CN1514016A - Method of gene type determination - Google Patents

Abstract

A method for testing the genotype features that the terminals 5' of the polymorphic primer and genotype primer respectively and partly have specific exogenous sequences and their terminals 3' have the sequences complementary with target sequence. There is a non-natural structure between exogenous sequence and complementary sequence. Its advantages are use of universal gene chip and universal genotype probe, high correctness, and low cost.

Description

A kind of method of genotype detection

The present invention relates to a kind of biotechnology, more specifically to a kind of method of genotype detection.

The genetic material of all living species is a nucleic acid, comprise Yeast Nucleic Acid (RNA) and thymus nucleic acid (DNA), the essentially consist of decision nucleic acid function feature is four kinds of bases, and they are respectively by A, C, G, T representative in DNA, and they are respectively by A, C, G, U representative in RNA.These are called nature Nucleotide or natural base in Nucleotide or the base that nature can find and be present in the nucleic acid, can join non-natural Nucleotide or non-natural base or other improper nucleic acid component in the nucleic acid chains by chemical synthesis process in addition, so the non-naturality organization definition of nucleic acid is: be not present in natural structure (can only synthetic) or other structure (in DNA, also belonging to non-natural structure) except that normal four kinds of Nucleotide (or four kinds of bases) as U.Base is formed chain-like structure with serial arrangement in nucleic acid, RNA is generally single-stranded structure, DNA is generally duplex structure, the T of base A and another chain (U) matches by hydrogen bond in duplex structure, in like manner the C of G and another chain matches, the whole double-stranded DNA of nature (in the biology) all matches fully, and the stability of duplex structure will reduce when situation about not exclusively matching occurs, and two nucleic acid chains that match on sequence are called complementary strand.Nucleotide sequence can change during evolution with under the influence of ambient conditions, the modal base that is changed to is replaced by another base, in same species, if any different base compositions, then this a kind of phenomenon is called single base (Nucleotide) polymorphism (SNP) to the same position of same nucleotide sequence (single base position) between different individualities.In particular individual, a kind of based composition of pleomorphism site is a kind of genotype (gene is the functional region of nucleic acid) in this site, a monoploid biont can only have a genotype on a polymorphic point, in amphiploid biont, has identical genotype as two homologous genes, this individuality is a homozygote, if any different genotype, then this individuality is a heterozygote.Genotype detection is significant to genetic diagnosis, species evaluation, drug screening etc.DNA can be replicated under the effect of polysaccharase etc., promptly copy complementary strand by template strand, duplicating of DNA needs a primer, primer is generally one and contains nucleic acid chains several or tens bases, nucleic acid chains has directivity and uses 5 ' end and 3 ' end expression respectively, sequence near 5 ' end is called 5 ' end parts, the 3 ' end parts that is called near 3 ' end, primer and template are held to have specifically 3 ' and are combined into duplex structure (this process is called molecular hybridization) according to the base pairing principle before the nucleic acid replication, after this polysaccharase can be from 3 ' end beginning of primer along 5 ' → 3 ' direction synthetic with the template new chain of complementary mutually, the new two strands that forms at high temperature (＞90 ℃) is separated into independently strand, the primer molecule of remainder can be again and the template corresponding making nucleic acid molecular hybridization when temperature descends, and can under the effect of polysaccharase, duplicate the nucleic acid chains that makes new advances, as above process repeatedly, a nucleic acid fragment can be amplified out ten million same molecule fragment, and as above process is pcr process (PCR).The part that one nucleic acid samples need increase is the target fragment, carry out the PCR reaction and generally corresponding primer will be arranged at target fragment two ends, to near the single-minded primer of the sequence the polymorphic site is polymorphism primer, specific gene type to a pleomorphism site has the narrow spectrum genotype primer that is, polymorphism primer can be used in combination with the genotype primer target fragment is carried out pcr amplification, and measures genotype.With the method for chemistry can the synthetic nucleic acid fragment as primer, the base sequence of primer is complementary mutually with the target fragment sequence, can have in the primer sequence in addition with nucleic acid samples in the non-complementary sequence of sequence, this and sample are not had a sequence of complementarity and are called exogenous array, and exogenous array can be used as the part-structure of primer.Gene chip is a kind of the different IPs acid molecule to be arranged in specific position respectively, and be used for the device of making nucleic acid molecular hybridization, the nucleic acid molecule that is fixed on the gene chip is a probe molecule, with probe molecule carry out specific hybrid for target molecule, probe molecule can by in the position of chip or with mark carry out target molecule identification, mark can have various ways, as radio-labeling, fluorescent mark, nanoparticle label, enzyme labelling, pigment mark etc., be the genotype probe in order to the nucleic acid probe molecules of tool mark of difference different genotype.Genotype detection method commonly used has nucleic acid sequencing method, gene chip hybridization method etc., and the slow cost height of nucleic acid sequencing method speed is not suitable for large-scale genotype detection; The result of use of gene chip hybridization method is decided by the preparation process of sample, and general specimen preparation process comprises multistep enzyme reaction, PCR reaction, labeled reactant etc., uses multistep enzyme reaction meeting to make cost up, genotype detection time growth etc.

The method that the purpose of this invention is to provide a kind of genotype detection, it can overcome the above-mentioned deficiency of prior art.

A kind of method of genotype detection, comprise and use polymorphism primer and genotype primer that the nucleic acid target fragment that contains pleomorphism site is increased, make amplified production and gene chip probe molecule and and general genotype probe hybridize, judge the genotype of pleomorphism site according to the signal of genotype probe on gene chip, 5 ' the end parts that it is characterized in that polymorphism primer and genotype primer has single-minded exogenous array respectively, 3 ' end parts of polymorphism primer and genotype primer has respectively and target sequence complementary sequence mutually, and non-naturality structure is arranged between exogenous array and the complementary sequence.

Advantage of the present invention is available general gene chip, need not in reactions such as the laggard row labels of PCR, utilizes that general genotype probe can save time, material-saving, attenuating consumption reduce cost, and can reduce the false positive that occurs in detecting and improve and detect quality.What of the genotype of animal, plant, microorganism and virus or its nucleic acid amount are present method can be used for measuring, and use present method and also can make the test kit of commercial value or be engaged in the molecular diagnosis service.

Below by embodiment the present invention is described.

Make the gene chip of diagnosis inherited disease, the inherited disease of being diagnosed can be hypertension, diabetes etc.Molecular probe on the gene chip is and the exogenous array of polymorphism primer complementary nucleic acid molecule mutually, its length is made up of 10-50 Nucleotide, each probe is fixed in the surface of chip respectively with known method, prepare a test kit, test kit comprises and is used for primer that polymorphism target fragment is increased, the segmental primer of a polymorphism target that increases comprises single-minded polymorphism primer and two single-minded genotype primers, test kit also comprises the enzyme of the reagent PCR reaction of nucleic acid purification, damping fluid etc., test kit comprises that two or more have different marker genotypes probes respectively in addition, be labeled as the structure of the nanometer range of fluorochrome or other available chemistry or physical method identification, test kit also comprises the damping fluid that is used for molecular hybridization.Adopt following schedule of operation when carrying out genotype detection: 1. extract DNA from a patients'blood, general one sample of bleeding can extract the DNA of tens of or hundreds of nanograms, the damping fluid that the DNA of extraction is water-soluble or suitable; 2. with the PCR reaction reagent DNA nucleic acid that extracts is carried out pcr amplification, with a polymorphism primer and two single-minded genotype primers one polymorphism target fragment is increased, many group primers can use (composite PCR reaction) in same test tube, the compound degree can be two groups or two groups of above primers, product after the amplification has the polymorphism exogenous array of strand and the genotype exogenous array of strand, and the volume of reaction is generally at the 10-100 microlitre; 3. the reaction product of a plurality of PCR can merge, and carries out suitable purifying, processings (can remove unreacted primer in case of necessity) such as concentrates, and combines with the damping fluid of hybridizing usefulness then, and final volume is in 0.1-1 milliliter scope.Comprise the different marker genotypes probes of tool in the hybridization solution; 4. carry out chip hybridization and cleaning with known method and instrument, and with the signal on laser scanner or other the corresponding instrument acquisition chip; 5. according to the ratio of the intensity of position, feature and the signal of signal on chip, can measure the genotype of pleomorphism site.The doctor of hospital can make diagnosis and carry out suitable treatment according to patient's genotype.

Can be made into according to this method: 1. test kit, test kit comprises relevant reaction primer and biological chemical reagent; 2. gene chip; 3. Xiang Guan computer software (operation instruction and data analysing method).

This method is used for the mensuration relevant with nucleic acid, mainly comprises: the 1. diagnosis of human inheritance's disease; 2. human or vegeto-animal genotype detection; 3. living species is identified.(different crowd is to the reaction of medicine also to can be used for drug screening; Police criminal detection (searching criminal); Fields such as living species improvement.

Claims (1)

1. A kind of method of genotype detection, comprise and use polymorphism primer and genotype primer that the nucleic acid target fragment that contains pleomorphism site is increased, make amplified production and gene chip probe molecule and and general genotype probe hybridize, judge the genotype of pleomorphism site according to the signal of genotype probe on gene chip, 5 ' the end parts that it is characterized in that polymorphism primer and genotype primer has single-minded exogenous array respectively, 3 ' end parts of polymorphism primer and genotype primer has respectively and target sequence complementary sequence mutually, and non-naturality structure is arranged between exogenous array and the complementary sequence.