WO2014101276A2 - 确定核酸样本的两个预定位点是否具有已知突变的方法、试剂盒以及引物 - Google Patents

确定核酸样本的两个预定位点是否具有已知突变的方法、试剂盒以及引物 Download PDF

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WO2014101276A2
WO2014101276A2 PCT/CN2013/001453 CN2013001453W WO2014101276A2 WO 2014101276 A2 WO2014101276 A2 WO 2014101276A2 CN 2013001453 W CN2013001453 W CN 2013001453W WO 2014101276 A2 WO2014101276 A2 WO 2014101276A2
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primer
nucleic acid
group
optionally
predetermined sites
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PCT/CN2013/001453
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French (fr)
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WO2014101276A3 (zh
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许华曦
卜国军
张含
张云武
刘家珍
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厦门人瑞生物医药科技有限公司
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Publication of WO2014101276A3 publication Critical patent/WO2014101276A3/zh

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of biomedicine.
  • the invention relates to methods, kits and primers for determining whether two predetermined sites of a nucleic acid sample have known mutations. Background technique
  • ApoE is an apolipoprotein whose genetic polymorphism is mainly controlled by two SNP loci, rs429358 and rs7412, which determine the three alleles of ⁇ by different nucleic acid combinations. : ⁇ 2 ( rs429358 is T, rs7412 is ⁇ ), ⁇ 3 (rs429358 is T, rs7412 is C), ⁇ 4 (rs429358 is C, rs7412 is C), and these three types of alleles encode three ApoE proteins, respectively.
  • the ApoE gene has three homozygous ⁇ 2/ ⁇ 2, ⁇ 3/ ⁇ 3, and ⁇ 4/ ⁇ 4 and ⁇ 2/ ⁇ 3, ⁇ 2/ ⁇ 4 and ⁇ 3/ ⁇ 4 species 3 ⁇ 4 zygote .
  • ApoE genotyping methods include: traditional DNA sequencing, allele-specific oligonucleotide probe hybridization, polymerase chain reaction-single strand conformation polymorphism, polymerase chain reaction-restriction enzyme The fragment length polymorphism method, the multiplex amplification hindered mutation system PCR technique, the high-resolution dissolution curve method, and the two-color fluorescence fraction/probe method.
  • the present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a method for efficiently determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the invention proposes a method of determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the method comprises: performing PCR amplification on the nucleic acid sample using a first primer and a second primer, and adding a nucleic acid probe to the PCR amplification system, the nucleic acid probe corresponding to two At least a portion of the nucleic acid sequence between predetermined sites; and detecting luminescence of the reaction system to determine if a predetermined mutation is present at a predetermined site of the nucleic acid sample, wherein the proximal 3' end of the first primer comprises One of the two predetermined sites is a base corresponding to a known mutation, and the near 3' end of the first primer comprises a base corresponding to another known mutation of the two predetermined sites, the first primer And one of the second primers serves as a forward primer, and the other of the first primer and the first primer serves as a reverse primer, and the nucleic acid probe
  • the method of the embodiment of the present invention it is possible to effectively determine the presence of a corresponding known mutation at two predetermined sites of the detected nucleic acid sample by detecting the fluorescence generated by the PCR amplification system, and further effectively To determine the allelic type of ApoE, for the prevention of ApoE genotype-related diseases such as Alzheimer's disease and cardiovascular and cerebrovascular diseases? The diagnosis of the disease and the prognosis of the disease are of great significance.
  • the above determination of two predetermined sites of the nucleic acid sample has a known mutation
  • the method can also have the following additional technical features:
  • the nucleic acid sample is derived from human tissue or cells, recombinant nucleic acid or transgenic biological tissue cells, preferably human whole genome DNA.
  • a nucleic acid sample of the above-described source can be efficiently determined to have a mutation type, so that an individual who provides a nucleic acid sample can be efficiently analyzed.
  • the predetermined sites are SNPs rs429358 and rs7412.
  • SNPs rs429358 and rs7412 have known mutations at two predetermined positions in the nucleic acid sample, thereby effectively determining the ApoE allelic typing.
  • the proximal 3' end of the first primer comprises a base corresponding to a known mutation of rs429358
  • the proximal 3' end of the second primer comprises a known mutation with rs7412 Corresponding base.
  • the 3' terminal base of the first primer is a base corresponding to a known mutation of rs429358
  • the 3' terminal base of the second primer is a rs7412 Know the base corresponding to the mutation.
  • the first primer serves as a forward primer and the second primer serves as a reverse primer.
  • the first primer is at least one selected from the group consisting of: 5'-CGGACATGGAGGACGTGT-3', 5'-GCGGACATGGAGGACGTGT-3', 5'-CGCGGACATGGAGGACGTGT-3', 5'-CGGACATGGAGGACGTGC -3 ' , 5 ' - GCGGACATGGAGGACGTGC - 3 ', 5' _CGCGGACATGGAGGACGTGC-3'.
  • the second primer is at least one selected from the group consisting of: 5'-TGGTACACTGCCAGGCA-3 ' , 5 ' - CTGGTACACTGCCAGGCA-3 ', 5, - CCTGGTACACTGCCAGGCA-3 ', 5' -TGGTACACTGCCAGGCG-3 ' , 5 ' - CTGGTACACTGCCAGGCG -3 ', 5 ' - CCTGGTACACTGCCAGGCG- 3 '.
  • a nucleic acid sample is detected by a method for determining whether two predetermined sites of a nucleic acid sample have a known mutation according to an embodiment of the present invention, and the primer can be selectively combined with the template according to the base pairing principle during the PCR reaction.
  • different alleles of ApoE can be specifically amplified, and the type of ApoE allele can be further effectively determined.
  • the nucleic acid probe has a 1:degree of 20 nucleotides.
  • the sequence of the nucleic acid probe is 5'-CAGCTCCTCGGTGCTCTGGC-3'.
  • the luminescent group of the nucleic acid probe is a catastrophic group.
  • the fluorophore is FAM.
  • the regulatory group is a quenching group.
  • the quenching base is closed to BHQ1.
  • the nucleic acid-like wood is detected by a method for determining whether two predetermined sites of the nucleic acid sample have known mutations according to an embodiment of the present invention, and by detecting the presence or absence of a fluorescent signal in the amplification system corresponding to each genotype, It can be judged whether or not the ApoE gene of the subtype is contained in the sample to be tested.
  • the invention proposes a kit for determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the kit can be applied to the method of determining whether two predetermined sites of a nucleic acid sample have known mutations as described above.
  • detecting a nucleic acid sample using a kit for determining whether two predetermined sites of a nucleic acid sample have known mutations according to an embodiment of the present invention, it is possible to effectively determine whether two predetermined sites of the nucleic acid sample have known mutations , and further steps can effectively determine the allelic classification of ApoE, which is very important for the prevention and early diagnosis of Alzheimer's disease and cardiovascular and cerebrovascular diseases.
  • the features and advantages previously described for determining whether two predetermined sites of a nucleic acid sample have known mutations of course The kit is applicable and will not be described again.
  • the invention proposes a set of PCR primers that determine whether two predetermined sites of a nucleic acid sample have known mutations.
  • the PCR primer can be applied to the aforementioned method and kit for determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the primers can be selectively bound to the template according to the base pairing principle during PCR amplification, and the PCR amplification can be efficiently amplified in a predetermined direction and can specifically amplify different alleles.
  • PCR primers are of course applicable to the method and the advantages of the description of the primers used for determining whether the two predetermined sites of the nucleic acid-like wood have known mutations and the reagents used in the kit, and will not be described again.
  • a method of determining whether two predetermined sites of a nucleic acid-like wood have known mutations can achieve at least one of the following advantages -
  • a method of determining whether two predetermined sites of a nucleic acid sample have known mutations according to an embodiment of the present invention, the method being simple to operate, requiring only two steps of PCR amplification and fluorescence detection;
  • a method for determining whether two predetermined sites of a nucleic acid sample have a known mutation according to an embodiment of the present invention, the method is easy to implement an automatic operation, and the human error in the manual operation process can be greatly reduced, so that The result is more accurate;
  • a method for determining whether two predetermined sites of a nucleic acid-like wood have known mutations the detection result of the method is intuitive, and the sample to be tested can be qualitatively determined according to the presence or absence of the fluorescence signal. Whether or not there is an ApoE allotype corresponding to the primer pair of the amplification system does not require a cumbersome analysis process.
  • 1 is a schematic diagram showing the principle of different allele-specific amplification of ApoE by different primer pairs for determining whether two predetermined sites of a nucleic acid sample have known mutations, in accordance with one embodiment of the present invention, in which: 1 shows a schematic diagram of a DNA fragment of the ApoE gene containing SXP rs42358 and rs7412;
  • 2 is a schematic diagram of an ApoE gene PCR amplification system for determining whether two predetermined sites of a nucleic acid sample have known mutations, in which 1 represents an ApoE-based DNA template, in accordance with one embodiment of the present invention.
  • the antisense strand indicates a forward amplification primer
  • 3 indicates a DNA molecule probe coupled with a fluorescent group (F) and a quenching group (Q)
  • 4 indicates an ApoE gene DNA template JH sense strand
  • 5 indicates a reverse To the amplification primer
  • 6 represents a DNA polymerase
  • 3 is a schematic diagram showing the working principle of a fluorescent probe for determining whether two predetermined sites of a nucleic acid sample have known mutations, in which 1 represents an ApoE gene DNA template antisense, according to an embodiment of the present invention.
  • Chain 2 indicates a forward amplification primer
  • 3 indicates a fluorophore (F) detached DNA molecule probe
  • 4 indicates an ApoE gene DNA template sense strand
  • 5 indicates a reverse amplification primer
  • 6 indicates a DNA polymerase
  • 4 is a graph showing an example sample detection fluorescence signal reading result of a method of determining whether two predetermined sites of a nucleic acid sample have known mutations, in accordance with an embodiment of the present invention.
  • FIG. 5 is a bar graph analysis of an example sample detection fluorescence signal reading for determining whether two predetermined sites of a nucleic acid sample have known mutations, in accordance with one embodiment of the present invention. [0027] FIG. detailed description
  • the invention proposes a method of determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the method of determining whether two predetermined sites of a nucleic acid sample have known mutations may comprise the steps of:
  • S100 PCR amplification of the nucleic acid sample by using the first primer and the second primer, and adding the nucleic acid probe to the PCR amplification system
  • a PCR primer is designed for the nucleic acid sequence of one of two predetermined sites of the nucleic acid sample: a first primer, a nucleic acid sequence design for another predetermined site of two predetermined sites of the nucleic acid sample
  • a PCR primer First. Primer, and a nucleic acid probe is added, and then the nucleic acid sample is subjected to PCR amplification. Thus, a PCR amplified sample is obtained for further fluorescence detection.
  • the inventors have found that, at the time of PCR amplification, if the near 3' end of the first primer contains a base corresponding to a known mutation of one of two predetermined sites of the nucleic acid sample, the near 3' of the second primer
  • the terminal comprises a base corresponding to a known mutation of another predetermined site of the nucleic acid sample, and one of the first primer and the second primer serves as a forward primer, and the other of the first primer and the second primer serves as a reverse primer
  • the primer can be selectively bound to the template according to the base pairing principle, and the PCR amplification can be efficiently amplified in a predetermined direction, and the specific allele can be specifically amplified.
  • the 3' terminal base of the first primer is a base corresponding to a known mutation of rs429358
  • the 3' terminal base of the second primer is a base corresponding to a known mutation of rs7412. base.
  • the first primer serves as a forward primer and the second primer serves as a reverse primer.
  • the first primer is at least one selected from the group consisting of: 5'-CGGACATGGAGGACGTGT-3', 5'-GCGGACATGGAGGACGTGT-3', 5'-CGCGGACATGGAGGACGTGT-3', 5'-CGGACATGGAGGACGTGC-3' , 5' -GCGGACATGGAGGACGTGC-3', 5' -CGCGGACATGGAGGACGTGC-3 ⁇
  • the second primer is at least one selected from the group consisting of: 5' - TGGTACACTGCCAGGCA-3', 5, -CTGGTACACTGCCAGGCA-3', 5 '-CCTGGTACACTGCCAGGCA -3 ', 5 ' - TGGTACACTGCCAGGCG- 3 ' , 5 ' -CTGGTACACTGCCAGGCG-3 ', 5 ' -CCTGGTACACTGCCAGGCG-3 ' .
  • primers are selectively bound to the template according to the base
  • a separate one is designed for the nucleic acid sequence of SNP rs429358 which is T or C.
  • PCR forward primer hereinafter referred to as pl l2T or pl l2C
  • P158T or pl58C PCR reverse primer
  • the nucleic acid sequence between ApoE different genotypes SNP rs429358 and rs7412 can be specifically amplified by PCR in the following primer pairs: Primers P112T and pl58T can specifically amplify ⁇ 2 gene, primer ⁇ 112 ⁇ And pl58C can specifically amplify the ⁇ 3 type genomic, and the primers P112C and pl58C can specifically amplify the ⁇ 4 type gangue.
  • PCR can specifically amplify different alleles fel, thereby effectively determining whether two predetermined sites of the nucleic acid-like wood have known mutations, and further effectively determining the type of the ApoE allele of the nucleic acid sample towel. .
  • a nucleic acid fluorescent probe having a specificity is designed for the nucleic acid sequence between two predetermined sites of the nucleic acid sample, and the nucleic acid probe corresponds to at least a part of the corresponding nucleic acid sequence between the two predetermined sites.
  • the inventors have found that if the nucleic acid probe has a luminescent group, a luminescent group is formed at the 5' end of the nucleic acid probe, a depressing group, and a deuterated group is formed on the nucleic acid probe. 'End, and the regulatory group can attenuate the luminescence of the luminescent group, and when the corresponding nucleic acid sequence containing the nucleic acid probe is PCR-amplified, the probe can specifically bind to the amplification template, at which time the nucleic acid is polymerized The exonuclease activity of the enzyme cleaves the 5' end group of the probe, separating the luminescent group and the regulatory group, thereby emitting a detectable signal.
  • the luminescent group of the nucleic acid probe is a fluorophore, preferably the fluorophore is an F layer.
  • the regulatory group of the nucleic acid probe is a fluorescent group, preferably the regulatory group is BHQ1.
  • the length of the nucleic acid probe is not particularly limited, and for example, the length of the nucleic acid probe may be 20 nucleotides.
  • a nucleic acid fluorescent probe having specificity is designed for the nucleic acid sequence between SNP rs429358 and rs7412.
  • the sequence of the nucleic acid probe is 5 'FAM-CAGCTCCT CGGTGCTCTGGC-BHQ13 '. Therefore, when detecting the ApoE genotype of the nucleic acid-like wood, it is only necessary to add the nucleic acid fluorescent probe designed according to the above method to the PCR amplification system, and PCR amplification is performed by using different primers corresponding to the E2/E3/E4 genotypes. Increase the DNA template to be tested. After the end of the PCR reaction, the presence or absence of a fluorescent signal in the amplification system corresponding to each of the base types can be determined to determine whether or not the ApoE gene of the subtype is contained in the nucleic acid sample to be tested.
  • the type and source of the nucleic acid sample are not particularly limited, and for example, may be at least a part of human genomic DNA, and may be derived from any human tissue or cell, recombinant nucleic acid or transgenic biological tissue cells.
  • the method according to an embodiment of the present invention it is possible to effectively determine whether two predetermined sites of the nucleic acid sample have known mutations, and further effectively determine the allelic classification of ApoE for Alzheimer's disease and heart and brain The prevention and early diagnosis of vascular diseases is of great significance.
  • nucleic acid sample means that at least a portion of the human genomic DNA is involved, and the nucleic acid sample to be detected may be derived from any human tissue or cell, recombinant nucleic acid or transgenic biological tissue cells.
  • first and second are merely for convenience of describing the invention, and are not to be construed as limiting the invention.
  • near 3' end means that the base at the 3' end is within a predetermined range, for example, 10, 9, 8, 7, and 6 , 5, 4, 3, 2, 1 or 0 (ie 3' end).
  • S200 detecting luminescence of the reaction system to determine whether a predetermined mutation exists in a predetermined site of the nucleic acid sample
  • the PCR amplification sample obtained in step S100 ⁇ is subjected to fluorescence detection.
  • fluorescence detection is performed, whether the nucleic acid sample to be tested has the qualitative determination according to the presence or absence of the fluorescence signal in the PCR amplification system PCR amplification system primer pair corresponding ApoE allele genotyping. Thereby, it is possible to determine whether a predetermined mutation exists in a predetermined site of the nucleic acid sample, and further to effectively determine the type of the ApoE allele.
  • a PCR amplification sample of human genomic DNA is subjected to fluorescence detection, and if a fluorescent signal is detected only in the ⁇ 2 amplification system, the ApoE genotype of the sample to be tested is ⁇ 2/ ⁇ 2 ; Similarly, if a fluorescent signal is detected only in the ⁇ 3 (or ⁇ 4 ) amplification system, the sample-derived human ApoE genotype is ⁇ 3 / ⁇ 3 (or ⁇ 4 / ⁇ 4); If the fluorescence signal is detected in both the ⁇ 2 and ⁇ 3 amplification systems, the ApoE basis of the sample to be tested is ⁇ 2 / ⁇ 3; the same reason if ⁇ 2 and ⁇ 4 are simultaneously expanded When the fluorescence signal is detected in the increasing system, the ApoE genotype of the sample to be tested is ⁇ 2 / ⁇ 4; the same is true if both ⁇ 2 and ⁇ 4 (or ⁇ 3 and ⁇ 4
  • the ApoE genotype of the sample to be tested is ⁇ 2 / ⁇ 4 (or ⁇ 3 / ⁇ 4 ).
  • the means for PCR amplification and fluorescence detection is not particularly limited, and PCR amplification and fluorescence detection can be performed by any known method and apparatus.
  • PCR thermal cycle, fluorescence detection and various application analysis software can be combined by using the 7500 Fast Real-Time PCR system of Applied Biosystems, USA, and the reaction of each cycle of PCR can be dynamically observed.
  • the PCR amplification product in the tube is gradually increased, and the result can be obtained immediately after the end of the PCR experiment, without gel electrophoresis analysis, without purification of the PCR product, which has the advantages of time saving, high sensitivity, good accuracy, and the like, for example, related technicians
  • the primers, the PCR temperature, and the like can be adjusted accordingly according to the actual needs of the present invention, and are not described herein again, and these are all within the scope of the present invention.
  • the invention proposes a kit for determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the kit can be applied to the method of determining whether two predetermined sites of a nucleic acid sample have known mutations as described above.
  • detecting a nucleic acid sample using a kit for determining whether two predetermined sites of a nucleic acid sample have known mutations according to an embodiment of the present invention, it is possible to effectively determine whether two predetermined sites of the nucleic acid sample have known mutations And further can effectively determine the allelic typing of ApoE.
  • the features and advantages previously described for determining whether two predetermined sites of a nucleic acid sample have known mutations are of course applicable to the kit and will not be described again.
  • the invention proposes a group of PCR primers for determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • the primers can be applied to the methods and kits described above for determining whether two predetermined sites of a nucleic acid sample have known mutations.
  • primers can be selectively bound to the template according to the base pairing principle during PCR amplification, and PCR amplification can be efficiently amplified in a predetermined direction and can specifically amplify different alleles.
  • the PCR primers are of course applied to the methods and advantages of the methods for determining whether two predetermined sites of a nucleic acid sample have a known mutation and the primers used in the kit, and will not be described again.
  • the method, kit and primer for determining whether two predetermined sites of a nucleic acid sample have known mutations in the present invention may be related to the prevention and early diagnosis of diseases such as Alzheimer's disease and cardiovascular and cerebrovascular diseases, and related technologies. It is of course possible for the personnel to extend it to other fields of application, and the details are not described herein, and these are all within the scope of the present invention.
  • the reaction solution was stored at 4 ° C.
  • the primers were all synthesized in Shanghai Boshang Biotechnology Co., Ltd., and the purification method was: PAGE purification.
  • the ⁇ ⁇ reaction solution was stored at 4 ° C.
  • the primers were synthesized in Shanghai Boshang Biotechnology Co., Ltd., and the purification method was: PAGE purification.
  • the primers were synthesized in Shanghai Boshang Biotechnology Co., Ltd., and the purification method was: PAGE purification.
  • the DNA molecular probe was synthesized by Infineon (Shanghai) Trading Co., Ltd., and the purification method was: HPLC purification.
  • PCR premix TaqMan Genotyping Master purchased from Applied Biosystems, USA
  • R0X reference fluorescence is not added to the PCR reaction, it can be used as a fluorescent internal reference to correct changes in fluorescence signal due to changes in concentration and volume in the PCR reaction.
  • sterile deionized water deionized water, water from British ELGA (Model P urel a b3000), and after the resulting autoclaved.
  • PCR plate MicroAmp® Opt ical96- Well Reaction Plate was purchased from Applied Biosystems, USA.
  • PCR sealing film MicroAmp® 96- Well Optical Adhesive Film was purchased from PI Applied Biosystems.
  • Disposable medical sterile syringe disposable medical sterile syringe (5ml specification) was purchased from Yuxiang Medical Sanitary Material Co., Ltd., Xinxiang City, Henan province.
  • red blood cell lysate weighed 3.735g ammonium chloride, tris (tris) l.3g dissolved in water and diluted to 500ml, 0.22m filter (purchased from Millipore, USA) filter sterilization , stored at 4 ° C;
  • ⁇ 3/ ⁇ 3 DNA known samples PCR-amplified and sequenced validated D. ⁇ A samples from ⁇ 3/ ⁇ 3 genomic DNA.
  • ⁇ 4/ ⁇ 4DNA known sample PCR-amplified and sequenced DNA samples were amplified from ⁇ 4/ ⁇ 4 genomic DNA.
  • Pipette A micropipette (10 UL, 20 L, 100 ⁇ , 1000 L) was purchased from Eppendorf, Germany.
  • Vortex mixer purchased from Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd.
  • Full-wavelength scanning multi-function reader The Thermo Scientific Varioskan Flash full-wavelength scanning multi-function reader was purchased from Thermo Scientific.
  • Real-Time PCR instrument 7500 Fast Real-Time PCR system, purchased in the United States - Applied Biosystems, USA.
  • the DNA sequence of the ApoE gene is provided by the National Center for Biotechnology Information (NCBI) website, and the primers are designed as follows according to the DNA sequence information including the ApoE gene polymorphism sites SNP rs429358 and SNP rs7412:
  • a forward PCR amplification primer directed against the SNP rs429358 nucleic acid in the form of T, the DNA sequence of which is: 5'-GCGGACATGGAGGACGTGT-3' and 5'-CGGACATGGAGGACGTGT-3';
  • a reverse PCR amplification primer directed against the SNP rs7412 nucleic acid in the form of C, the DNA sequence of which is: 5'-CTGGTACACTGCCAGGCG-3'.
  • a fluorescent probe is selected, and a FAM fluorescent group is selected.
  • a BHQ1 quenching group the FAM fluorophore is coupled to the 5' end of the complementary nucleic acid sequence
  • the BHQ1 quenching group is coupled to the 3' end of the complementary nucleic acid sequence
  • the fluorescent probe is: 5 'FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 '.
  • the collected human blood was taken out from the refrigerator at 4 ° C and gently mixed, and then 300 ul of blood was pipetted and transferred to a 1.5 ml centrifuge tube containing 900 ul of red blood cell lysate, and inverted 5-6 times.
  • the supernatant is aspirated, leaving about 10-20 ul at the bottom of the tube.
  • the concentration of the extracted DM is measured by a DNA concentration measurement program in a full-wavelength scanning multi-function reader, and a part of the DNA solution is diluted to 2 ng/ml with sterile deionized water as a template for subsequent PCR detection. Store the DNA solution at 2 ⁇ 8 °C or _20 °C.
  • ⁇ genotypes ⁇ 2/ ⁇ 2 , ⁇ in the population there are 6 different ⁇ genotypes ⁇ 2/ ⁇ 2 , ⁇ in the population, respectively. 3/ ⁇ 3, ⁇ 4/ ⁇ 4, ⁇ 2/ ⁇ 3, ⁇ 2/ ⁇ 4 and ⁇ 3/ ⁇ 4 detection. 6 known ⁇ genotype H ⁇ A known samples ( ⁇ 2/ ⁇ 2DNA known samples, ⁇ 3/ ⁇ 3DNA known samples, ⁇ 4/ ⁇ 4DNA known wood, ⁇ 2/ ⁇ 3DNA Wood, ⁇ 2/ ⁇ 4DNA known as wood and ⁇ 3/ ⁇ 4DNA known samples) as a control
  • ⁇ 2 reagent comprises:
  • Fluorescent probe (100 ⁇ ) 0.05u 1
  • ⁇ 3 reagent comprises:
  • a forward primer ⁇ 112 ⁇ corresponding to the ApoE ⁇ 3 allele type SNP site rs429358SNP nucleic acid form (the example ⁇ nucleic acid sequence is 5'-CGGACATGGAGGACGTGT-3 ').
  • Fluorescent probe in the example, the probe form is 5'FAM-CAGCTCCTCGGTGCTCTGGC-BHQ 13 ').
  • Fluorescent probe (100 ⁇ ) 0.05 ⁇ 1
  • the towel ⁇ 4 reagent comprises:
  • Fluorescent probe in the example, the probe form is 5'FAM-CAGCTCCTCGGTGCTCTGGC-BHQ 13 ').
  • Fluorescent probe (100 ⁇ ) 0.05 ⁇ 1
  • the PCR reaction system for ⁇ 2 gene detection is:
  • DNA sample to be tested (2ng/ u 1) : 5 ⁇ 1
  • the PCR reaction system for ⁇ 3 gene detection is:
  • DNA sample to be tested (2ng/ ⁇ 1) : 5 ⁇ 1
  • the PCR reaction system for ⁇ 4 gene detection is:
  • PCR plate into the 7500 Fast Real-Time PCR system instrument, open the 7500software V2.0.6 software, select the Genotyping module in the software, and set the corresponding test content in the Plate setup to the PCR plate _ I: per hole. Then set the PCR reaction program and reaction system in the Run method. After setting these items, you can run the program. The program pre-reads the background fluorescence intensity value and then performs PCR amplification at 60 °C before the default PCR reaction. To eliminate background fluorescence values. After the PCR reaction is completed, the instrument automatically collects fluorescence data under 60 conditions. Table 1 shows the standard thermal cycling procedure for this example PCR reaction process.
  • ⁇ 2, ⁇ 4 Kegang detection system is a negative result, the fluorescence values are 0. 1933465 and 0. 40723515; in the known samples of DNA with ApoE genotype ⁇ 4 / ⁇ 4, ⁇ 4 gene detection system is positive As a result, the fluorescence value was 2. 59521461.
  • the ⁇ 2, ⁇ 3 gene detection system was a negative result, and the fluorescence values were 0. 17685843 and 0. 22782183, respectively.
  • a sample of the deuterium-free deionized water in the ⁇ 2, ⁇ 3 and ⁇ 4 gene detection systems, has a fluorescence value of 0. 17888165, 0. 22742152 and -0. 0582402, respectively.
  • the maximum negative result fluorescence value in this example is 0. 51656318, and the fluorescence values of all positive results are much larger than the maximum negative result fluorescence values.
  • the value of the maximum value of the fluorescence value of the negative result is defined as the cut-off value of the positive and negative results of the fluorescent signal. In this example, the cutoff value is 1. 03312636.
  • the shape chart can be used as a figure, and the column chart is shown in FIG. 5, and a straight line is drawn between the positive and negative result boundary values, and it can be visually seen that all the positive results are above the straight line. All negative results are below the straight line.
  • the results of the DNA samples to be tested in the ⁇ 2 and ⁇ 3 gene detection systems were judged to be positive according to the above analysis method, and the results obtained in the ⁇ 4 gene detection system were judged to be negative results according to the above analysis method. From this, it was found that the ApoE-based 141 type of the DNA sample to be tested in this example was of the ⁇ 2 / ⁇ 3 type.
  • the primer ⁇ 112 ⁇ is only matched with the nucleic acid sequence of SNP rs429358
  • the primer pl l2C is only matched with the nucleic acid sequence of SNP rs429358 being C
  • the primer pl58T is only T with SNP rs7412.
  • the PCR system containing the primer pair ⁇ 112 ⁇ / ⁇ 158 ⁇ can only amplify the ApoE allele (ie ⁇ 2 ) with SNP rs429358 as T and SNP rs7412 as T; the PCR system containing primer pair pl l2T/pl58C can only expand Increasing SNP rs429358 is the ApoE allele of T and SNP rs7412 as C (ie ⁇ 3 ); the PCR system containing primer pair l l2C/pl58C can only amplify the ApoE allele with SNP rs429358 as C and SNP rs7412 as C (ie ⁇ 4). Therefore, using the above three primer pairs, different alleles of ApoE can be specifically amplified, and the type of human ApoE allele in the DNA sample can be determined.
  • the two segments of the DNA molecule probe 3 from which the fluorophore (F) is detached are coupled with a fluorescent group ffl and a quenching group, respectively, at this time due to the quenching group. It can absorb the excitation energy of the fluorophore, so only the background fluorescence signal can be detected in the system.
  • the fluorescent molecularly closed (F) detached DNA molecule probe 3 can specifically complement the ApoE gene DNA template antisense strand 1 and the matching sequence is located between the DNA sequences corresponding to the forward amplification primer 2 and the reverse amplification primer 5.
  • the DNA molecule probe 3 detached from the amplification primer 2 and the fluorescent group (F) will bind to the ApoE-based DNA template antisense strand 1, and the reverse amplification primer 5 will be combined with ApoE.
  • the Keegan DA template sense strand 4 binds and begins to amplify the template DNA under the action of DNA polymerase 6.
  • the DNA polymerase 6 starts from the forward amplification primer 2 or the reverse amplification primer 5 in the PCR reaction process, and the ApoE gene DNA template antisense strand 1 or ApoE
  • the gene DNA template sense strand 4 is a template that initiates the extension of DNA.
  • DNA polymerase 6 mediates DNA elongation, it encounters the fluorophore (F) detached on the antisense strand 1 of the ApoE gene DNA template.
  • DNA polymerase 6 will utilize its exonuclease activity from fluorescein
  • the 5' end of the DNA molecule probe 3 from which the photo group (F) is detached cleaves the nucleotide on the probe, causing the fluorophore to be detached from the probe and separated from the quenching group.
  • a fluorescent signal excited by the fluorophore is detected, and thereby the detection sample contains the ApoE allele type corresponding to the primer pair.

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Abstract

本发明公开了确定核酸样本的两个预定位点是否具有已知突变的方法、试剂盒以及引物。其中,该方法包括:利用第一引物和第二引物对核酸样本进行PCR扩增,并且在PCR扩增体系中加入核酸探针,核酸探针对应两个预定位点之间至少一部分对应的核酸序列;检测反应体系的发光,以便确定核酸样本的预定位点是否存在已知突变,第一引物的近3'端包含与两个预定位点之一已知突变对应的碱基,第二引物的近3'端包含与另一个已知突变对应的碱基,核酸探针具有:发光基团,发光基团形成于核酸探针的5'端,调节基团,调节基团形成于核酸探针的3'端,并且调节基团可以调节发光基团的发光。利用该方法能够有效地确定核酸样本的两个预定位点是否具有已知突变。

Description

确定核酸样本的两个预定位点是否具有已知突变的方法、
试剂盒以及引物 技术领域
[0001 ] 本发明涉及生物医学领域。 具体而言,本发明涉及确定核酸样本的两个预定位点 是否具有已知突变的方法、试剂盒以及引物。 背景技术
[0002] ApoE是一种载脂蛋白,其遗传多态性主要受控于两个 SNP位点 rs429358和 rs7412,这两个 SNP位点通过不同的核酸组合决定了 ΛροΕ的三种等位基因型: ε 2 ( rs429358为 T, rs7412为 Τ )、 ε 3 ( rs429358为 T, rs7412为 C )、 ε 4 ( rs429358为 C, rs7412为 C ), 这三种类型等位基因分别编码三种 ApoE蛋白亚型,即 ApoE2 (其蛋白序列 的 112位和 158位均为半胱氨酸)、 ApoE3 (其蛋白序列的 112位为半胱氨酸, 158位为精氨 酸)、 ApoE4 (其蛋白序列的 112位和 158位均为精氨酸)。 因此, ApoE基因有 ε 2/ ε 2、 ε 3/ ε 3、和 ε 4/ ε 4三种纯合子型与 ε 2/ ε 3、 ε 2/ ε 4和 ε 3/ ε 4 种 ¾合子型。 目前 ApoE 基因分型的方法主要有:传统 DNA测序法、等位基因特异性寡核苷酸探针杂交法、聚合酶链 反应-单链构象多态性法、聚合酶链反应 -限制性酶切片段长度多态性法、多重扩增受阻突 变体系 PCR技术法、高分辨率溶解曲线法以及双色荧光分 /·探针法等。
[0003] 然而,目前确定核酸样本的两个预定位点是否具有已知突变的方法,仍有待改进。 发明内容
[0004] 本发明旨在至少解决现有技术中存在的技术问题之一。 为此,本发明提出了可以 有效确定核酸样本的两个预定位点是否具有已知突变的方法。
[0005] 在本发明的一个方面,本发明提出了一种确定核酸样本的两个预定位点是否具有 已知突变的方法。 根据本发明的实施例,该方法包括:利用第一引物和第二引物对所述核 酸样本进行 PCR扩增,并且在所述 PCR扩增体系中加入核酸探针,所述核酸探针对应两个预 定位点之间至少一部分的核酸序列;以及检测反应体系的发光,以便确定所述核酸样本的 预定位点是否存在巳知突变,其中,所述第一引物的近 3 '端包含与所述两个预定位点之一 已知突变对应的碱基,所述第一引物的近 3 '端包含与所述两个预定位点另一个己知突变对 应的碱基,所述第一引物和第二引物之一作为正向引物,所述第一引物和所述第一.引物的 另一个作为反向引物,所述核酸探针具有:发光基团,所述发光基团形成于所述核酸探针的 5 '端,调节基团,所述调节基团形成于所述核酸探针的 3 '端,并且所述调节基团可以调节所 述发光基团的发光。
[0006] 根据本发明实施例的方法,能够通过检测 PCR扩增体系所产生的荧光,有效地确 定所检测的核酸样本的两个预定位点同时存在相应的已知突变,并进一步可以有效地确定 ApoE的等位基因分型,对于老年痴呆症及心脑血管等与 ApoE基因型相关疾病的预防、? -期 诊断以及疾病预后具有非常重要的意义。
[0007] 根据本发明的 实施例,上述确定核酸样本的两个预定位点是否具有已知突变 的方法还可以具有下列附加技术特征:
[0008] 根据本发明的一个实施例,所述核酸样本来源于人体组织或细胞、重组核酸或转 基因生物组织细胞,优选地采用人类全基因组 DNA。 根据本发明的实施例,可以对上述来源 的核酸样本进行有效地确定突变类型,从而可以对提供核酸样本的个体进行有效地分析。
[0009] 根据木发明的一个实施例,所述预定位点是 SNP rs429358和 rs7412。 由此,利用 根据本发明实施例,可以有效地确定核酸样本中在两个预定位点 SNP rs429358和 rs7412 是否具有已知突变,从而可以有效地确定 ApoE等位基因分型。
[0010] 根据本发明的一个实施例,所述第一引物的近 3'端包含与 rs429358的已知突变 对应的碱基,所述第二引物的近 3'端包含与 rs7412的己知突变对应的碱基。 由此,利 ffl根 据本发明实施例的确定核酸样本的两个预定位点是否具有已知突变的方法对核酸样本进 行检测,在 PCR反应时引物可以与模板按照碱基配对原则选择性结合,从而可以特异地扩 增出 ApoE的不同等位基因,并进一步可以有效地确定 ApoE等位基因的类型,
[0011] 根据本发明的一个实施例,所述第一引物的 3'端碱基为与 rs429358的己知突 变对应的碱基,所述第二引物的 3'端碱基为与 rs7412的己知突变对应的碱基。 根据本 发明的 ·个实施例,所述第 ·引物作为正向引物,所述第二引物作为反向引物。 根据本 发明的一个实施例,所述第一引物为选 Θ下列的至少之一:5' -CGGACATGGAGGACGTGT-3', 5 ' -GCGGACATGGAGGACGTGT-3 ' , 5 ' -CGCGGACATGGAGGACGTGT-3 ' , 5 ' -CGGACATGGAGGACGTGC-3 ' , 5 ' - GCGGACATGGAGGACGTGC - 3 ', 5' _CGCGGACATGGAGGACGTGC-3'。 所述 第 二 引 物 为 选 自 下 列 的 至 少 之 一: 5' -TGGTACACTGCCAGGCA-3 ' , 5 ' - CTGGTACACTGCCAGGCA-3 ', 5, - CCTGGTACACTGCCAGGCA-3 ', 5' -TGGTACACTGCCAGGCG-3 ' , 5 ' - CTGGTACACTGCCAGGCG-3 ', 5 ' - CCTGGTACACTGCCAGGCG- 3 '。 由此,利用根据本发明实施例的确定核酸样本的两个预定位点是否具有已知突变的方法对 核酸样本进行检测,在 PCR反应过程中,引物可以与模板按照碱基配对原则选择性结合,从 而可以特异地扩增出 ApoE的不同等位基因,并进一步可以有效地确定 ApoE等位基因的类 型。
[0012] 根据本发明的- ·个实施例,所述核酸探针的1:度为 20个核苷酸。根据本发明的 - 个实施例,所述核酸探针的序列为 5'- CAGCTCCTCGGTGCTCTGGC-3'。 根据本发明的一个实施 例,所述核酸探针的发光基团为灾光基团。根据本发明的一个实施例,所述荧光基团为 FAM。 根据本发明的一个实施例,所述调节基团为淬灭基团。根据本发明的一个实施例,所述淬灭 基闭为 BHQ1。 由此,利用根据本发明实施例的确定核酸样本的两个预定位点是否具有己知 突变的方法对核酸样木进行检测,通过检测各基因型对应的扩增体系中有无荧光信号,即 可判断待测样品中是否含有该亚型的 ApoE基因。
[0013] 在本发明的又一个方面,本发明提出了一种确定核酸样本的两个预定位点是否具 有已知突变的试剂盒。 根据本发明的实施例,所述试剂盒可以应用于前面所述的确定核酸 样本的两个预定位点是否具有已知突变的方法。 由此,利用根据本发明实施例的确定核酸 样本的两个预定位点是否具有已知突变的试剂盒对核酸样本进行检测,可以有效地确定核 酸样本的两个预定位点是否具有已知突变,并进 -步可以有效地确定 ApoE的等位基因分 型,对于老年痴呆症和心脑血管疾病的预防以及早期诊断具有非常重要的意义。另外,前面 针对确定核酸样本的两个预定位点是否具有已知突变的方法所描述的特征和优点,也当然 地适用该试剂盒,不再赘述。
[0014] 在本发明的又一个方面,本发明提出了一组确定核酸样本的两个预定位点是否具 有己知突变的 PCR引物。 根据本发明的实施例,所述 PCR引物可以应用于前面所述的确定 核酸样本的两个预定位点是否具有已知突变的方法以及试剂盒。 由此, PCR扩增过程中引 物可以与模板按照碱基配对原则选择性结合, PCR扩增可以按照预定方向进行有效扩增,并 且能够特异地扩增出不同等位基因。 另外,前面针对确定核酸样木的两个预定位点是否具 有已知突变的方法及试剂盒所用引物的描述的特征和优点,也当然地适用该 PCR引物,不 再赘述。
[0015] 根据木发明的实施例的确定核酸样木的两个预定位点是否具有己知突变的方法 可以实现下列优点至少之一 -
[0016] 1、根据本发明的实施例的确定核酸样本的两个预定位点是否具有已知突变的方 法,该方法操作简便,仅需 PCR扩增和荧光检测两个步骤;
[0017] 2、根据本发明的实施例的确定核酸样本的两个预定位点是否具有巳知突变的方 法,该方法容易实现自动化操作,可以大大减小手工操作过程中的人为误差,使得到的结果 更加准确;
[0018] 3、根据本发明的实施例的确定核酸样本的两个预定位点是否具有已知突变的方 法,该方法快速省时,完成整个检测的时间不到 1. 5小时,耗时少于现有的方法;
[0019] 4、根据本发明的实施例的确定核酸样本的两个预定位点是否具有已知突变的方 法,该方法的成本较低,仅需一个荧光探针和常规 PCR体系即可;
[0020] 5、根据本发明的实施例的确定核酸样木的两个预定位点是否具有已知突变的方 法,该方法的检测结果直观,根据荧光信号的有无即可定性判断待测样本中是否具有该扩 增体系引物对所对应的 ApoE等位基冈型,无需繁琐的分析过程。
[0021 ] 本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变 得明显,或通过本发明的实践了解到。 附图说明
[0022] 本发明的上述和 I或附加的方面和优点从结合下面附图对实施例的描述中将变 得明显和容易理解,其中:
[0023] 图 1是根据本发明一个实施例的确定核酸样本的两个预定位点是否具有已知突 变的方法的不同引物对 ApoE不同等位基因特异性扩增原理示意图,在该图中, 1表示含 SXP rs42358和 rs7412的 ApoE基因 DNA片段示意图;
[0024] 图 2是根据本发明一个实施例的确定核酸样本的两个预定位点是否具有已知突 变的方法的 ApoE基因 PCR扩增体系示意图,在该图中, 1表示 ApoE基闲 DNA模板反义链, 2 表示正向扩增引物, 3表示偶联有荧光基团(F )和淬灭基团(Q )的 DNA分子探针, 4表示 ApoE 基因 DNA模板 JH义链, 5表示反向扩增引物, 6表示 DNA聚合酶;
[0025] 图 3是根据本发明一个实施例的确定核酸样本的两个预定位点是否具有已知突 变的方法的荧光探针工作原理示意图,在该图中, 1表示 ApoE基因 DNA模板反义链, 2表示 正向扩增引物, 3表示荧光基团(F )脱离的 DNA分子探针, 4表示 ApoE基因 DNA模板正义链, 5表示反向扩增引物, 6表示 DNA聚合酶; [0026] 图 4是根据本发明 -个实施例的确定核酸样本的两个预定位点是否具有己知突 变的方法的实例样品检测荧光信号读值结果图;
[0027] 图 5是根据本发明一个实施例的确定核酸样本的两个预定位点是否具有已知突 变的方法的实例样品检测荧光信号读值的柱型分析图。 具体实施方式
[0028] 下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终 相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。 卜面通过参考附 图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对木发明的限制。
[0029] 在本发明的描述中,需要理解的是,术语"厚度"、 "上"、"下 "等指示的方位或位置 关系为基于附图所不的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指 示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理 解为对本发明的限制。
[0030] 在本发明的一个方面,本发明提出了一种确定核酸样本的两个预定位点是否具有 已知突变的方法。 根据本发明的实施例,确定核酸样本的两个预定位点是否具有已知突变 的方法可以包括以下步骤:
[0031] S100 :利用第一引物和第二引物对核酸样本进行 PCR扩增,并且在 PCR扩增体系中 加入核酸探针
[0032] 在该歩骤中,针对核酸样本的两个预定位点之一的核酸序列设计一条 PCR引物: 第一引物,针对核酸样本的两个预定位点另一个预定位点的核酸序列设计一条 PCR引物: 第一.引物,并且加入核酸探针,然后对核酸样本进行 PCR扩增。 此,获得 PCR扩增样本,以 便进一步对其进行荧光检测。
[0033] 发明人发现,在 PCR扩增时,如果第一引物的近 3 '端包含与核酸样本的两个预定 位点之一的已知突变对应的碱基,第二引物的近 3'端包含 核酸样本的另一个预定位点的 己知突变对应的碱基,以及第一引物和第二引物之一作为正向引物,第一引物和所述第二 引物的另 个作为反向引物,则在 PCR扩增吋引物可以与模板按照碱基配对原则选择性结 合, PCR扩增可以按照预定方向进行有效扩增,并且能够特异地扩增出不同等位基因。
[0034] 根据本发明的实施例,第一引物的 3'端碱基为与 rs429358的已知突变对应的 碱基,第二引物的 3'端碱基为与 rs7412的已知突变对应的碱基。 根据本发明的实施 例,第一引物作为正向引物,第二引物作为反向引物。 根据本发明的实施例,第一引物 为选自下列的至少之一: 5' -CGGACATGGAGGACGTGT-3 ' , 5 ' -GCGGACATGGAGGACGTGT-3 ', 5 ' -CGCGGACATGGAGGACGTGT-3 ' , 5 ' -CGGACATGGAGGACGTGC-3 ' , 5' -GCGGACATGGAGGACGTGC-3',5' -CGCGGACATGGAGGACGTGC-3 Ό 所 述 第 二 引 物 为 选 自 下 列 的 至 少 之 一: 5' - TGGTACACTGCCAGGCA-3',5, -CTGGTACACTGCCAGGCA- 3', 5 ' -CCTGGTACACTGCCAGGCA-3 ', 5 ' - TGGTACACTGCCAGGCG- 3 ' , 5 ' -CTGGTACACTGCCAGGCG- 3 ', 5 ' -CCTGGTACACTGCCAGGCG-3 ' . 由此, PCR扩增时引物 ^以与模板按照碱基配对原则选择性 结合, PCR扩增可以按照预定 5'到 3'方向进行有效扩增,并且能够特异地扩增出不同等位 基因。
[0035] 根据本发明的具体实施例,针对 SNP rs429358为 T或 C的核酸序列分别设计一条 PCR正向引物(以下称为 pl l2T或 pl l2C ),针对 SNP rs7412为 T或 C的核酸序列分别设计 一条 PCR反向引物(以下称为 P158T或 pl58C)。 根据 ApoE基因型的 别可得知, ApoE不同 基因型 SNP rs429358和 rs7412之间的核酸序列可由如下引物对在 PCR反应中特异扩增: 引物 P112T和 pl58T能够特异扩增 ε 2型基因,引物 ρ112Τ和 pl58C能够特异地扩增 ε 3 型基冈,以及引物 P112C和 pl58C能够特异地扩增 ε 4型基冈。 由此, PCR能够特异地扩增 出不同等位基 fel,从而可以有效地确定核酸样木的两个预定位点是否具有已知突变,进一 步可以有效地确定核酸样本巾 ApoE等位基因的类型。
[0036] 在该步骤屮,针对核酸样本的两个预定位点之间的核酸序列设计一段具有特异性 的核酸荧光探针,核酸探针对应两个预定位点之间至少一部分对应的核酸序列。
[0037] 发明人发现,如果核酸探针具有:发光基团,发光基团形成于所述核酸探针的 5 ' 端,凋节基团,凋节基团形成于所述核酸探针的 3 '端,并且所述调节基团可以凋节所述发光 基团的发光,则当含有核酸探针对应核酸序列被 PCR扩增时,探针能够特异结合到扩增模 板上,此时核酸聚合酶的外切酶活性能够切下探针 5 '端基团,使发光基团和调节基团分离, 从而发出可检测的信号。
[0038] 根据本发明的具体实施例,核酸探针的发光基团为荧光基团,优选荧光基团为 F層。 根据本发明的实施例,核酸探针的调节基团为荧光基团,优选调节基团为 BHQ1。 根据 本发明的实施例,核酸探针的长度不受特别限制,例如,核酸探针的长度可以为 20个核苷 酸。 根据本发明的具体实施例,针对 SNP rs429358和 rs7412之间的核酸序列设计一段具 有特异性的核酸荧光探针。 根据本发明的一个实施例,核酸探针的序列为 5 ' FAM-CAGCTCCT CGGTGCTCTGGC- BHQ13 '。 由此,当检测核酸样木的 ApoE基因型时,仅需在 PCR扩增休系中加 入按上述方法设计的核酸荧光探针,利用 E2/E3/E4基因型各自对应的不同引物分别 PCR扩 增待测 DNA模板。 PCR反应结束后,检测各基冈型对应的扩增体系中有无荧光信号,即可判 断待测核酸样本中是否含有该亚型的 ApoE基因。
[0039] 根据本发明的实施例,核酸样本的类型及来源并不受特别限制,例如,可以是人基 因组 DNA的至少一部分,可以来源于任何人体组织或细胞、重组核酸或转基因生物组织细 胞。由此,利用根据本发明实施例的方法,可以有效地确定核酸样本的两个预定位点是否具 有已知突变,进一步可以有效地确定 ApoE的等位基因分型,对于老年痴呆症和心脑血管疾 病的预防以及早期诊断具有非常重要的意义。
[0040] 在本发明中所使用的术语"核酸样本"的含义是指在任何涉及人基因组 DNA的至 少一部分,检测的核酸样本可以来源于任何人体组织或细胞、重组核酸或转基因生物组织 细胞。 在本发明中,除非特别说明,"第一与、第二 "仅是为了便于描述木发明,不能理解为 对本发明的限制。 在本发明中,除非特别说明,"近 3' 端"指的是,距离 3' 末端的碱基为预 定范围以内的碱基,例如距离 10个, 9个, 8个, 7个, 6个, 5个, 4个, 3个, 2个, 1个或者 0个 (即 3' 末端)。
[0041 ] S200:检测反应体系的发光,以便确定核酸样本的预定位点是否存在已知突变
[0042] 在该步骤屮,对步骤 S100屮所得到的 PCR扩增样本进行荧光检测,在荧光检测时, 根据 PCR扩增体系中有无荧光信号即可定性判断待测核酸样本中是否具有该 PCR扩增体系 引物对所对应的 ApoE等位基因分型。 由此,可以确定核酸样本的预定位点是否存在已知突 变,并进一步可以有效地确定 ApoE等位基因的类型。 [0043] 根据本发明的具体实施例,对人体基因组 DNA的 PCR扩增样本进行荧光检测,如果 仅在 ε 2扩增体系中检测到荧光信号,则该样品来源的待测人 ApoE基因型为 ε 2/ ε 2 ;同 理如果仅在 ε 3 (或 ε 4 )扩增体系中检测到荧光信号,则该样品來源的待测人 ApoE基因型 为 ε 3/ ε 3 (或 ε 4/ ε 4 );如果同时在 ε 2和 ε 3扩增体系中检测到荧光信号,则该样品来 源的待测人 ApoE基闲型为 ε 2/ ε 3;同理如果同时在 ε 2和 ε 4扩增体系中检测到荧光信 号,则该样品来源的待测人 ApoE基因型为 ε 2/ ε 4;同理如果同时在 ε 2和 ε 4 (或 ε 3和 ε 4 )扩增体系中检测到荧光信号,则该样本来源的待测人 ApoE基因型为 ε 2/ ε 4 (或 ε 3/ ε 4 )。由此,可以有效地确定核酸样本的两个预定位点是否具有己知突变,进一步 Β」'以有效 地确定 ApoE的等位基因分型,对于老年痴呆症和心脑血管疾病的预防、早期诊断以及疾病 预后具有非常重要的意义。
[0044] 根据本发明的实施例, PCR扩增及荧光检测的手段并不受特别限制,可以借助任何 已知的方法与设备来进行 PCR扩增及荧光检测。 例如根据本发明的具体实施例,利用美国 Appl ied Biosystems公司的 7500Fast Real-Time PCR system,可以将 PCR热循环、荧光检 测及各种应用分析软件结合在一起,可以动态观察 PCR每一循环各反应管中 PCR扩增产物 逐渐增加的情况, PCR实验结束后可以马上得到结果,无需凝胶电泳分析,无需纯化 PCR产 物,具有时间省、灵敏度高、准确性好等优点,再例如,相关技术人员可以根据具体实际需要 的不同,对引物以及 PCR温度等进行相应的调整,在此不再赘述,这些均在本发明的权利保 护范围之内。
[0045] 在本发明的又一个方面,本发明提出了一种确定核酸样本的两个预定位点是否具 有已知突变的试剂盒。 根据本发明的具休实施例,该试剂盒可以应用于前面所述的确定核 酸样本的两个预定位点是否具有已知突变的方法。 由此,利用根据本发明实施例的确定核 酸样本的两个预定位点是否具有己知突变的试剂盒对核酸样本进行检测,可以有效地确定 核酸样本的两个预定位点是否具有已知突变,并进一步可以有效地确定 ApoE的等位基因 分型。 另外,前面针对确定核酸样本的两个预定位点是否具有已知突变的方法所描述的特 征和优点,也当然地适用该试剂盒,不再赘述
[0046] 在本发明的又 个方面,本发明提出了 ·组确定核酸样本的两个预定位点是否具 有已知突变的 PCR引物。 根据本发明的具体实施例,该引物可以应用于前面所述的确定核 酸样本的两个预定位点是否具有已知突变的方法以及试剂盒。 由此, PCR扩增过程中引物 可以与模板按照碱基配对原则选择性结合, PCR扩增可以按照预定方向进行有效扩增,并且 能够特异地扩增出不同等位基因。 另外,前面针对确定核酸样本的两个预定位点是否具有 巳知突变的方法及试剂盒所用引物的描述的特征和优点,也当然地适用该 PCR引物,不再 赘述。
[0047] 本法明的确定核酸样本的两个预定位点是否具有已知突变的方法、试剂盒及引物 可以应 ffl在老年痴呆症以及心脑血管等疾病的预防以及早期诊断方面,相关技术人员当然 也可以将其扩大至其它其应用领域,在此不予赘述,这些均在本发明的权利保护范围之内。
[0048] 下面通过具体的实施例,对本发明进行说明,需要说明的是这些实施例仅仅是为 了说明目的,而不能以任何方式解释成对本发明的限制。另外,在下列实施例中如果没有特 别说明,则所采用的设备和材料均为市售可得的。
[0049] —般方法: [0050] 1材料与试剂:
[0051] 1)醒 弓 | 物 pll2T :5' -GCGGACATGGAGGACGTGT-3',Tm=59.5 °C , GC含 量 =63.2%, 5.6nM/0D/管,加无菌去离子水 55.6 μ 1配制成 100 μ M反应液,于 4 °C储藏; 5' -CGGACATGGAGGACGTGT-3 ' , Tm=57.2°C, GC含量 =61.1%, 5.9nM/0D/管,加无菌去离子水 58.9 μ 1配制成 100 IX Μ反应液,于 4°C储藏。 引物均合成于上海博尚生物技术有限公司,纯 化方式为: PAGE纯化。
[0052] 2 ) DNA引物 pll2C :5, - CGGACATGGAGGACGTGC-3 ', Tm-59.5 °C , GC含量 =66.7%, 5.9nM/0D/管,加无茼 £·离子水 59 μ 1配制成 100 μ Μ反应液,于 4°C储藏。 引物均合成于上 海博尚生物技术有限公司,纯化方式为: PAGE纯化。
[0053] 3) DNA引物 pl58T :5' - CCTGGTACACTGCCAGGCA- 3 ', Tm=59.5 °C, GC含量 =63· 2%, 5.7nM/0D/管,加无菌去离子水 57.2 μ 1配制成 100 μ Μ反应液,于 4°C储藏。 引物均合成于 上海博尚生物技术有限公司,纯化方式为: PAGE纯化。
[0054] 4)DNA引物 pl58C :5'_CTGGTACACTGCCAGGCG- 3',Tm 59· 5°C, GC含量 =66.7%, 6nM/ 0D/管,加无菌去离子水 60 μ 1配制成 100 μ Μ反应液,于 4°C储藏。 引物均合成于上海博尚 生物技术有限公司,纯化方式为: PAGE纯化。
[0055] 5)荧光 DNA分子探针:5' FAM-CAGCTCCTCGGTGCTCTGGC- BHQ13', Tra=65°C , GC含量
=70%, 5. InM/OD/管,加无菌去离子水 51 μ 1配制成 100 u M反应液,于 4°C避光储藏。 ¾光
DNA分子探针合成于英潍捷基 (上海)贸易有限公司,纯化方式为: HPLC纯化。
[0056] 6)PCR预混液:购于美国 Applied Biosystems公司的 TaqMan Genotyping Master
Mix (包括 DNA聚合酶, dNTPs,R0X参比荧光), R0X参比荧光在 PCR反应中不会加入扩增,它 作为荧光内参能够矫正在 PCR反应中由于浓度和体积改变引起的荧光信号变化。
[0057] 7)无菌去离子水:去离子水取自英国 ELGA纯水仪(型号 Purelab3000),并经高压 蒸汽灭菌后所得。
[0058] 8) 1.5ml离心管:购于美国 Axygen公司。
[0059] 9) PCR板: MicroAmp® Opt ical96- Well Reaction Plate购于美国 Applied Biosystems公司。
[0060] 10)PCR封板膜: MicroAmp® 96- Well Optical Adhesive Film购于美 PI Applied Biosystems公司。
[0061] 11)一次性医用无菌注射器:一次性医用无菌注射器(5ml规格)购于河南新乡市 宇安医用卫材有限公司。
[0062] 12 )人血液基因组 DNA提取相关试剂:
[0063] a、红细胞裂解液:称取 3.735g氯化铵、三羟甲基氨基甲烷(Tris)l.3g加水溶解并 稀释至 500ml,0.22m滤膜(购于美国 Millipore公司)过滤除菌,于 4°C保存;
[0064] b. Nuclei Lysis Solution (购于美国 Promega公司);
[0065] c> Precipitation Solution (购于美国 Promega公司)。
[0066] 13) ε 2/ ε 2DNA已知样本:从 ε 2/ ε 2基因组 DNA巾 PCR扩增并测序验证的 DNA 样品。
[0067] 14) ε 3/ ε 3DNA已知样本:从 ε 3/ ε 3基因组 DNA中 PCR扩增并测序验证的 D.\A 样品。 [0068] 15) ε 4/ ε 4DNA己知样本:从 ε 4/ ε 4基因组 DNA中 PCR扩增并测序验证的 DNA 样品。
[0069] 16) ε 2/ ε 3D A已知样本:从 ε 2/ ε 3基因组 DNA中 PCR扩增并测序验证的 DXA 样品。
[0070] 17) ε 2/ ε 4DNA已知样本:从 ε 2/ ε 4基丙组 DNA中 PCR扩增并测序验证的 D.\A 样品。
[0071] 16) ε 3/ ε 4DNA已知样本:从 ε 3/ ε 4基因组 DNA巾 PCR扩增并测序验证的 D A 样品。
[0072] 2仪器与设备:
[0073] 1)移液器:微量移液器 (10 UL, 20 L, 100 μί, 1000 L)购于德国 Eppendorf 公 司。
[0074] 2)离心机:小型高速离心机(型 · :5424),购于德国 Eppendorf 公司。
[0075] 3)离心机:Universal32R型控温高速离心机,购于德国 Hettich zentrifugen公 司。
[0076] 4)涡旋混合器:购于江苏海门其林贝尔仪器制造有限公司。
[0077] 5)全波长扫描式多功能读数仪: Thermo Scientific Varioskan Flash全波长扫 描式多功能读数仪,购于 Thermo Scientific公司。
[0078] 6) Real-Time PCR仪器:7500Fast Real-Time PCR system,购丁 -美国 Applied Biosystems公司。
[0079] 7)分析软件 :7500software V2.0.6,购于美国 Applied Biosystems公司。
[0080] 在下列实施例中(详细见下面的实施例 1-3),确定核酸样本的两个预定位点是否 具有已知突变的方法的步骤如下:
[0081] 1引物设计:
[0082] ApoE基因的 DNA序列由美国国家生物科技信息中心(NCBI)网站提供,根据包含 ApoE基因多态性位点 SNP rs429358和 SNP rs7412的 DNA序列信息,对引物进行如下设计:
[0083] 1)针对 SNP rs429358位点核酸形式为 T的正向 PCR扩增引物(pll2T), 其 DNA序 列为:5' -GCGGACATGGAGGACGTGT-3 '和 5' - CGGACATGGAGGACGTGT- 3 ';
[0084] 2)针对 SNP rs429358位点核酸形式为 C的正向 PCR扩增引物(pll2C), 其 DNA序 列为:5' -CGGACATGGAGGACGTGC-3 ';
[0085] 3)针对 SNP rs7412位点核酸形式为 T的反向 PCR扩增引物(ρ158Τ) , 其 DNA )Υ- 列为:5' - CCTGGTACACTGCCAGGCA- 3';
[0086] 4)针对 SNP rs7412位点核酸形式为 C的反向 PCR扩增引物(pl58C), 其 DNA序 列为 :5' - CTGGTACACTGCCAGGCG- 3'。
[0087] 2荧光 ϋΝλ分子探针设计:
[0088] 根据美国国家生物科技信息巾心(NCBI)网站提供的 ApoE基因 DNA序列信息,该 基冈 SNP rs429358位点与 SNP rs7412位点之间的 DNA序列为: tgcggccgcctggtgcagtacc gcggcgaggtgcaggccatgctcggccagagcaccgaggagctgcgggtgcgcctcgcctcccacctgcgcaagctg cgtaagcggctcctccgcgatgccgatgacctgcagaagcgc,选取该序歹 lj巾的一段作为探针的互补核 酸序列部分,该序列为 5' -CAGCTCCTCGGTGCTCTGGC-3 选用荧光探针,选取 FAM荧光基团 以及 BHQl淬灭基团, FAM荧光基团偶联在互补核酸序列的 5 '端, BHQ1淬灭基团偶联在互补 核酸序列的 3 '端,荧光探针为:5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 '。
[0089] 以下部分以人血液基因组 DNA样本为检测样品进行阐述:
[0090] 3操作流程
[0091 ] 1 )样本采集:
[0092] 使用一次性医用无菌注射器( 5ml )抽取人静脉血 1ml于含有 1. 5mg EDTA钠抗凝 血剂的管中,放置 4°C冰箱储藏待用。
[0093] 2 )人血液基因组醒提取:
[0094] a、从 4°C冰箱取出采集的人血液并轻轻混匀,然后用移液器取 300ul血液转移到 含有 900ul红细胞裂解液的 1. 5ml离心管中,上下颠倒 5_6次。
[0095] b、室温放置 10分钟(期 W颠倒 2-3次),室温条件下 13000-16000g离心 20秒。
[0096] c、吸去上清,剩下约 10-20ul于管底。
[0097] d、涡旋振荡重悬白细胞,加入 Nuc le i Lysi s Solut ion,吹吸 5-6次,可选步骤加入 1. 5ul的 RNA酶混匀,37°C条件下放置 15分钟。
[0098] e、加入 lOOul Precipi tat ion Solut ion,涡旋振荡 10-20秒。
[0099] f、室温条件下 13000_16000g离心 10分钟。
[0100] g、转移上清到新的 1. 5ml离心管,加入 300ul异内醇,上下轻轻颠倒直到有白色絮 状 DNA出现。
[0101 ] h、室温条件下 13000- 16000g离心 1分钟。
[0102] i、吸去上清,加入 70%乙醇,轻轻的上下颠倒几次洗涤 DNA,室温条件下 13000-16000g离心 1分钟。
[0103] j、轻轻吸去乙醇,将 1. 5ml离心管倒置在干净的吸水纸上 10-15分钟。
[0104] k、加入 lOOul无菌去离了水溶解 DNA,65°C水浴 1小时,也可以 4°C过夜。
[0105] 1、利用全波长扫描式多功能读数仪中的 DNA浓度测量程序测量所提 DM的浓度, 取一部分 DNA溶液用无菌去离子水稀释至 2ng/ml,作为后续 PCR检测模板,剩余 DNA溶液存 放于 2〜 8 °C或 _20°C。
[0106] 3 ) PCR反应体系配制:
[0107] 根据 ApoE基因的 个多态性,与二种检测试剂( ε 2试剂、 ε 3试剂和 ε 4试剂), 分别用于人群中存在 6种不同 ΑροΕ基因型 ε 2/ ε 2, ε 3/ ε 3, ε 4/ ε 4, ε 2/ ε 3, ε 2/ ε 4 和 ε 3/ ε 4的检测。 6种已知 ΑροΕ基因型 H\A己知样本( ε 2/ ε 2DNA已知样本、 ε 3/ ε 3DNA 己知样本、 ε 4/ ε 4DNA己知样木、 ε 2/ ε 3DNA己知样木、 ε 2/ ε 4DNA已知样木和 ε 3/ ε 4DNA已知样本)作为对照
[0108] 其中 ε 2试剂包括:
[0109] a、对应 ΑροΕ ε 2等位基因型 SNP位点 rs429358SNP核酸形式的正向引物 pi 12T (该 实例巾核酸序列为 5 ' -GCGGACATGGAGGACGTGT-3 ' )。
[01 10] b、对应 ΑροΕ ε 2等位基闲型 SNP位点 r s 7412反向引物核酸形式的反向引物 p 158T (该实例中核酸序列为 5 ' -CCTGGTACACTGCCAGGCA-3 ' )。
[01 1 1 ] c、荧光探针(该实例巾探针形式为 5 ' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ13 ' )。
[01 12] d、PCR预混液 TaqMan Genotyping Master Mix。 [0113] 其各成分具体剂量为:
[0114] TaqMan Genotyping Master Mix 4.925 μ 1
[0115] ε 2正向引物 pll2T (ΙΟΟμΜ) 0.0125μ 1
[0116] ε 2正向引物 ρ158Τ (ΙΟΟμΜ) 0.0125 μ 1
[0117] 荧光探针(100 μΜ) 0.05u 1
[0118] 其中 ε 3试剂包括:
[0119] a、对应 ApoE ε 3等位基因型 SNP位点 rs429358SNP核酸形式的正向引物 ρ112Τ (该 实例屮核酸序列为 5' -CGGACATGGAGGACGTGT-3 ' )。
[0120] b、对应 ΑροΕ ε 3等位基因型 SNP位点 rs7412反向引物核酸形式的反向引物 pl58C (该实例中核酸序列为 5' -CTGGTACACTGCCAGGCG-3 ' )。
[0121] c、荧光探针(该实例中探针形式为 5' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ 13 ' )。
[0122] d、 PCR预混液 TaqMan Genotyping Master Mix。
[0123] 其各成分具体剂量为:
[0124] TaqMan Genotyping Master Mix 4.925 μ 1
[0125] ε 3正向引物 pll2T (ΙΟΟμΜ) 0.0125 μ 1
[0126] ε 3正向引物 pl58C (ΙΟΟμΜ) 0.0125 1
[0127] 荧光探针(100 μΜ) 0.05μ 1
[0128] 其巾 ε 4试剂包括:
[0129] a、对应 ApoE ε 4等位基因型 SNP位点 rs429358SNP核酸形式的正向引物 pi 12C (该 实例中核酸序列为 5' -CGGACATGGAGGACGTGC-3 ' )。
[0130] b、对应 ΑροΕ ε 4等位基因型 SNP位点 rs7412反向引物核酸形式的反向引物 pl58C (该实例中核酸序列为 5' -CTGGTACACTGCCAGGCG-3 ' )o
[0131] c、荧光探针(该实例中探针形式为 5' FAM-CAGCTCCTCGGTGCTCTGGC-BHQ 13 ' )。
[0132] d、PCR预混液 TaqMan Genotyping Master Mix。
[0133] 其各成分具体剂量为:
[0134] TaqMan Genotyping Master Mix 4.925 μ 1
[0135] ε 4止向引物 pll2C (100μΜ) 0.0125 1
[0136] ε 4正向引物 pl58C (ΙΟΟμΜ) 0.0125 μ 1
[0137] 荧光探针(100 μΜ) 0.05 μ 1
[0138] 使用 ApoE基因分型检测试剂需要 10 μ 1反应体系就可以充分检测到信号。
[0139] ε 2基因检测的 PCR反应体系为:
[0140] 2试齐11: 5μ 1
[0141] 待测 DNA样品(2ng/ u 1) : 5 μ 1
[0142] ε 2阳性对照检测体系 1:
[0143] £ 2试齐 5μ 1
[0144] ε 2/ ε 2DNA已知样本(2pg/ul): 5 μ 1
[0145] ε 2阳性对照检测体系 2:
[0146] £ 2试齐1」: 5μ 1
[0147] ε 2/ ε 3DNA已知样本(2pg/ul ): 5 μ 1 :0148] ε 2阳性对照检测体系 3:
:0149] 52试齐1」: 5μ 1
:0150] ε 2/ ε 4DNA已知样本( 2pg/ul ): 5 μ 1
:0151] ε 2阴性对照检测体系 1:
:0152] £ 2试齐!1: 5μ 1
:0153] ε 3/ ε 3DNA已知样本( 2pg/ul ): 5 μ 1
:0154] ε 2阴性对照检测体系 2 :
:0155] £ 2试齐1」: 5μ 1
:0156] ε 4/ ε 4DNA已知样木( 2pg/ul ): 5μ 1
:0157] ε 2阴性对照检测体系 3:
:0158] 2试齐|]: 5μ 1
:0159] ε 3/ ε 4DNA已知样本(2pg/ul): 5μ 1
:0160] ε 3基因检测的 PCR反应体系为:
:0161] 63试齐1」: 5μ 1
:0162] 待测 DNA样品(2ng/ μ 1) : 5 μ 1
:0163] ε 3阳性对照检测体系 1 :
:0164] 3试齐1」: 5μ 1
:0165] ε 3/ ε 3DNA已知样本( 2pg/ul ): 5 μ 1
:0166] ε 3阳性对照检测体系 2 :
:0167] £ 3试齐 5μ 1
:0168] ε 2/ ε 3DNA已知样本( 2pg/ul ): 5 μ 1
:0169] ε 3阳性对照检测体系 3 :
:0170] 3试齐[]: 5μ 1
:0171] ε 3/ ε 4DNA已知样本( 2pg/ul ): 5 μ 1
:0172] ε 3阴性对照检测体系 1:
:0173] 63试齐1」: 5μ 1
[0174] ε 2/ ε 2DNA已知样本( 2pg/ul ): 5 μ 1
[0175] ε 3阴性对照检测体系 2 :
[0176] ε 3试剂: 5μ 1
[0177] ε 4/ ε 4DNA已知样本(2pg/ul): 5μ 1
[0178] ε 3阴性对照检测体系 3 :
[0179] £ 3试齐!」: 5μ 1
[0180] ε 2/ ε 4DNA已知样本( 2pg/ul ): 5 μ 1
[0181] ε 4基因检测的 PCR反应体系为:
[0182] £ 4试齐[1: 5μ 1
[0183] 待测 DNA样品(2ng/ 1): 5u 1
[0184] ε 4阳性对照检测体系 1:
[0185] 64试齐1」: 5u 1
[0186] ε 4/ ε 4DNA已知样本( 2pg/ul ): 5μ 1 [0187] ε 4阳性对照检测体系 2:
[0188] £ 4试齐!1 : 5μ 1
[0189] ε 2/ ε 4DNA已知样本( 2pg/ul ): 5 μ 1
[0190] ε 4阳性对照检测体系 3 :
[0191] ε4试齐lJ : 5μ 1
[0192] ε 3/ ε 4DNA已知样木( 2pg/ul ): 5μ 1
[0193] ε 4阴性对照检测体系 1:
[0194] £4试齐^ 5μ 1
[0195] ε 2/ ε 2DNA已知样木(2pg/ul): 5μ 1
[0196] ε 4阴性对照检测体系 2:
[0197] 4试齐|]: 5μ 1
[0198] ε 3/ ε 3DNA已知样本(2pg/ul): 5u 1
[0199] ε 4阴性对照检测体系 3 :
[0200] ε 4试剂: 5 μ 1
[0201] ε 2/ ε 3DNA已知样本(2pg/ul): 5μ 1
[0202] 4) PCR扩增反应:
[0203] 将待测样品即人血液提取的基因组 DNA (2ng/ μ 1 )与已知 ΑροΕ基因型的 DNA已 知样品分别加入 ε 2, ε 3和 ε 4基因检测的 10 μ 1 PCR反应体系巾。用灭菌的去离子水作 为整个 PCR体系的阴性对照。 在 PCR板中配制好所有 PCR反应体系后,用 PCR封板膜封闭 好 PCR板,残留在 PCR板孔壁上液体 ffl Universal32R型控温高速离心机离心到管底并排除 气泡。然后,将该 PCR板放入 7500Fast Real-Time PCR system仪器中,打开 7500software V2.0.6软件,选择软件中的 Genotyping模块,在 Plate setup中设置对应放入 PCR板_ I:每 孔的检测内容。 然后在 Run method中设置 PCR反应程序及反应体系,这些项目设置完毕 后,便可运行程序,该程序默认 PCR反应之前先在 60°C条件下预读取本底荧光强度值再进 行 PCR扩增以消除背景荧光值。 待 PCR反应完成之后,仪器可自动在 60Γ条件卜―收集荧光 数据。 表 1为该实例 PCR反应过程的标准热循环程序。
[0204] 表 1PCR反应程序
[0205]
Figure imgf000013_0001
[0206] 5)数据分析与结论: [0207] 在 Real-Time PCR仪读完荧光值之后导出荧光数值,结果如图 4所示。 在 ApoE基因型为 ε 2/ ε 2的DNA已知样品中, ε 2基因检测体系为阳性结果,其荧光值为 2. 23623729 ο ε 3, ε 4基因检测体系为阴性结果,其荧光值分别为 0. 21861 1和 0. 51656318; 在 ApoE基因型为 ε 3/ ε 3的 DNA已知样品中, ε 3基因检测体系为阳性结果,其荧光 值为 2. 50921822。 ε 2, ε 4基冈检测体系为阴性结果,其荧光值分别为 0. 1933465和 0. 40723515;在 ApoE基因型为 ε 4/ ε 4的 DNA已知样品中, ε 4基因检测体系为阳性结果, 其荧光值为 2. 59521461。 ε 2, ε 3基因检测体系为阴性结果,其荧光值分别为 0. 17685843 和 0. 22782183。 作为阴性对照的无茼去离子水样品,在 ε 2, ε 3和 ε 4基因检测体系屮, 其荧光值分别为 0. 17888165, 0. 22742152和 -0. 0582402。 这样,在这一实例中最大的阴性 结果荧光值为 0. 51656318,而所有阳性结果的荧光值均远大于最大的阴性结果荧光值。 在 此定义以最大阴性结果荧光值的 2倍数值作为荧光信号阳性和阴性结果的分界值,该实例 中这一分界值为 1. 03312636。
[0208] 根据图 4中的荧光值可以作杵形图,柱形图如图 5所示,在阳性与阴性结果分界值 处划一直线,可以直观的看出所有的阳性结果都在直线上方,而所有阴性结果都在直线下 方。 该实例中待测 DNA样品在 ε 2和 ε 3基因检测体系得到结果根据上述分析方法判断为 阳性结果,而在 ε 4基因检测体系得到结果根据上述分析方法判断为阴性结果。 由此得知, 该实例中待测 DNA样品的 ApoE基 141型为 ε 2/ ε 3型。
[0209] 实施例 1
[0210] 在实施例中,如图 1所示,引物 ρ112Τ仅与 SNP rs429358为 Τ的核酸序列匹配, 引物 pl l2C仅与 SNP rs429358为 C的核酸序列匹配,引物 pl58T仅与 SNP rs7412为 T的 核酸序列匹配,引物 P158C仅与 SNP rs7412为 C的核酸序列匹配。 ώ此,含引物对 ρ112Τ/ ρ158Τ的 PCR体系仅能扩增 SNP rs429358为 T同时 SNP rs7412为 T的 ApoE等位基冈(即 ε 2 );含引物对 pl l2T/pl58C的 PCR体系仅能扩增 SNP rs429358为 T同时 SNP rs7412为 C的 ApoE等位基因(即 ε 3 );含引物对 l l2C/pl58C的 PCR体系仅能扩增 SNP rs429358为 C同时 SNPrs7412为 C的 ApoE等位基因(即 ε 4)。 所以,利用上述三种引物对,可以特异地 扩增出 ApoE的不同等位基因,并确定 DNA样品中人 ApoE等位基因的类型。
[021 1 ] 实施例 2
[0212] 在实施例中,如图 2所不,荧光基团(F )脱离的 DNA分子探针 3的两段分别偶联有 荧光基 ffl和淬灭基团,此时由于淬灭基团能吸收荧光基团的激发能量,因此,体系中只能检 测到本底荧光信号。 荧光基闭(F )脱离的 DNA分子探针 3能与 ApoE基因 DNA模板反义链 1特异互补匹配,且匹配序列位于正向扩增引物 2和反向扩增引物 5对应的 DNA序列之间, 当 PCR扩展反应开始后, ιΗ向扩增引物 2和荧光基团(F)脱离的 DNA分子探针 3将与 ApoE 基闵 DNA模板反义链 1结合,反向扩增引物 5将与 ApoE基冈 D A模板正义链 4结合,在 DNA 聚合酶 6的作用下开始扩增模板 DNA。
[0213] 实施例 3
[0214] 在实施例屮,如图 3所示, DNA聚合酶 6在 PCR反应过程屮从正向扩增引物 2或反 向扩增引物 5开始,以 ApoE基因 DNA模板反义链 1或 ApoE基因 DNA模板正义链 4为模板, 起始 DNA的延伸,当 DNA聚合酶 6在介导 DNA延伸的过程中遇到结合在 ApoE基因 DNA模板 反义链 1上的荧光基团(F )脱离的 DNA分子探针 3,DNA聚合酶 6将利用其外切酶活性从荧 光基团(F)脱离的 DNA分子探针 3的 5 '端逐 ^刀掉探针上的核苷酸,致使荧光基团从探针 上脱离并与淬灭基团分离,此时即能检测到荧光基团激发出的荧光信号,并由此确定检测 样品含有该引物对所对应的 ApoE等位基因类型。
[0215] 尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例 性的,不能理解为对本发明的限制,木领域的普通技术人员在不脱离木发明的原理和宗旨 的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型,这些均落在本 发明的权利保护范围之内。

Claims

权 利 要 求 书
1. 一种确定核酸样本的两个预定位点是否具有已知突变的方法,其特征在于,包括: 利用第一引物和第二引物对所述核酸样本进行 PCR扩增,并且在所述 PCR扩增体系屮 加入核酸探针,所述核酸探针对应两个预定位点之间至少一部分的核酸序列;以及
检测反应体系的发光,以便确定所述核酸样本的预定位点是否存在巳知突变, 其中,
所述第一引物的近 3 '端包含与所述两个预定位点之一己知突变对应的碱基, 所述第二引物的近 3 '端包含与所述两个预定位点另一个己知突变对应的碱基, 所述第一引物和第一引物之一作为正向引物,所述第一引物和所述第二引物的另一个 作为反向引物,
所述核酸探针具有 - 发光基团,所述发光基团形成于所述核酸探针的 5 '端,
调节基团,所述调节基团形成丁万述核酸探针的 3 '端,并且所述调节基团可以调节所 述发光基闭的发光,
任选地,所述核酸样本来源于人体组织或细胞、重组核酸或转基因生物组织细胞, 任选地,所述预定位点是 SNP rs429358和 rs7412,
任选地,所述述第一引物的近 3 '端包含与 rs429358的已知突变对应的碱基,所述第二 引物的近 3 '端包含与 rs7412的已知突变对应的碱基,
任选地,所述第一引物的 3 '端碱基为与 rs429358的已知突变对应的碱基,所述第二引 物的 3 '端碱基为与 rs7412的已知突变对应的碱基,
任选地,所述第 ·引物作为正向引物,所述第二引物作为反向引物,
任选地,所述第一引物为选 Θ下列的至少之一:
5 ' - CGGACATGGAGGACGTGT-3 '
5 ' - GCGGACATGGAGGACGTGT - 3,
5 ' -CGCGGACATGGAGGACGTGT-3 '
5 ' - CGGACATGGAGGACGTGC- 3 ' ·
5 ' - GCGGACATGGAGGACGTGC- 3,
5 ' - CGCGGACATGGAGGACGTGC- 3 ',
所述第二引物为选自下列的至少之一:
5 ' -TGGTACACTGCCAGGCA-3 '
5 ' -CTGGTACACTGCCAGGCA-3,
5 ' -CCTGGTACACTGCCAGGCA-3 '
5 ' - TGGTACACTGCCAGGCG- 3,
5 ' -CTGGTACACTGCCAGGCG-3 '
5 ' -CCTGGTACACTGCCAGGCG-3 ',
任选地,所述核酸探针的长度为 20个核苷酸。
2. 根据权利要求 1所述的方法,其特征在于,所述核酸探针的序列为:
5 ' -CAGCTCCTCGGTGCTCTGGC- 3 '。
3. 根据权利要求 1所述的方法,其特征在于,所述核酸探针的发光基团为荧光基团, 任选地,所述荧光基团为 FAM, 任选地,所述调节基团为淬灭基团,
任选地,所述淬灭基团为 BHQ1。
4. 一种确定核酸样本的两个预定位点是否具有已知突变的试剂盒,其特征在于,包 括:
第一引物,所述第一引物的近 3 '端包含与所述两个预定位点之一已知突变对应的碱 基,
第二引物,所述第二引物的近 3 '端包含与所述两个预定位点另一个已知突变对应的碱 基,
核酸探针,所述核酸探针与两个预定位点之间至少一部分对应的核酸序列,并且所述 核酸探针具有:
发光基团,所述发光基团形成于所述核酸探针的 5 '端,
调节基团,所述调节基团形成于所述核酸探针的 3 '端,并且所述调节基团可以调节所 述发光基团的发光,
任选地,所述预定位点是 SNP rs429358和 rs7412 ,
任选地,所述述第- -引物的近 3 '端包含与 rs429358的已知突变对应的碱基,所述第二 引物的近 3 '端包含与 rs7412的巳知突变对应的碱基,
任选地,所述第一引物的 3 '端碱基为与 rs429358的已知突变对应的碱基,所述第二引 物的 3 '端碱基为与 rs7412的已知突变对应的碱基,
任选地,所述第一引物作为正向引物,所述第二引物作为反向引物,
任选地,所述第一引物为选自下列的至少之一:
5 ' -CGGACATGGAGGACGTGT-3 '
5 ' -GCGGACATGGAGGACGTGT-3 '
5, -CGCGGACATGGAGGACGTGT-3 '
5 ' -CGGACATGGAGGACGTGC-3 '
5 ' - GCGGACATGGAGGACGTGC- 3 '
5 ' -CGCGGACATGGAGGACGTGC- 3 ',
所述第二引物为选 下列的至少之一:
5 ' - TGGTACACTGCCAGGCA- 3 '
5 ' -CTGGTACACTGCCAGGCA-3 '
5 ' -CCTGGTACACTGCCAGGCA-3,
5 ' -TGGTACACTGCCAGGCG-3 '
5 ' -CTGGTACACTGCCAGGCG-3 '
5 ' - CCTGGTACACTGCCAGGCG- 3,,
任选地,所述核酸探针的长度为 20个核苷酸。
5. 根据权利要求 4所述的试剂盒,其特征在于,所述核酸探针的序列为:
5 ' - CAGCTCCTCGGTGCTCTGGC- 3 '。
6. 根据权利要求 4所述的试剂盒,其特征在于,所述核酸探针的发光基团为荧光基团, 任选地,所述荧光基团为 FAM,
任选地,所述调节基团为淬灭基团, 任选地,所述淬灭基团为 BHQ1。
7. 一组确定核酸样本的两个预定位点是否具有巳知突变的 PCR引物,其特征在于,包 括:
第一引物,所述第一引物的近 3'端包含与所述两个预定位点之一巳知突变对应的碱 基,
第二引物,所述第二引物的近 3'端包含与所述两个预定位点另一个已知突变对应的碱 基,
核酸探针,所述核酸探针与两个预定位点之间至少一部分对应的核酸序列,并且所述 核酸探针具有:
发光基团,所述发光基团形成于所述核酸探针的 5'端,
调节基团,所述凋节基团形成于所述核酸探针的 3'端,并且所述凋节基团可以调节所 述发光基团的发光,
任选地,所述预定位点是 SNP rs429358和 rs7412,
任选地,所述述第一引物的近 3'端包含与 rs429358的已知突变对应的碱基,所述第— 引物的近 3'端包含与 rs7412的已知突变对应的碱基,
仟选地,所述第一引物的 3'端碱基为与 rs429358的巳知突变对应的碱基,所述第二引 物的 3'端碱基为与 rs7412的已知突变对应的碱基,
任选地,所述第一引物作为正向引物,所述第二引物作为反向引物,
任选地,所述第一引物为选自下列的至少之一:
5' -CGGACATGGAGGACGTGT-3 '
5' -GCGGACATGGAGGACGTGT-3 '
5' -CGCGGACATGGAGGACGTGT-3 '
5' -CGGACATGGAGGACGTGC-3 '
5 ' - GCGGACATGGAGGACGTGC- 3,
5 ' - CGCGGACATGGAGGACGTGC- 3 ',
所述第二引物为选自下列的至少之 -:
5' -TGGTACACTGCCAGGCA-3 '
5' - CTGGTACACTGCCAGGCA- 3'
5' -CCTGGTACACTGCCAGGCA-3 '
5' -TGGTACACTGCCAGGCG-3 '
5' -CTGGTACACTGCCAGGCG-3 '
5' -CCTGGTACACTGCCAGGCG— 3,。
PCT/CN2013/001453 2012-12-31 2013-11-27 确定核酸样本的两个预定位点是否具有已知突变的方法、试剂盒以及引物 WO2014101276A2 (zh)

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CN106011298A (zh) * 2015-11-12 2016-10-12 厦门人瑞生物医药科技有限公司 一种ApoE试剂盒、引物及其用途
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