed by a 0.3 ℃ reduction in annealing temperature per cycle for 11 cycles.
In another preferred embodiment, the amplification product obtained in step (ii) contains the 1-15 exon sequences of the PROS1 gene.
In another preferred embodiment, the amplification product further comprises a partial intron sequence of the PROS1 gene.
In a fifth aspect of the present invention, there is provided a method for diagnosing a genetic protein S deficiency, comprising the steps of:
(i) providing a sample to be detected from a subject, and extracting DNA in the sample to be detected, so as to obtain the DNA of the sample to be detected;
(ii) detecting the DNA of the sample to be detected by using the kit of the third aspect of the invention, thereby obtaining the PROS1 gene exon sequence of the DNA of the sample to be detected; and
(iii) (iii) aligning the sequence obtained in step (ii) with a reference sequence of the PROS1 gene;
wherein, the existence of mutation in the PROS1 gene exon sequence of the test sample DNA compared with the PROS1 gene reference sequence indicates that the subject has hereditary protein S deficiency or the probability of hereditary protein S deficiency is higher than that of the general population.
In another preferred embodiment, the subject is a human.
In another preferred embodiment, the sample to be tested is selected from the group consisting of: a blood sample, a saliva sample, a buccal swab, or a combination thereof.
In another preferred embodiment, the general population includes healthy people.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows an electrophoretogram of PCR amplification using primers of group A (experimental group).
FIG. 2 shows an electropherogram of PCR amplification using primers from group B (control).
FIG. 3 shows the results of PCR amplification of exon 1 of the PROS1 gene in reaction systems with and without DMSO; the left side of the sample is an amplified band added with DMSO, and the right side is an amplified band without DMSO.
FIG. 4 shows the sequencing results of an intron SNP amplified using the kit of the present invention; wherein A is a partial sequencing result of an amplified PROS1 gene exon 1 sequence; b is the sequencing result of intron 1 region of the exon 2 sequence of the amplified PROS1 gene, wherein the arrow position shows the polymorphic site SNP (rs 8178610G/A) of the intron region, and the hybridization is carried out; c is the sequencing result of intron 3 region of the exon 4 sequence of the amplified PROS1 gene, wherein the arrow position shows the polymorphic site SNP (rs 8178623G/A) of the intron region, and the hybridization is carried out.
Detailed Description
The present inventors have conducted extensive and intensive studies to find a new SNP site associated with a genetic Protein S Deficiency (PSD) for the first time and have developed a corresponding detection technique. The primer pair aiming at the SNP can amplify partial intron sequences containing specific SNPs, and is favorable for realizing more comprehensive detection of PROS1 gene mutation.
In addition, the inventor researches and develops a primer pair set and a corresponding kit which are rapid, comprehensive and simple and convenient to operate and can be used for detecting the human PROS1 gene exon and the specific SNP for the first time through a large amount of screening and optimization, and a method for detecting the PROS1 gene exon and the specific SNP by using the kit. The kit comprises a plurality of pairs of primer pairs for specifically amplifying the PROS1 gene exons, and can efficiently and once amplify all the PROS1 gene exons. In addition, part of the primer pairs can amplify partial intron sequences containing specific SNPs, and the more comprehensive detection of the PROS1 gene mutation is facilitated. On the basis of this, the present invention has been completed.
Experiments show that the optimized primer pair set can be efficiently and specifically amplified under the same amplification condition, the number of amplification products is consistent, and high-quality sequencing and exon and specific SNP analysis can be conveniently carried out at the later stage. The detection method is simpler and more convenient to operate, and can meet the amplification reaction of all exons only by setting one PCR amplification program.
Term(s) for
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject.
Gene
The PROS1 gene refers to the gene encoding the protein s (ps) in humans, which has 15 exons and 14 introns, spans 101kb, and is located on the long arm of chromosome 3, position 11.1.
The mutation site of the PROS1 gene in the sample DNA is obtained by amplifying 15 exon and partial intron sequences of the PROS1 gene in the sample DNA by using a specific primer pair and comparing the sequences with a PROS1 gene reference sequence. The PROS1 gene reference sequence is the gene sequence with NCBI accession number NG _ 009813.1.
As used herein, the term "PSE" means the pro s1 gene exon, e.g., "PSE 1" is pro s1 gene exon 1, "PSE 5/6" is pro s1 gene exons 5 and 6.
Specific primer pair set for amplifying PROS1 gene exon
The invention provides a group of specific primer pair sets for amplifying PROS1 gene exons, wherein the primer pair sets comprise 1-15 pairs of primers, and the sequences of the primers are as follows:
primer pair 1: PSE1 SP: GGGATCCTGTCCTCTTGAACC (SEQ ID NO: 1) and PSE1 AP: GGCTGCAGCTCTAGAGAAGAAAA (SEQ ID NO: 2);
and (3) primer pair 2: PSE2 SP: CACCAAACCCGCTCTCTGAATTGTT (SEQ ID NO: 3) and PSE2 AP: AAGATGGAACAGAAGGAAGTA (SEQ ID NO: 4);
and (3) primer pair: PSE3 SP: TTTACCATCCTAAATAGAGC (SEQ ID NO: 5) and PSE3 AP: CCAAATTTCTCAACATGTG (SEQ ID NO: 6);
and (3) primer pair 4: PSE4 SP: GGGCAACATCTTGTTTCTGCT (SEQ ID NO: 7) and PSE4 AP: TCTGAAGCTGATAACCTTTAGGAACA (SEQ ID NO: 8);
and (3) primer pair 5: PSE5/6 SP: AGGCTCACTTTTTAATTTAG (SEQ ID NO: 9) and PSE5/6 AP: GCACTTACATATCATTTTTCC (SEQ ID NO: 10);
and (3) primer pair 6: PSE7 SP: GATTTAATGTTTTTTGGTCC (SEQ ID NO: 11) and PSE7 AP: GCCAATGCTTTTAAATATCAG (SEQ ID NO: 12);
and (3) primer pair 7: PSE8 SP: TTGTGAGGTAATCTCAATGAA (SEQ ID NO: 13) and PSE8 AP: CGTCTGTATTTTCCTGACTTAG (SEQ ID NO: 14);
and (3) primer pair 8: PSE9 SP: CATTAGTAACCAAACAAAAATG (SEQ ID NO: 15) and PSE9 AP: AATGACTCAAAAAGGTTTTAGG (SEQ ID NO: 16);
and (3) primer pair 9: PSE10 SP: TGACTCTTTGTCTGTGTTACTG (SEQ ID NO: 17) and PSE10 AP: TCAGGATGCTTCAGGGATT (SEQ ID NO: 18);
a primer pair 10: PSE11 SP: CCTAAGATAGAATGGCTCAC (SEQ ID NO: 19) and PSE11 AP: GATCATTTCAAGTTGTCACAC (SEQ ID NO: 20);
a primer pair 11: PSE12 SP: GTTAAAAACCATTAAGATCCCA (SEQ ID NO: 21) and PSE12 AP: TGGGCACACAGTAGATACTC (SEQ ID NO: 22);
primer pair 12: PSE13 SP: ATCATTGAGAAAGGGAATGG (SEQ ID NO: 23) and PSE13 AP: CCTTAATGATGTGCTGGAGAT (SEQ ID NO: 24);
a primer pair 13: PSE14 SP: TTCCCTTAAATTTAGAGGCT (SEQ ID NO: 25) and PSE14 AP: GGCTGGGATAGCCAAATGA (SEQ ID NO: 26);
primer pair 14: PSE15SP 1: CATGTATATCGGCGTCACTTAACA (SEQ ID NO: 27) and PSE15AP 1: CACCATCTCTTCTGCCTTCATC (SEQ ID NO: 28);
primer pair 15: PSE15SP 2: TGATGAAGGCAGAAGAGATG (SEQ ID NO: 29) and PSE15AP 2: AGTCTACCACCCAACCATA (SEQ ID NO: 30).
Wherein the primer pair 1 specifically amplifies PROS1 gene exon 1; the primer pair 2 specifically amplifies PROS1 gene exon 2; the primer pair 3 specifically amplifies PROS1 gene exon 3; the primer pair 4 specifically amplifies PROS1 gene exon 4; the primer pair 5 specifically amplifies PROS1 gene exon 5 and exon 6; the primer pair 6 specifically amplifies PROS1 gene exon 7; the primer pair 7 specifically amplifies PROS1 gene exon 8; the primer pair 8 specifically amplifies PROS1 gene exon 9; the primer pair 9 specifically amplifies PROS1 gene exon 10; the primer pair 10 specifically amplifies PROS1 gene exon 11; the primer pair 11 specifically amplifies PROS1 gene exon 12; the primer pair 12 specifically amplifies PROS1 gene exon 13; the primer pair 13 specifically amplifies PROS1 gene exon 14; the primer pair 14 and/or the primer pair 15 specifically amplify the PROS1 gene exon 15.
The primer pair set of the present invention can amplify a partial intron sequence of the PROS1 gene in addition to 15 exon sequences of the PROS1 gene.
Preferably, the sequence amplified by the primer pair 2 comprises a small part of the sequences of intron 1 and intron 2 in addition to the sequence of exon 2, a polymorphic site SNP (rs 8178610G/A) exists in the amplified intron 1 sequence, and a polymorphic site SNP (rs 531436627G/A) exists in the intron 2 sequence; the sequence amplified by the primer pair 4 comprises a small part of the sequences of the intron 3 and the intron 4 besides the sequence of the exon 4, and a polymorphic site SNP (rs 8178623G/A) exists in the amplified intron 3 sequence. These additional polymorphic sites, i.e., rs 8178610G/A, rs 531436627G/A, rs 8178623G/A, help to provide additional diagnostic basis for genetic Protein S Deficiency (PSD).
Therefore, the primer pair set provided by the invention can detect not only the exon of the PROS1 gene, but also part of the intron of the PROS1 gene, and the detection range is more comprehensive and wider. The primer pair can be used for preparing a kit for detecting the PROS1 gene exon, and the kit can be used for (a) detecting the PROS1 gene mutation and the mutation site thereof; and (b) diagnosing a genetic Protein S Deficiency (PSD).
Kit of the invention
The invention also provides a kit for detecting the PROS1 gene exon, which comprises: (a) a PROS1 gene reference sequence; (b) a set of specific primer pairs for amplifying an exon of the PROS1 gene; and (c) PCR amplification reagents.
In addition, the kit may further comprise (d) a label or instructions for use in detecting an exon of the PROS1 gene.
Wherein, the specific primer pair for amplifying the PROS1 gene exon is the specific primer pair 1-15 of the first aspect of the invention. The PROS1 gene reference sequence is a gene sequence with NCBI accession number NG _ 009813.1. The PCR amplification reagent comprises: 2 × Taq Master Mix, Mg2+And DMSO, ddH2O。
In the kit of the present invention, the specific primer pair for amplifying the exon of the PROS1 gene is configured as a dry powder or a liquid. The primer pairs 1 to 15 are arranged in separate containers.
Method for detecting PROS1 gene exon
The invention also provides a method for detecting the PROS1 gene exon, which comprises the following steps:
(i) providing a sample DNA to be detected;
(ii) carrying out PCR amplification on the DNA of the sample to be detected by using the primer pair set of the first aspect of the invention, thereby obtaining an amplification product containing a PROS1 gene exon sequence; and
(iii) sequencing the amplification product containing the exon sequence of the PROS1 gene, thereby obtaining sequence information of the exon sequence of the PROS1 gene; and
(iv) and (iv) comparing the sequence information obtained in the step (iii) with a PROS1 gene reference sequence, thereby obtaining the mutation site of the PROS1 gene in the DNA of the sample to be detected.
Wherein in step (ii), the PCR amplification comprises the steps of:
(i) respectively preparing a class I PCR reaction system R1 and a class II PCR reaction system R2;
wherein, the I-type PCR reaction system R1 contains the following components: 10ng-1 mug sample to be tested, 0.1-0.5 mug primer pair 1, 2 XTaq Master Mix, 0.5-2mM Mg2+1-4% by volume of DMSO and ddH2O；
The type II PCR reaction system R2 contains the following components: 10ng-1 ug of sample DNA to be tested, 0.1-0.5 uM of primer pair 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, 2 XTaq Master Mix, 0.5-2mM Mg2+And ddH 2O; and
(ii) the PCR reaction systems R1 and R2 were subjected to PCR amplification according to the following procedure:
5min at 95 ℃; the following procedure was performed for 12 cycles: 25s at 94 ℃, 40s at 62 ℃ (-0.3 ℃), 45s at 72 ℃; the following procedure was performed for 24 cycles: 25s at 94 ℃, 30s at 58 ℃ and 45s at 72 ℃; 5min at 72 ℃; 25 ℃ for 25 s.
Thereby obtaining amplification products of nucleic acid sequences containing exons 1, 2, 3, 4, 5 and 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, respectively, of the PROS1 gene.
Wherein 62 ℃ (-0.3 ℃) represents 0.3 ℃ reduction in temperature per cycle, i.e., the annealing temperature during PCR is 62 ℃ for the first cycle, and then 0.3 ℃ reduction in annealing temperature per cycle for 11 cycles.
In another preferred embodiment, the class I PCR reaction system R1 is formulated as follows: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, DMSO 0.8. mu.L, plus ddH2O to the total volume of 20 mu L;
wherein, the upstream primer and the downstream primer are the upstream primer and the downstream primer of the primer pair 1.
In another advantageIn an alternative example, the class II PCR reaction system R2 is prepared according to the following method: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, plus ddH2O to the total volume of 20 mu L;
wherein the upstream primer and the downstream primer comprise upstream primers and downstream primers of primer pairs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 shown in Table A.
The main advantages of the invention are:
(1) high efficiency and comprehensiveness: the kit can detect 15 exon sequences of the PROS1 gene at one time and can detect partial intron sequences of the PROS1 gene;
(2) the sensitivity is high: the kit can be used for detecting PROS1 gene exons of genomic DNA with lower concentration;
(3) the specificity is strong: the kit of the invention designs 15 pairs of specific primers, thus effectively improving the specificity of detection;
(4) the accuracy is high: the method analyzes the specific fragment amplified by the PCR by a sequencing method, and ensures the accuracy of the result;
(5) simple and quick: the kit is simple and convenient to operate, 15 pairs of primers are separately packaged, and only 2 xMix, the genome DNA and H of a sample to be detected2And O is mixed and added into the PCR reaction tube, so that the reaction error is greatly reduced.
(6) Can be efficiently and specifically amplified under the same amplification condition, and the number of amplification products is consistent, thereby being convenient for high-quality sequencing and analysis in the later period.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.
Example 1
Preparation of test samples
In this example, a sample from the oral cavity is taken as an example to describe a preparation method of a test sample.
Extraction of buccal swab genomic DNA was performed as follows:
1. oral sampling
The sampling adopted is an oral swab sampling method, and a sampling reagent comprises two parts: oral swab, saliva preserving fluid.
About 15 minutes before sampling, advising the patient to rinse the mouth with clear water to remove impurities such as food residues in the oral cavity; after the gargling of the patient is confirmed, the oral swab package is opened, and oral cells are uniformly scraped from the inner wall of the oral cavity (the times are about 30 times).
The preservation tube cover containing the saliva preservation solution is opened, the swab is placed and broken along the swab head, the cover is closed, and corresponding marks are made for subsequent detection. (the collected sample can be stored for 1 week at room temperature)
2. Sample DNA extraction
DNA extraction was performed using a nucleic acid extraction kit (magnetic bead method) from the Molerian science and technology (Shanghai) Co., Ltd. The method comprises the following steps:
(1) and placing the preservation tube with the collected sample on a constant-temperature shaking and mixing instrument, and selecting parameters of 56 ℃, 15min and 800rpm/min to carry out pretreatment on the oral swab sample.
(2) Opening the nucleic acid extraction kit, taking out the calandria, shaking up and down for 6-10 times to uniformly mix the magnetic beads in the seventh hole of the calandria, and carefully tearing off the sealing film;
(3) and adding 400 mu L of sample (the sample stored in the storage tube is 400 mu L) into the first hole, adding 80 mu L of precipitation buffer into the ninth hole, placing the calandria into a drawer of the CTC-2000 nucleic acid extraction instrument, installing a magnetic rod sleeve, and selecting a program to run.
(4) After the program is finished, collecting the extracted DNA at the ninth hole, and carrying out subsequent experiments or storing at-20 ℃.
Example 2
SNP loci associated with genetic Protein S Deficiency (PSD)
Based on genomic sequencing data (about 177) for clinical PSD patients (127 cases) and normal persons (50 cases), the present invention performed SNP locus correlation analysis on the PROS1 genomic sequence (including introns and exons).
The result shows that two SNP sites of SNP (rs 8178610G/A) and SNP (rs 8178623G/A) are related to PSD pathogenesis or susceptibility, and the two SNP sites are SNP sites in the PROS1 gene intron region.
Example 3
Verification of PSD-related SNP
In this example, primers for SNP (rs 8178610G/A) and SNP (rs 8178623G/A) were designed and 33 samples were used for validation. The basic cases for the 33 samples (cases) of this validation set are as follows: protein S activity was slightly reduced to about 70% of normal human protein S activity.
The primer pair used for amplifying the SNP (rs 8178610G/A) was primer pair 2 (SEQ ID NOS: 3 and 4).
The primer pair used for amplifying the SNP (rs 8178623G/A) was primer pair 4 (SEQ ID NOS: 7 and 8).
And respectively carrying out PCR amplification by using the primers by using the sample DNA as a template, and sequencing and analyzing the amplified products.
Sequencing results show that the sequences amplified by using the primer pair 2 of the invention comprise the sequence of the PROS1 exon 2 and the sequence of a part of PROS1 intron 1, wherein a polymorphic site SNP (rs 8178610G/A) exists in the intron 1 region, and the sequences are heterozygous (shown in B in figure 4).
Among the sequences amplified using primer pair 4 of the present invention, the sequence comprising exon 4 of PROS1 and a part of intron 3 of PROS1, a polymorphic site SNP (rs 8178623G/A) was present in the intron 3 region of PROS1, and they were heterozygous (shown in FIG. 4C).
An exemplary sequencing profile for SNPs without polymorphic sites is shown in fig. 4, panel a.
Further, statistics were made on the number of SNPs occurring at polymorphic sites in 33 samples examined, and the results are shown in Table 1 below.
TABLE 1
According to the statistical result, the following results are obtained: a total of 33 samples were tested, and the mutation rate of the SNP (rs 8178610) was 75.76%, wherein heterozygous mutations accounted for 51.52% of the total population; the mutation rate of the SNP (rs 8178623) is 75.76%, wherein the heterozygous mutation accounts for 48.48% of the total population.
The results show that SNP (rs 8178610G/A) and SNP (rs 8178623G/A) can be related to genetic Protein S Deficiency (PSD), and when SNP (rs 8178610G/A) or SNP (rs 8178623G/A) is detected, the probability that the detected object has genetic Protein S Deficiency (PSD) is high.
Meanwhile, 10 normal samples are subjected to sequencing analysis, and the sequencing result shows that the sequences obtained by amplification by using the primer pair 2 of the invention comprise a sequence of PROS1 exon 2 and a part of sequence of PROS1 intron 1, wherein the SNP (rs 8178610G/A) site of an intron 1 region shows that 10 samples are all wild homozygous (GG) and have no mutation; among the sequences amplified using primer pair 4 of the present invention, the sequence including exon 4 of PROS1 and a part of intron 3 of PROS1 showed that 10 samples were wild homozygous (GG) and were not mutated at the SNP (rs 8178623G/A) site in intron 3 region of PROS 1.
Example 4
Amplification of PROS1 for exon detection and SNP detection of samples
1. Configuring a reaction system
(1) Preparing a primer solution with the concentration of 10 mu M, wherein the primer solution comprises the following primer pairs 1-15:
primer pair 1: PSE1 SP: GGGATCCTGTCCTCTTGAACC (SEQ ID NO: 1) and PSE1 AP: GGCTGCAGCTCTAGAGAAGAAAA (SEQ ID NO: 2);
and (3) primer pair 2: PSE2 SP: CACCAAACCCGCTCTCTGAATTGTT (SEQ ID NO: 3) and PSE2 AP: AAGATGGAACAGAAGGAAGTA (SEQ ID NO: 4);
and (3) primer pair: PSE3 SP: TTTACCATCCTAAATAGAGC (SEQ ID NO: 5) and PSE3 AP: CCAAATTTCTCAACATGTG (SEQ ID NO: 6);
and (3) primer pair 4: PSE4 SP: GGGCAACATCTTGTTTCTGCT (SEQ ID NO: 7) and PSE4 AP: TCTGAAGCTGATAACCTTTAGGAACA (SEQ ID NO: 8);
and (3) primer pair 5: PSE5/6 SP: AGGCTCACTTTTTAATTTAG (SEQ ID NO: 9) and PSE5/6 AP: GCACTTACATATCATTTTTCC (SEQ ID NO: 10);
and (3) primer pair 6: PSE7 SP: GATTTAATGTTTTTTGGTCC (SEQ ID NO: 11) and PSE7 AP: GCCAATGCTTTTAAATATCAG (SEQ ID NO: 12);
and (3) primer pair 7: PSE8 SP: TTGTGAGGTAATCTCAATGAA (SEQ ID NO: 13) and PSE8 AP: CGTCTGTATTTTCCTGACTTAG (SEQ ID NO: 14);
and (3) primer pair 8: PSE9 SP: CATTAGTAACCAAACAAAAATG (SEQ ID NO: 15) and PSE9 AP: AATGACTCAAAAAGGTTTTAGG (SEQ ID NO: 16);
and (3) primer pair 9: PSE10 SP: TGACTCTTTGTCTGTGTTACTG (SEQ ID NO: 17) and PSE10 AP: TCAGGATGCTTCAGGGATT (SEQ ID NO: 18);
a primer pair 10: PSE11 SP: CCTAAGATAGAATGGCTCAC (SEQ ID NO: 19) and PSE11 AP: GATCATTTCAAGTTGTCACAC (SEQ ID NO: 20);
a primer pair 11: PSE12 SP: GTTAAAAACCATTAAGATCCCA (SEQ ID NO: 21) and PSE12 AP: TGGGCACACAGTAGATACTC (SEQ ID NO: 22);
primer pair 12: PSE13 SP: ATCATTGAGAAAGGGAATGG (SEQ ID NO: 23) and PSE13 AP: CCTTAATGATGTGCTGGAGAT (SEQ ID NO: 24);
a primer pair 13: PSE14 SP: TTCCCTTAAATTTAGAGGCT (SEQ ID NO: 25) and PSE14 AP: GGCTGGGATAGCCAAATGA (SEQ ID NO: 26);
primer pair 14: PSE15SP 1: CATGTATATCGGCGTCACTTAACA (SEQ ID NO: 27) and PSE15AP 1: CACCATCTCTTCTGCCTTCATC (SEQ ID NO: 28);
primer pair 15: PSE15SP 2: TGATGAAGGCAGAAGAGATG (SEQ ID NO: 29) and PSE15AP 2: AGTCTACCACCCAACCATA (SEQ ID NO: 30).
(2) The DNA of the sample to be tested prepared in example 1 was used at a concentration of 10-100 ng/. mu.L;
(3) a20. mu.l reaction was prepared as follows:
reaction system of PSE 1: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, DMSO 0.8. mu.L, plus ddH2O to the total volume of 20 mu L;
reaction system of PSE2, PSE3, PSE4, PSE5/6, PSE7, PSE8, PSE9, PSE10, PSE11, PSE12, PSE13, PSE14, PSE 15: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, plus ddH2O to a total volume of 20. mu.L.
PCR amplification
PCR amplification was performed according to the following procedure: 5min at 95 ℃; the following procedure was performed for 12 cycles: 25s at 94 ℃, 40s at 62 ℃ (-0.3 ℃), 45s at 72 ℃; the following procedure was performed for 24 cycles: 25s at 94 ℃, 30s at 58 ℃ and 45s at 72 ℃; 5min at 72 ℃; 25 ℃ for 25 s.
4. Sequencing
And after the running of the PCR program is finished, sequencing the PCR product.
5. Analysis of results
And (4) analyzing the mutation condition of the PROS1 gene by aligning the sequencing result with a known sequence.
For exon 1 of the PROS1 gene (PSE 1), no significant band could be amplified under the same conditions when no DMSO was added to the reaction system (FIG. 3).
Comparative example 1
Comparison of the kit of the present invention with the existing primer set 1
A, B two experiments were performed simultaneously, and 15 pairs of PROS1 exon primers (group A, experimental group) in the kit and the exon primer set 1 (group B, control group) of PROS1 in the literature were used, respectively, and the sample to be tested was the DNA prepared in example 1.
The exon primer sequences for PROS1 in the literature are shown below:
PSE1F：TGTTATCACTTCCCCTCTCG (SEQ ID NO：31)
PSE1R：TAGGAGCTGCAGCTCTAGA (SEQ ID NO：32)
PSE2F：GTCATACAATTCATAGGCAG (SEQ ID NO：33)
PSE2R：CAGAAGGAAGTACAGGCTGG (SEQ ID NO：34)
PSE3F：AGGTTTGCTAAGATATGTTTT (SEQ ID NO：35)
PSE3R：AGAGTTAGACAGGAACATAT (SEQ ID NO：36)
PSE4F：CCATGAATTCAGATCAAGTA (SEQ ID NO：37)
PSE4R：GGTGTACTTTACCTACAGAG (SEQ ID NO：38)
PSE5/6F：GGCTTCAGGATTTTTATTATAGTA (SEQ ID NO：39)
PSE5/6R：CTAACTGGGATTATTCTCACAT (SEQ ID NO：40)
PSE7F：CACAAATCAAGGGTTCTTTGG (SEQ ID NO：41)
PSE7R：GATCAGTAATGATACCACCA (SEQ ID NO：42)
PSE8F：ATAAGATTGAACATTTAGGG (SEQ ID NO：43)
PSE8R：CAGGTGAGAAGTTAAGCATT (SEQ ID NO：44)
PSE9F：TAGTAACCAAACAAAAATGC (SEQ ID NO：45)
PSE9R：CCCTTATCTGCTTAACCTCT (SEQ ID NO：46)
PSE10F：AGCTTTCTGTATTTCTTACTC (SEQ ID NO：47)
PSE10R：ACAGACTGCATCAAAGTGGG (SEQ ID NO：48)
PSE11F：GTAATACTTGGTTATTTGGTAAT (SEQ ID NO：49)
PSE11R：CACACATATTCAAATCTATTAC (SEQ ID NO：50)
PSE12F：CCTATACTCATAATCGAGCC (SEQ ID NO：51)
PSE12R：TGGGCACACAGTAGATACTC (SEQ ID NO：52)
PSE13F：CTGATGCACTTTAGGAGTGC (SEQ ID NO：53)
PSE13R：GTAAATACTGCTATGTATAC (SEQ ID NO：54)
PSE14F：GCTTATATTGAATCTTTGCTCTC (SEQ ID NO：55)
PSE14R：ATATGCCAATAAATGTCGGT (SEQ ID NO：56)
PSE15F：CAAGATGCTAAAAGTCTTGG (SEQ ID NO：57)
PSE15R：GATAGCAAGAGAAGTAAGAATTTC(SEQ ID NO：58)
1. preparation of primers
The primers provided by the invention are prepared to be 10 mu M in concentration, and the exon primers of PROS1 in the literature are prepared to be 10 mu M in concentration.
2. Preparing a reaction system
Group a (experimental group) the reaction system was prepared as follows:
reaction system of PSE 1: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, DMSO 0.8. mu.L, plus ddH2O to the total volume of 20 mu L;
reaction system of PSE2, PSE3, PSE4, PSE5/6, PSE7, PSE8, PSE9, PSE10, PSE11, PSE12, PSE13, PSE14, PSE 15: adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, plus ddH2O to a total volume of 20. mu.L.
Group B (control) the reaction system was prepared as follows:
adding 2 XTaq Master Mix10 μ L, upstream primer (10 μ M) 0.5 μ L, downstream primer (10 μ M) 0.5 μ L, sample DNA 10ng-1 μ g to be tested, Mg2+(25mM) 1. mu.L, plus ddH2O to a total volume of 20. mu.L.
PCR amplification
Experimental group (group a) PCR amplification was performed according to the PCR amplification conditions described in example 2.
The control group (group B) was PCR amplified according to the following PCR amplification conditions:
PSE11, PSE13, PSE 15: 5min at 95 ℃; the following procedure was followed for 36 cycles: 30s at 95 ℃, 30s at 54 ℃ and 30s at 72 ℃;10 min at 72 ℃;
PSE3, PSE8, PSE 10: 5min at 95 ℃; the following procedure was followed for 36 cycles: 30s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃;10 min at 72 ℃;
PSE1, PSE2, PSE4, PSE7, PSE 9: 5min at 95 ℃; the following procedure was followed for 36 cycles: 30s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃;10 min at 72 ℃;
PSE5/6, PSE12, PSE 14: 5min at 95 ℃; the following procedure was followed for 36 cycles: 30s at 95 ℃, 30s at 62 ℃ and 30s at 72 ℃;10 min at 72 ℃.
Results
The amplification products obtained by PCR amplification of group A (experimental group) and group B (control group) were subjected to agarose gel electrophoresis, and the results are shown in FIGS. 1 and 2, respectively.
Using the same template, PCR amplification was performed for group a (experimental group) and group B (control group) using the PCR amplification conditions described above, respectively, and it can be seen that:
(1) the group A (experimental group) can not only amplify the whole exon region of PROS1, but also amplify the region corresponding to the specific SNP, namely SNP (rs 8178610G/A) and SNP (rs 8178623G/A), and can be used for auxiliary diagnosis of PSD. In contrast, although most of the exon regions of PROS1 were amplified in group B (control), some of the exon regions were not amplified, and the amplified region in group B did not include the SNP (rs 8178610G/a) and SNP (rs 8178623G/a) sites, and thus did not provide information about these specific SNPs.
(2) Group B is more complex to operate than group A, only 1 PCR program is required for 15 exons of group A, while 4 different PCR programs are required for group B;
(3) as can be seen from the two electrophoretograms shown in FIG. 1 and FIG. 2, the amplification effect of each exon in group A is obviously better than that of group B, the band is clearer and more obvious, and the requirement of sequencing is better met;
(4) for each exon, the amplified fragments of group A are generally larger than those of group B, and the primers of group A can detect the complete exon sequences and partial intron sequences, while group B can detect only partial exon sequences.
The lengths of the amplified fragments obtained by amplifying the groups A and B are shown in Table 2 below.
TABLE 2
Comparative example 2
Comparison of the kit of the present invention with the existing primer set 2
Another set of primer pairs 2 (see Wenman Wu et al, Haematologica. 2021 Jan 1;106(1): 264-268.) disclosed for amplification of the exon of the PROS1 gene was subjected to sequence analysis and compared with the set of primer pairs used in the kit of the present invention.
The sequence information and analysis results of the primers in the primer set 2 are shown in the following table 3.
TABLE 3
The primers and amplification products shown in Table 3 were analyzed, and the results showed that:
(a) the primer pair set 2 is used for amplifying PROS1 gene exon, SNP (rs 8178610G/A) and SNP (rs 8178623G/A) sites cannot be detected in a targeted mode, and therefore information of specific SNPs cannot be provided.
(b) The primers shown in Table 3 have large length differences (the longest primer 33nt is different from the shortest primer 18nt by 15nt), and the GC content fluctuates greatly. The primer disclosed by the invention (the difference between the longest primer 26nt and the shortest primer 19nt is only 7nt), the GC content fluctuation is small, and the GC value is relatively uniform.
(c) The amplification products using primer set 2 are not conducive to sequencing analysis. The primer pair set 2 is used for amplifying PROS1 gene exons, and the obtained amplification products have great length difference with each other. The longest amplification product (4808 bp) and the shortest amplification product (445 bp) differ in length by a factor of 10, and there are many amplification products of larger lengths. This primer design approach does not or substantially does not allow amplification of a set of primer pairs under the same PCR procedure, and it takes a long time to amplify certain products of larger length. In addition, amplification products with non-uniform lengths are not conducive to subsequent sequencing analysis, and some amplification products (e.g., 4808bp amplification products) need to be segmented and then spliced, which will increase sequencing cost and reduce sequencing efficiency and quality.
(d) Comparison of PCR reaction System and reaction procedure
The PCR reaction system and reaction procedure used for the primer set of the present invention (example 4) and the primer set 2 (comparative example 2) were compared.
Regarding the reaction system, two reaction systems were used for primer set 2:
the use of primers E2-3, 5-6, 7-8, 9, 11, 12-13, and 14 included 1 XPCR bufferWash, 0.2. mu.M of each of the upstream and downstream primers, 50 ng of sample DNA, 0.2mM dNTP, 5mM MgCl2And 0.3U Hotstar Taq DNA polymerase, the total volume of which is 10 mu L; for the primer pairs E1-1, E4, E10, E15-1 and E15-2, a reaction system comprising 1 XGC buffer I, 0.2mM dNTP, 0.2. mu.M each of the upstream and downstream primers, 1U of HotStarTaq polymerase and 50 ng of sample DNA in a total volume of 20. mu.L was used.
Regarding the reaction procedure, two reaction procedures were used for primer set 2:
for primer pairs E5-6, 7-8 and 12-13, the reaction program was 95 ℃ for 15min, and the following program was run for 35 cycles: at 96 deg.C for 10s, at 68 deg.C for 6min, and then at 72 deg.C for 2 min; for the other primer pairs, the reaction program was 95 ℃ for 15min, and the following program was run for 11 cycles: 94 ℃ 20s, 62 ℃ (1 ℃ reduction per cycle) 40s, 72 ℃ 2min, then 24 cycles with the following procedure: 94 ℃ for 20s, 56 ℃ for 30s, 72 ℃ for 2min, and finally 72 ℃ for 2 min.
As can be seen, primer set 2 is more complex in formulating the reaction system and formulating the reaction procedure. Although the primer pair set of the present invention also requires the preparation of two reaction systems, only one primer pair needs to be prepared separately, and all primer pairs can be subjected to PCR amplification under the same reaction procedure. Furthermore, the amplification time required using the primer set and method of the present invention is about 1.2 hours, whereas with primer set 2, about 4 hours is required to complete amplification of all exons.
Therefore, the primer pair set greatly improves the efficiency of amplification reaction, simplifies the operation process and is more convenient in practical application.
In conclusion, the kit of the invention can amplify and detect the novel PSD-related SNP sites, can detect 15 exon sequences of the PROS1 gene at one time, has longer length of the amplified fragment compared with the existing primer set 1, has more uniform length compared with the existing primer set 2, and has the advantages of high efficiency, comprehensiveness, high specificity, high sensitivity and the like.
In addition, unexpectedly, the 15 primer pairs can be efficiently and specifically amplified under the same PCR amplification condition, the number of amplification products is consistent, the quality of amplified sequences is better, the number of amplified sequences is more, the detection sensitivity and specificity are greatly improved, and high-quality sequencing and analysis can be conveniently carried out in the later period.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Thymus elegans medical science and technology (Shanghai) Co., Ltd
<120> kit and method for detecting PROS1 gene exon
<130> P2021-0543
<160> 84
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
gggatcctgt cctcttgaac c 21
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ggctgcagct ctagagaaga aaa 23
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
caccaaaccc gctctctgaa ttgtt 25
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
aagatggaac agaaggaagt a 21
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
tttaccatcc taaatagagc 20
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
ccaaatttct caacatgtg 19
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
gggcaacatc ttgtttctgc t 21
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
tctgaagctg ataaccttta ggaaca 26
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
aggctcactt tttaatttag 20
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
gcacttacat atcatttttc c 21
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
gatttaatgt tttttggtcc 20
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
gccaatgctt ttaaatatca g 21
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
ttgtgaggta atctcaatga a 21
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
cgtctgtatt ttcctgactt ag 22
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
cattagtaac caaacaaaaa tg 22
<210> 16
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
aatgactcaa aaaggtttta gg 22
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
tgactctttg tctgtgttac tg 22
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tcaggatgct tcagggatt 19
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
cctaagatag aatggctcac 20
<210> 20
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
gatcatttca agttgtcaca c 21
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
gttaaaaacc attaagatcc ca 22
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
tgggcacaca gtagatactc 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
atcattgaga aagggaatgg 20
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
ccttaatgat gtgctggaga t 21
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
ttcccttaaa tttagaggct 20
<210> 26
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
ggctgggata gccaaatga 19
<210> 27
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
catgtatatc ggcgtcactt aaca 24
<210> 28
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
caccatctct tctgccttca tc 22
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
tgatgaaggc agaagagatg 20
<210> 30
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
agtctaccac ccaaccata 19
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
tgttatcact tcccctctcg 20
<210> 32
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
taggagctgc agctctaga 19
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
gtcatacaat tcataggcag 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
cagaaggaag tacaggctgg 20
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
aggtttgcta agatatgttt t 21
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
agagttagac aggaacatat 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
ccatgaattc agatcaagta 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
ggtgtacttt acctacagag 20
<210> 39
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
ggcttcagga tttttattat agta 24
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
ctaactggga ttattctcac at 22
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
cacaaatcaa gggttctttg g 21
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
gatcagtaat gataccacca 20
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
ataagattga acatttaggg 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
caggtgagaa gttaagcatt 20
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
tagtaaccaa acaaaaatgc 20
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
cccttatctg cttaacctct 20
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
agctttctgt atttcttact c 21
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
acagactgca tcaaagtggg 20
<210> 49
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
gtaatacttg gttatttggt aat 23
<210> 50
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
cacacatatt caaatctatt ac 22
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
cctatactca taatcgagcc 20
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
tgggcacaca gtagatactc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
ctgatgcact ttaggagtgc 20
<210> 54
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 54
gtaaatactg ctatgtatac 20
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 55
gcttatattg aatctttgct ctc 23
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 56
atatgccaat aaatgtcggt 20
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 57
caagatgcta aaagtcttgg 20
<210> 58
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 58
gatagcaaga gaagtaagaa tttc 24
<210> 59
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
attcccttct tttcttcatg ttct 24
<210> 60
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ccacccagga ccctcattt 19
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tccgaaaagc ttcctggaaa tg 22
<210> 62
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cgctgggtgt ctgtcggtac t 21
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
caccaaaccc gctctctgaa ttgtt 25
<210> 64
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
aacagtggaa ttcaccacac atgccta 27
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
atttgagggc aacatcttgt ttctg 25
<210> 66
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ttgggaataa aaattctgaa gctgata 27
<210> 67
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
atttaaagaa aggagttgtg tgttttt 27
<210> 68
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
agatgaaggg ctgatgttgg a 21
<210> 69
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tggtccaaag gccaatctgt t 21
<210> 70
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
cagaacgtct gtattttcct gacttagc 28
<210> 71
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
acgtaattct cctcaaaata tcctca 26
<210> 72
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
cttcatttct gaactcaatc taagtacg 28
<210> 73
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cctggggaaa agattccaag a 21
<210> 74
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ttggcaattg aggagttttc c 21
<210> 75
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ggcgggtgga tcatgagg 18
<210> 76
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
ggcacacagt agatactcaa taatgtttca ca 32
<210> 77
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gtgaatgtgc aagggagaag g 21
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gtactcggat ggctccaagc 20
<210> 79
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
tccggttgtt ttacggaatt t 21
<210> 80
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tcgcaaggtc aggagatcaa g 21
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
tcggcgtcac ttaacatgta ttt 23
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gcaatcttac ctccttactt ctttg 25
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ctgcagtctg tcaggatgag at 22
<210> 84
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
aatggccctg ttatttggag t 21
Claims (10)
1. A set of primer pairs specific for amplifying an exon of the PROS1 gene, wherein the set of primer pairs comprises:
(Z2) primer pair 2 comprising the sequence set forth in SEQ ID NO: 3 and the upstream primer shown as SEQ ID NO: 4, a downstream primer; and
(Z4) primer pair 4 comprising the sequence set forth in SEQ ID NO:7 and the upstream primer shown as SEQ ID NO: 8, a downstream primer;
wherein, the sequence amplified by the primer pair 2 has a polymorphism site SNP1 related to the deficiency of the genetic protein S: rs 8178610G/A; the sequence amplified by using the primer pair 4 has a polymorphism site SNP2 related to the deficiency of the genetic protein S: rs 8178623G/A.
2. The set of primer pairs of claim 1, further comprising a primer pair selected from the group consisting of:
(Z1) primer pair 1, comprising the sequence set forth in SEQ ID NO: 1 and the upstream primer shown as SEQ ID NO: 2, or a reverse primer;
(Z3) primer pair 3, comprising the sequence set forth in SEQ ID NO: 5 and the upstream primer shown as SEQ ID NO: 6 as shown in the figure;
(Z5) primer pair 5 comprising the sequence set forth in SEQ ID NO: 9 and the upstream primer shown as SEQ ID NO: 10, a downstream primer;
(Z6) primer pair 6 comprising the sequence set forth in SEQ ID NO: 11 and the upstream primer shown as SEQ ID NO: 12, a downstream primer;
(Z7) primer pair 7, comprising the sequence set forth in SEQ ID NO: 13 and the upstream primer shown as SEQ ID NO: 14, a downstream primer;
(Z8) primer pair 8 comprising the sequence set forth in SEQ ID NO: 15 and the sequence shown in SEQ ID NO: 16, or a reverse primer;
(Z9) primer pair 9 comprising the sequence set forth in SEQ ID NO: 17 and the upstream primer shown as SEQ ID NO: 18, a downstream primer;
(Z10) primer pair 10 comprising the sequence set forth in SEQ ID NO: 19 and the upstream primer shown as SEQ ID NO: 20, a downstream primer;
(Z11) primer pair 11 comprising the sequence set forth in SEQ ID NO: 21 and the upstream primer shown as SEQ ID NO: 22;
(Z12) primer pair 12 comprising the sequence set forth in SEQ ID NO: 23 and the upstream primer shown as SEQ ID NO: 24, a downstream primer;
(Z13) primer pair 13 comprising the sequence set forth in SEQ ID NO: 25 and the sequence shown in SEQ ID NO: 26, a downstream primer;
(Z14) primer pair 14 comprising the sequence set forth in SEQ ID NO: 27 and the upstream primer shown as SEQ ID NO: 28, a downstream primer;
(Z15) primer pair 15 comprising the sequence set forth in SEQ ID NO: 29 and the upstream primer shown in SEQ ID NO: 30, or a reverse primer as shown.
3. The primer set of claim 2, wherein the primer set comprises 15 primers of primer pairs 1-15.
4. Use of a set of primer pairs according to any one of claims 1 to 3 for the preparation of a diagnostic reagent or kit for:
(a) detecting PROS1 gene mutation and mutation sites thereof; and/or
(b) Diagnosing a genetic protein S deficiency.
5. A kit for detecting the exon of the PROS1 gene, which is characterized by comprising: the primer pair set of any one of claims 1-3 and instructions for use.
6. The kit of claim 5, wherein the kit further comprises PCR amplification reagents comprising: 2 × Taq Master Mix, Mg2+DMSO, and ddH2O。
7. A non-diagnostic method for detecting an exon of the PROS1 gene, comprising the steps of:
(S1) providing a sample DNA to be tested;
(S2) performing PCR amplification on the test sample DNA using the primer set according to any one of claims 1 to 3, thereby obtaining an amplification product containing a sequence of an exon of the PROS1 gene; and
(S3) sequencing the amplification product containing the pro S1 gene exon sequence, thereby obtaining sequence information of the pro S1 gene exon sequence; and
(S4) comparing the sequence information obtained in the step (S3) with a reference sequence of the PROS1 gene, thereby obtaining a mutation site of the PROS1 gene in the test sample DNA.
8. The method of claim 7, wherein in step (S2), the PCR amplification comprises the steps of:
(i) respectively preparing a class I PCR reaction system R1 and a class II PCR reaction system R2;
wherein R1 contains the following components: 10ng-1 mug sample to be tested, 0.1-0.5 mug primer pair 1, 2 XTaq Master Mix, 0.5-2mM Mg2+1-4% by volume of DMSO and ddH2O；
R2 contains the following components: 10ng-1 ug of sample to be tested, 0.1-0.5 uM of primer pair 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, 2 XTaq Master Mix, 0.5-2mM Mg2+And ddH2O; and
(ii) PCR amplification was performed for R1 and R2 according to the following procedure:
5min at 95 ℃; the following procedure was performed for 12 cycles: 25s at 94 ℃, 40s at 62 ℃ (-0.3 ℃), 45s at 72 ℃; the following procedure was performed for 24 cycles: 25s at 94 ℃, 30s at 58 ℃ and 45s at 72 ℃; 5min at 72 ℃; 25 ℃ for 25 s;
wherein 62 ℃ (-0.3 ℃) represents 0.3 ℃ reduction in temperature per cycle, i.e. the annealing temperature in the first cycle is 62 ℃ during PCR, and the annealing temperature is reduced by 0.3 ℃ for 11 cycles after each cycle;
thereby obtaining amplification products of nucleic acid sequences containing exons 1, 2, 3, 4, 5 and 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, respectively, of the PROS1 gene.
9. The method of claim 7, wherein the test sample DNA is derived from a sample selected from the group consisting of: a blood sample, a saliva sample, a buccal swab, or a combination thereof.
10. The method of claim 7, wherein the test sample DNA is derived from a subject having or suspected of having a genetic protein S deficiency.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111109673.7A CN113549689B (en) | 2021-09-23 | 2021-09-23 | Kit and method for detecting PROS1 gene exon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111109673.7A CN113549689B (en) | 2021-09-23 | 2021-09-23 | Kit and method for detecting PROS1 gene exon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113549689A true CN113549689A (en) | 2021-10-26 |
| CN113549689B CN113549689B (en) | 2021-12-24 |
Family
ID=78134563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111109673.7A Active CN113549689B (en) | 2021-09-23 | 2021-09-23 | Kit and method for detecting PROS1 gene exon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113549689B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657243A (en) * | 2022-05-12 | 2022-06-24 | 广州知力医学诊断技术有限公司 | Primer and kit for detecting genetic anticoagulant protein deficiency and fibrinogen abnormal high-frequency gene mutation |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996021042A2 (en) * | 1995-01-04 | 1996-07-11 | Trustees Of Boston University | Primers for the pcr amplification of metastatic sequences |
| CN106591461A (en) * | 2016-12-29 | 2017-04-26 | 天津协和华美医学诊断技术有限公司 | Detection kit for detecting hereditary thrombophilia related gene group |
| CN107012238A (en) * | 2017-05-04 | 2017-08-04 | 上海交通大学医学院附属瑞金医院 | Easy thrombus related gene mutation detection kit |
| CN110714077A (en) * | 2019-08-13 | 2020-01-21 | 上海艾汭得基因科技有限公司 | Primer for simultaneously detecting BRCA1/2 exon sequence and chemotherapy drug site and application |
| CN110951871A (en) * | 2019-11-21 | 2020-04-03 | 广州瑞博奥生物科技有限公司 | PCA3 and PSA RNA detection kit and amplification system |
-
2021
- 2021-09-23 CN CN202111109673.7A patent/CN113549689B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996021042A2 (en) * | 1995-01-04 | 1996-07-11 | Trustees Of Boston University | Primers for the pcr amplification of metastatic sequences |
| CN106591461A (en) * | 2016-12-29 | 2017-04-26 | 天津协和华美医学诊断技术有限公司 | Detection kit for detecting hereditary thrombophilia related gene group |
| CN107012238A (en) * | 2017-05-04 | 2017-08-04 | 上海交通大学医学院附属瑞金医院 | Easy thrombus related gene mutation detection kit |
| CN110714077A (en) * | 2019-08-13 | 2020-01-21 | 上海艾汭得基因科技有限公司 | Primer for simultaneously detecting BRCA1/2 exon sequence and chemotherapy drug site and application |
| CN110951871A (en) * | 2019-11-21 | 2020-04-03 | 广州瑞博奥生物科技有限公司 | PCA3 and PSA RNA detection kit and amplification system |
Non-Patent Citations (6)
| Title |
|---|
| MASAYA HIROSE等: ""Protein S Gene Mutation in a Young Woman with Type III Protein S Deficiency and Venous Thrombosis During Pregnancy"", 《JOURNAL OF THROMBOSIS AND THROMBOLYSIS 》 * |
| P. H. REITSMA等: ""Three Novel Mutations in Five Unrelated Subjects with Hereditary Protein S Deficiency Type I"", 《J CLIN INVEST》 * |
| SEBASTIAN DUEBGEN等: ""Genotype and Laboratory and Clinical Phenotypes of Protein S Deficiency"", 《AM J CLIN PATHOL》 * |
| WENMAN WU等: ""Factor IX alteration p.Arg338Gln (FIX Shanghai) potentiates FIX clotting activity and causes thrombosis"", 《HAEMATOLOGICA》 * |
| 叶絮等: ""蛋白S基因突变导致深静脉血栓形成的研究"", 《南方医科大学学报》 * |
| 金佩佩等: ""两个终止密码子导致的遗传性蛋白C和蛋白S缺陷症"", 《诊断学理论与实践》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657243A (en) * | 2022-05-12 | 2022-06-24 | 广州知力医学诊断技术有限公司 | Primer and kit for detecting genetic anticoagulant protein deficiency and fibrinogen abnormal high-frequency gene mutation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113549689B (en) | 2021-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiang et al. | A polymorphism in the promoter region of catalase is associated with blood pressure levels | |
| CN110541025B (en) | Detection method, primer composition and kit for Duchenne muscular dystrophy gene defect | |
| Jin et al. | Detecting genetic variations in hereditary retinal dystrophies with next-generation sequencing technology | |
| CN115141884B (en) | Novel ATP7B mutant gene and diagnostic reagent thereof | |
| CN113549689B (en) | Kit and method for detecting PROS1 gene exon | |
| Zhang et al. | Evaluation of genetic association and expression reduction of TRPC1 in the development of diabetic nephropathy | |
| CN108715893B (en) | SNP markers related to radioactive brain injury caused by radiotherapy and application thereof | |
| Harold et al. | Sequence variation in the CHAT locus shows no association with late-onset Alzheimer's disease | |
| CN103421908B (en) | Kit used for screening CYP21A2 gene of Chinese population | |
| CN104726604B (en) | Decayed-sample degradation DNA (deoxyribonucleic acid) detection method and application thereof | |
| CN108753945B (en) | SNP (single nucleotide polymorphism) locus related to obesity and/or hypertriglyceridemia of Chinese children and application thereof | |
| CN107058486B (en) | Primer group and kit for detecting glycolytic related genotyping of nasopharyngeal carcinoma | |
| Aytekin et al. | Evaluation of GenoFlow thrombophilia array test kit in its detection of mutations in factor V Leiden (G1691A), prothrombin G20210A, MTHFR C677T and A1298C in blood samples from 113 Turkish female patients | |
| Rao et al. | Adrenergic polymorphism and the human stress response | |
| Ostrovsky et al. | Characterization of HPSE gene single nucleotide polymorphisms in Jewish populations of Israel | |
| Friend et al. | Examination of the role of nitric oxide synthase and renal kallikrein as candidate genes for essential hypertension | |
| CN114875148A (en) | Familial multiple lipoma detection kit and application of primer group | |
| Poblete‐Gutiérrez et al. | The molecular basis of porphyria cutanea tarda in Chile: identification and functional characterization of mutations in the uroporphyrinogen decarboxylase gene | |
| CN104894261B (en) | Kit for predicting curative effect of ranibizumab on treatment of age-related macular degeneration | |
| JP5904501B2 (en) | Method for detecting type 2 diabetes | |
| Lau et al. | Two novel polymorphic sequences in the glucocerebrosidase gene region enhance mutational screening and founder effect studies of patients with Gaucher disease | |
| Vollmer et al. | Lipopolysaccharide-binding protein (LBP) gene polymorphisms: rapid genotyping by real-time PCR and association with infective endocarditis | |
| WO2010007063A2 (en) | Method of diagnosing dyslexia | |
| Lobato et al. | Detection of c.− 32T> G (IVS1− 13T> G) mutation of Pompe disease by real-time PCR in dried blood spot specimen | |
| WO2008057265A2 (en) | Single nucleotide polymorphisms and the identification of lactose intolerance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |