CN1215165A - Down's syndrome antenatal diagnosis method and reagent box - Google Patents
Down's syndrome antenatal diagnosis method and reagent box Download PDFInfo
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Abstract
The present invention discloses a prenatal diagnosis method of Down's syndrome. It is characterized by using PCR to amplify at least three lock on the No.21 chromosome and observing the polymorphism of these loci. Besides, said invention also discloses the reagent kit for said invented method, including the specific primer for PCR amplification.
Description
The present invention relates to a kind of method of pre-natal diagnosis Down's syndrome, particularly a kind of down's syndrome antenatal diagnosis method that utilizes PCR (PCR) also relates to a kind of kit that is used for diagnostic method of the present invention.
Down's syndrome (Down ' s syndrome), claim mongolism again, or trisomy 21, be one of modal chromosomal disorder.It also is the main cause that causes congenital feeblemindedness.Its Clinical symptoms is: serious feeblemindedness, unique face and physical abnormality (as wide eye distance, the low bridge of the nose, myasthenia, limbs are short and small, and connect hand etc.), congenital heart disease, alimentary canal deformity etc., the leukemic incidence of disease is more much higher than common people.
When being reproduction cell meiosis, this sick pathogenesis do not separate.And most cases be do not separate by egg cell (75%) or (15%) meiosis for the second time for the first time due to; Betide only occupy the minority (10%) of spermatoblast.So the incidence of disease that should disease is relevant with maternal age, female age is big more, and the incidence of disease is also high more.For example, be 25 years old person female age, and the incidence of disease is 1/1200; 35 years old persons, the incidence of disease is 1/300, and 45 years old person's incidence of disease can be up to 1/50.Average attack rate in the colony is 1/700～1/800.With general genopathy (show/recessive inheritance as autosome, or the property autosome showing/recessive inheritance) difference, this chromosomoid disease of Down's syndrome is accidental, and each pregnant woman has certain danger, and the phenomenon that does not generally have race or family to gather.
Conventional cytogenetic methods diagnosis need be got fetal tissue's (amniotic fluid or fine hair) usually, through reaching the cellular incubation in two weeks, passes through complicated chromosome sectioning again, shows band, takes pictures, and workers such as the row's of cutting caryogram are continuous could to wait until the cytogenetics result.Not only the cycle is long for conventional cytogenetic methods, trivial operations, and efficient is not high.
Once reported the method for looking into chromosome abnormality with PCR (polymerase chain reaction).For example, von Eggeling F etc., (Human Genetics.91 (6): 567-70,1993 Jul.); Celi FS. etc., (Genomics.21 (2): 304-10,1994 May 15.); Lee HH. etc., (HumanGenetics.99 (3): 364-7,1997 Mar.) have reported respectively and have utilized the means of natural (existing DNA in the genome) or artificial (utilizing biotechnology to process) amplification tester quantitatively to infer chromosome number purpose test method.(Bioessays.17 (7): 661-4,1995 Jul.) such as Adinolfi M. also report the method diagnosis of down syndrome with the polymorphic repetitive sequence of pcr amplification.But be subject to the above-mentioned relevant technologies of the following factor shaped article of failing to be converted into: the quantifying PCR method with contrast or internal reference will lean on the size of PCR product to distinguish wild type and tester that pcr amplification goes out.Because the PCR product varies in size, even if use same primer, it is very big that the product amount that amplifies also can difference, therefore may cause quantitative error and mistaken diagnosis chromosome trisome syndrome.In addition, the employed instrument and equipment of most reports all expensive (as use capillary electrophoresis apparatus, or the equipment of tens0000 dollars of value such as full-automatic dna sequencing instrument), or used harmful reagent such as emitting isotope.
As mentioned above, existing molecular genetics method is unsatisfactory owing to there is the instrument of many factors that influence the amplified production amount and needs costliness in the operation such as PCR hybridization.The purpose of this invention is to provide the simple and result of a kind of easy and simple to handle, equipment down's syndrome antenatal diagnosis method accurately.
Another object of the present invention provides the kit that is used for down's syndrome antenatal diagnosis.
Briefly say, the present invention relates to a kind of methods for prenatal diagnosis of Down's syndrome, comprise that (a) separates fetus cells from the sample of taking from the pregnant woman that contains fetus cells; (b) extract DNA from described fetus cells; (c) with PCR primer more than three pairs or three pairs from the above-mentioned DNA polymorphic site more than three or three on No. 21 chromosome that increases simultaneously; (d) electrophoretic separation PCR product and colour developing, the existence of observing at least one polymorphic site amplified production trizonal whether.
Sample is selected from described in (a) step of said method: blood, urine, tissue sample and amniotic fluid are preferably amniotic fluid.
In (c) step of said method, the heterozygosis rate of each polymorphic site is preferably greater than 80% greater than 70%, for example greater than 83%.
The invention still further relates to the kit that is used for above-mentioned diagnostic method, comprising three couples or more primers of three or three above polymorphic sites on No. 21 chromosome of being used to increase be used to carry out the reagent of pcr amplification.
The present invention adopts the method diagnosis of down syndrome of molecular genetics, and its diagnostic means is to be positioned at the pleomorphism site on the chromosome No. 21 with the PCR method amplification.So-called polymorphism is meant that many allele are arranged on this site in the colony.Normal person's No. 21 chromosomes have only two of father source and sources of parents, so only two allele should be arranged on each site.When suffering from Down's syndrome, No. 21 chromosomes that have more make the patient that three allele be arranged on each site, and three PCR products that molecular weight is different are also just arranged.The dna marker that we are used for diagnosing is the three or more polymorphic sites that are distributed in zones of different on No. 21 chromosome, and the PCR product in these sites of analysis-by-synthesis can be that 97% patient makes diagnosis qualitatively, even reaches more than 99.5%.
In a preferred embodiment of the invention, utilize three polymorphic sites on No. 21 chromosomes of three pairs of primer amplifications, the heterozygosis rate in each site is greater than 83%.
In a preferred embodiment of the invention, allele shows as the different dna fragmentation of four polynucleotide repetitive sequence multiplicity in each polymorphic site, and the difference of molecular weight size is enough to be distinguished with gel electrophoresis between them.
In a preferred embodiment of the invention, select the amplified production size of three polymorphic sites, make to amplify three groups of differentiable PCR products in gel electrophoresis.Be that the migration position of polymorphism amplified production on running gel in every group is adjacent mutually, the middle amplified production band of not being organized is separated.The amplified production size differs at least 20 nucleotide between preferred group, more preferably at least 40 nucleotide.
In a preferred embodiment of the invention, three pairs of PCR products are No. 1 and No. 2 sequences; No. 3 and No. 4 sequences; And No. 5 and No. 6 sequences.
In a preferred embodiment of the invention, the pcr amplification that also comprises a control site in the diagnostic method of the present invention.Preferred this site is the site of isozygotying.In one embodiment, this control site for sequence number 7 and 8 primers to the site on No. 10 chromosomes that increased.This contrast is as the contrast (AC) of amplified reaction.
In another embodiment of the present invention, comprise also in the diagnostic method of the present invention that is detected a contrast, for example by the X chromosome genomic DNA fragment of 9, No. 10 primers, as the contrast of electrophoresis and staining reaction to amplifying.
In an embodiment of the present invention, the primer is to being labeled, and for example uses the fluorophore mark or with biotin labeling etc.
Chromosome did not separate when the pathogenesis of Down's syndrome was reproduction cell meiosis.Homologue does not separate when having case more than 85% to be initial meiosis, due to sister chromatid did not separate when all the other cases of 15% were second meiotic division.
Initial meiosis does not separate and causes two No. 21 chromosomes of homology to rest in the same reproduction cell, after normal second meiotic division, two No. 21 sister's monomers is arranged in each reproduction cell.This abnormal egg cell after fertilization adds No. 21 chromosomes in a father source just to become trisomy 21.Because (wherein one is come the father to her to two No. 21 chromosomes of homology, another comes the mother to her) and come to a chromosomal inheritance source of sperm different, though the three is difficult to distinguish on cellular level, can lean on polymorphic dna marker to be differentiated on molecular level fully.So-called polymorphism is meant that a plurality of allele are all arranged on each gene loci in the colony, as on the A site A1 can being arranged, and A2, A3 ... etc. a plurality of allele.And with regard to certain normal individual in the colony, because he (she) has only two homologues, so on a certain site, only have two allele.These two allelic inhereditary materials are homozygote (Homozygous) when identical, are not heterozygote (Heterozygous) simultaneously.Three No. 21 chromosomes when the present invention relies on the method difference Down's syndrome of four polynucleotide repetitive sequences of detection highly polymorphic (high heterozygosis rate).Concrete grammar is by the pcr amplification polymorphic site.Site of one couple of PCR primers amplification, several allele on this site then show as the different dna fragmentation of four polynucleotide repetitive sequence multiplicity, because the difference of molecular weight size can be distinguished with gel electrophoresis.Site heterozygosis rate on our selected No. 21 chromosomes is all more than 83%, and the rate of isozygotying is 17%.So, with regard to any one site, 83% normal person is arranged because be heterozygote and behind the PCR electrophoresis, can see two band lines.The possibility that the combination of three kinds of allele is then arranged during Down's syndrome: the allele on (1) three No. 21 chromosome is all inequality, is pure heterozygote.A pair ofly can amplify the product of three different molecular weights, electrophoresis showed three bands at the PCR primer of specific site.Have in (2) three allele two identical, a difference is half heterozygosis.One couple of PCR primers can amplify the product of two different molecular weights, electrophoresis showed two bands.(3) three allele are identical, are homozygote.One couple of PCR primers can only amplify a kind of product, electrophoresis showed one band.With regard to a site, the probability that three bands occur is 68% (83% * 83%), and Down's syndrome can obtain etiologic diagnosis in this case; The probability that two bands appear in the down's disease people is 28% (83% * 17% * 2), because the amount of two kinds of PCR products has obvious difference (be 2: 1, two allele PCR product amounts of normal person are identical, are 1: 1) to assist diagnosis with quantitative methods; The probability that one band only appears in patient is 3% (17% * 17%), can not diagnose.
Characteristics of the present invention are exactly to improve the etiologic diagnosis ability with the method that increases the detection site number.If three site PCR results of analysis-by-synthesis, it is that pure heterozygote (electrophoresis three bands) obtains etiologic diagnosis because have a site at least that 97% case is then arranged.
Second meiotic division does not separate yet can cause Down's syndrome.Because unseparated is sister chromatid,, also just etiologic diagnosis can't have been done so, just can not have heterozygote to exist if in meiosis homologue taking place in the past exchanges.The present invention has exemplified three sites: No. 1 site GATA49E01; No. 2 site GATA8G04; No. 3 site GATA70B08.Three sites lay respectively at No. 21 chromosome apart from the kinetochore the rub place of (CM) of 45,55,80 millis.That is to say that three sites have 45%, 55%, 80% recombination fraction respectively, the chromosome recombination fraction improves at least 30% when adding Down's syndrome, so three sites are the possibility of pure heterozygote is respectively: 40.3%, 49.3%, and 71.65%.Three site PCR results of analysis-by-synthesis, it is that pure heterozygote (electrophoresis three bands) obtains etiologic diagnosis because have a site at least that 91.4% case is then arranged.
Fig. 1: PCR reaction rear electrophoresis separates the photo as a result after amplified production also develops the color in expression the present invention one instantiation.
No. 1 and No. 2 sequences are one couple of PCR primers, are used for increasing being positioned at a polymorphic repetitive sequence of genomic DNA on No. 21 chromosome.The amplified production size is between 124-152 base-pair.
No. 3 and No. 4 sequences are one couple of PCR primers, are used for increasing being positioned at a polymorphic repetitive sequence of genomic DNA on No. 21 chromosome.The amplified production size is between 162-186 base-pair.
No. 5 and No. 6 sequences are one couple of PCR primers, are used for increasing being positioned at a polymorphic repetitive sequence of genomic DNA on No. 21 chromosome.The amplified production size is between 209-227 base-pair.
No. 7 and No. 8 sequences are one couple of PCR primers, are used for increasing being positioned at one section conserved sequence of genomic DNA on No. 10 chromosome.The amplified production size is 158 base-pairs.
No. 9 and No. 10 sequences are one couple of PCR primers, are used for increasing being positioned at one section conserved sequence of genomic DNA on the X chromosome.The amplified production size is 236 base-pairs.
Following specific embodiment is used to illustrate the present invention, but does not limit the scope of the invention.
Used following reagent in this example:
1.DNA extract (being U.S. Gull Laboratory company product), DNA lysate (1.5ml distilled water).
2.PCR reagent: primer (synthetic): contain four pairs of primers, 1 on No. 21 chromosome that can increase by U.S. Research Genetics company, 2, control site (sequence is seen sequence table) on No. 3 sites and No. 10 chromosome, dNTPs (buying) from Boringer Manham company, Taq heat-resistant dna polymerase (Perkin Elmer), 10x PCR damping fluid (Genaco), mineral oil (Sigma).
3. electrophoresis reagent: TBE (damping fluid of Tris-boric acid-EDTA), 40% polyacrylamide (Fisher), urea (Fisher), TEMED (Sigma), ammonium persulfate (Sigma), point sample damping fluid.
4. chromogenic reagent: streptavidin AP conjugate (Schleicher ﹠amp; Schuell), sealing damping fluid (Schleicher ﹠amp; Schuell), cleaning fluid (1.5M NaCl), 20%SDS, chromogenic substrate tablet (Schleicher ﹠amp; Schuell).
I. extract foetal DNA
(1). adding the 30ml distilled water, that DNA extraction liquid is diluted to 1 times of preparation is standby.Get amniotic fluid 5-10 milliliter with the method for conventional abdomen wall puncture.By the density of macroscopic method, and be divided into level Four with cell in the guestimate amniotic fluid :+, cell is few, and amniotic fluid is almost transparent; ++, cell concentration is medium, and amniotic fluid has muddiness slightly; +++, cell is many, the amniotic fluid muddiness; ++ ++, cell polar is many, and amniotic fluid is very muddy.The purpose of giving the classification of amniocyte content is the dna profiling amount standardization of using for PCR making after, and patient reduces (reacting one referring to following relevant preparation PCR saves) as far as possible with difference between the patient.
(2). getting the 2ml amniotic fluid, to put into capacity be 2 milliliters micro-centrifuge tube, centrifugal (10,000 to 14,000 rev/mins) 20 seconds.Outwell supernatant, add 0.7ml DNA extraction liquid, cover tight test tube, with the cell granulations of oscillator suspension precipitation.The amniotic fluid of remainder is placed 4 ℃ of refrigerators to be preserved to make provision against emergencies and needs duplicate detection.
(3). with the central authorities that test tube is put into micro-wave oven, microwave (775 watts) heated for 10 seconds.Each heating is no more than 8 pipes.Heating back DNA extraction liquid can be by the as clear as crystal mist sample muddiness that becomes, and test tube also can be very warm.
(4). rock test tube gently with the mixing content, do not go but liquid is rocked to test tube lid the inside.If there is liquid the lid the inside, and is please centrifugal slightly.
(5). again test tube is put into micro-wave oven and heated 3 seconds (2325 watt-second).It is muddy that DNA extraction liquid can become again.Attention: if the constant muddiness of extract, the tube wall of centrifuge tube is not warm yet, illustrates that heating is not enough, causes the DNA extraction failure probably and causes false negative.
(6). the centrifuge tube that will heat is placed room temperature cooling 3 minutes (or placing 1 minute) on ice.Cooling helps to separate unnecessary proteins matter.
(7). centrifugal 1 minute, with protein precipitation with sex change.
(8). pipette 500 μ l supernatants and put into a new 1.5ml micro-centrifuge tube, do not remove disturbance protein precipitation particle as far as possible.Certain each patient specimen of attention is changed a rifle head when moving liquid.If the protein precipitation particle is stirred, centrifugal again 1 minute.
(9). in being placed with the micro-centrifuge tube of supernatant, add isopropyl alcohol 500 microlitres of molecular biology purity.Micro-centrifuge tube is put upside down ten for several times to mix inclusions.
(10). micro-centrifuge tube is put into hydro-extractor, centrifugal 1 minute.If the neck of micro-centrifuge tube all is to unify up, deposit seed will be in the same direction of test tube.Might observe a very little DNA deposit seed after centrifugal.
(11). carefully outwell supernatant, add the alcohol of 1ml 70% and use oscillator washing and precipitating particle.
(12). once more with centrifugal 1 minute of sample.Under the prerequisite of not disturbance deposit seed, inhale and remove supernatant (or outwell supernatant).Micro-centrifuge tube was inverted airing about 5 minutes.
(13). the estimation of carrying out for amniocyte density during according to the first step, each patient adds the DNA lysate of different amounts :+person adds 10 microlitres; ++ add 20 microlitres; +++, 30 microlitres; ++ ++, add 40 microlitres.
(14). do not use as sample at once, place in-20 refrigerators and preserve.
II. set up the PCR reaction
Contain 4 pairs of PCR primers in the PCR reagent 1, wherein three polymorphic sites on three pairs of No. 21 chromosome of can increasing were respectively sequence 1: 2; Sequence 3: 4 and sequence 5: 6; Another is to a non-pleomorphism site on No. 10 chromosome of then increasing (be used for being PCR over against according to), and promptly sequence is 7: 8.
(1). the good patient number of mark on the little centrifuge tube that PCR uses at first.Each patient will prepare a little centrifuge tube.In each patient's two pipes, add the DNA that 2 μ l extract respectively according to numbering from amniotic fluid.Notice that a patient's of every interpolation sample will change the liquid-transfering gun head one time.
(2). make the PCR reaction mixture in proportion according to number of patients.For example, single test does detection then by following prescription will for eight patients:
Primer mixed liquor (each primer contains 25ng) 130 μ l
dNTPs 32μl
Taq enzyme 2 μ l
10 times of damping fluid 20 μ l
Amount to 184 μ l
Add 23 μ l reaction mixtures after reaction mixture prepares in each has added the little centrifuge tube of dna profiling, the final volume of PCR reaction is 25 μ l.
(3). every pipe adds a mineral oil.Centrifugal one minute.By following condition layout pcr amplification program: do 95 ℃ earlier, 3 minutes one-periods; Then, 94 ℃, 1 minute; 61 ℃, 2 minutes; 72 ℃, 3 minutes.Increasing, the condition of changing is after 6 cycles: 94 ℃, and 45 seconds; 61 ℃, 45 seconds; 72 ℃, 45 seconds.24 all after dates of amplification add one 72 ℃ 7 minutes cycles again under this New Terms.Reaction stops the back sample and puts room temperature preservation.
III. electrophoretic separation PCR product
(1). prepare 10 times tbe buffer liquid.
(2). prepare 6% polyacrylamide sex change glue.
(3). according to producer's explanation glass plate is installed, the preparation gel.
(4) 0.6 times of TBE electrophoretic buffer of preparation.Made the temperature of glass plate reach about 50-55 ℃ in one hour pre-race of glue.About about 500 volts, the electrophoresis apparatus condition of different brands may be different greatly for voltage strength.
(5). in 25 ℃ every part PCR product, add point sample damping fluid (the PCR product that to contain a size that useful the 9th, No. 10 primer amplification go out in the point sample damping fluid be 236 base-pairs of 17 μ l.Be placed on purpose in the point sample damping fluid and be do electrophoresis and next step chromogenic reaction over against according to).94-100 ℃ five minutes (can in the PCR instrument, carry out heat denatured) of heating after vibration is mixed.
(6). thoroughly clean point sample borehole bottom urea with syringe, with heat denatured will after specimens point in each wellhole, at electrophoresis under 500 volts about about 35 minutes, the dyestuff (the light green colour band that pulls up lame) of macromolecule stopped electrophoresis after just having run out of at the bottom of the glue by the time.
(7). change film.The glass plate that clips gel is separated.Gel usually combine (in order to allow glue all follow a specific glass to walk at every turn, should at another piece one deck silicon that is coated with on glass) with glass plate wherein.Nylon membrane was soaked 5 minutes with deionized water, being placed on (because all associated products have all been gone to the Lower Half of glue, have only half need change film, the first half is taken the glue limit with preservative film and covered, to guarantee to change the film success) on the gel at the bottom of the moist neat glue of nylon membrane.Putting two on nylon membrane changes film filter papers, puts back to that layer glass of taking apart, puts upside down two blocks of glass and makes DNA in the gel rely on power for support and siphonic effect is transferred on the nylon membrane.Add a weight and on glass plate, change membrane process with quickening.Change membrane process and need at least 4 hours.
IV. chromogenic reaction
(1). Streptavidin-AP conjugate of getting 4.5 μ l is diluted in the sealing damping fluid of 1 times of 7.5ml.
(2). the nylon membrane that will have DNA is put into a plastic pocket, adds above-mentioned 7.5ml chromogenic reagent mixed liquor.Polybag is added heat-seal.Shake reaction one hour under the room temperature.
(3). the preparation cleaning fluid, nylon membrane is put into cleaning fluid that 50ml contains 1%SDS shake and cleaned 5 minutes.Repeated washing once.
(4). the cleaning fluid that does not contain SDS with 50ml cleaned 5 minutes.
(5). the substrate tablet is dissolved in the 30ml distilled water.The nylon membrane of wash clean is put into a plastic pocket again, added 15ml substrate (other 15m leaves with next time and uses), heating is sealed.Sapphire band line began to show in 20 minutes to 2 hours.
V. interpretation of result
Fig. 1 is the diagnosis of molecular biology result.DC:the PCR product that is gone out by the 9th, No. 10 primer amplification is used for doing electrophoresis and chromogenic reaction contrast; No. 1 the site shows zone:No., 2 the site shows zone; AC:amplified reaction contrast; No. 3 the site shows zone.As shown in the figure; Should at first observe DC during analysis; Two control site of AC.Each sample all should be seen product in the DC site; And electrophoresis is described; Changes film; And the colour developing supervisor is no problem.Each sample also all should be seen a fixed product of size weighing apparatus in the AC site; And DNA extraction is described; Pcr amplification reaction reagent etc. are all no problem.Observe three polymorphic sites then successively.There are two allele in each site during normal person's heterozygote; Shows as the two band lines that intensity equates.17% possibility of isozygotying is also arranged; Electrophoresis showed one band. (as No. 2 sites among the figure) are pure heterozygote if Down's syndrome patient is in certain site; And three allele inequality are just arranged; And then can see the three band lines that intensity equates behind the electrophoresis.As the site is half heterozygote; Then three allele have two identical; (as 1 for visual intensity is 2:1 two band lines; No. 3 site among the figure) behind the electrophoresis.As previously mentioned; According to pathogenetic difference; The PCR result in three sites of comprehensive observing can be respectively and do 97% (initial meiosis does not separate) or 91.4% (second meiotic division does not separate) etiologic diagnosis.In fact; if the quantitative Diagnosis when adding first heterozygosis ( by 2: 1 band line intensity ) , then diagnosis all can reach more than 99% under two kinds of mechanism. ( 1 ) ( ⅰ ) ： ( ⅱ ) ： ( ⅲ ) ：8 ( ⅳ ) ： ( A ) ： ( B ) ： ( C ) ： ( D ) ： ( E ) ：100081 ( ⅴ ) ： ( A ) ：3.5 ( B ) ：IBM ( C ) ：Microsoft Windows ( D ) ：Microsoft Word 6.0 ( 2 ) 1： ( ⅰ ) ( A ) ：27 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ：5′ ( ⅹⅰ ) ：1CATTCCCCTC TCAATTGTTT GTCTACC ( 2 ) 2： ( ⅰ ) ( A ) ：26 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ： ( ⅹⅰ ) ：2CGTATATTAT TGAGTCCTGT GAAATG ( 2 ) 3： ( ⅰ ) ( A ) ：27 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ：5′ ( ⅹⅰ ) ：3TTCAGGGCTA CATAGAGAAA CAGAACC ( 2 ) 4： ( ⅰ ) ( A ) ：20 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ： ( ⅹⅰ ) ：4ACACACACAC ACACACATGG ( 2 ) 5： ( ⅰ ) ( A ) ：26 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ：5′ ( ⅹⅰ ) ：5TATGTACGAT ACGTAATACT TGACAA ( 2 ) 6： ( ⅰ ) ( A ) ：23 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ： ( ⅹⅰ ) ：6AATATGTCCC AAAGGACCTG CTC ( 2 ) 7： ( ⅰ ) ( A ) ：22 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ：5′ ( ⅹⅰ ) ：7GAGTTTATTG GTACAGGTGC AG ( 2 ) 8： ( ⅰ ) ( A ) ：21 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ： ( ⅹⅰ ) ：8ATGGACAGTA GCCTATATGC C ( 2 ) 9： ( ⅰ ) ( A ) ：20 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ：5′ ( ⅹⅰ ) ：9TGTATGCCTT GGCAATTTAA ( 2 ) 10： ( ⅰ ) ( A ) ：20 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅰⅳ ) ： ( ⅹⅰ ) ：10CATGTTTCAC AGGAAAGTTC
Claims (20)
1. the methods for prenatal diagnosis of a Down's syndrome comprises that (a) separates fetus cells from the sample of taking from the pregnant woman that contains fetus cells; (b) extract DNA from described fetus cells; (c) with PCR primer more than three pairs or three pairs from the above-mentioned DNA polymorphic site more than three or three on No. 21 chromosome that increases simultaneously; (d) electrophoretic separation PCR product and colour developing, the existence of observing at least one polymorphic site amplified production trizonal whether.
2. according to the process of claim 1 wherein that described sample is selected from blood, urine, tissue sample and amniotic fluid.
3. according to the method for claim 2, wherein said sample is an amniotic fluid.
4. according to the process of claim 1 wherein that the heterozygosis rate of each polymorphic site is greater than 70%.
5. according to the method for claim 4, wherein the heterozygosis rate of each polymorphic site is greater than 80%.
6. according to each method of claim 1-5, wherein use three polymorphic sites of three pairs of PCR primer amplifications.
7. according to the method for claim 6, wherein three pairs of primers are as follows:
CATTCCCCTC?TCAATTGTTT?GTCTACC
CGTATATTAT?TGAGTCCTGT?GAAATG；
TTCAGGGCTA?CATAGAGAAA?CAGAACC
ACACACACAC ACACACATGC; With
TATGTACGAT?ACGTAATACT?TGACAA
AATATGTCCC?AAAGGACCTG?CTC。
8. according to the method for one of claim 1-5, wherein also comprise the amplification control site that one of pcr amplification isozygotys in (c) step.
9. method according to Claim 8, the PCR primer of the control site that wherein is used to increase is to being GAGTTTATTG GTACAGGTGC AG
ATGGACAGTA?GCCTATATGC?C。
10. according to the method for one of claim 1-5, wherein comprise also that in (c) step one of pcr amplification detects control site.
11., wherein be used to detect the PCR primer of control site to being according to the method for claim 10
TGTATGCCTT?GGCAATTTAA
CATGTTTCAC?AGGAAAGTTC。
12. a kit that is used for down's syndrome antenatal diagnosis is comprising at least three pairs of primers of at least three polymorphic sites on No. 21 chromosome of being used to increase be used to carry out the reagent of pcr amplification.
13. according to the kit of claim 12, wherein the heterozygosis rate of each polymorphic site is greater than 70%.
14. according to the kit of claim 13, wherein the heterozygosis rate of each polymorphic site is greater than 80%.
15., wherein use three polymorphic sites of three pairs of PCR primer amplifications according to each kit of claim 12-14.
16. according to the kit of claim 15, wherein: as follows to primer:
CATTCCCCTC?TCAATTGTTT?GTCTACC
CGTATATTAT?TGAGTCCTGT?GAAATG；
TTCAGGGCTA?CATAGAGAAA?CAGAACC
ACACACACAC ACACACATGC; With
TATGTACGAT?ACGTAATACT?TGACAA
AATATGTCCC?AAAGGACCTG?CTC。
17., comprise also that wherein the primer that is used for the amplification control site that one of pcr amplification isozygotys is right according to the kit of one of claim 12-14.
18. according to the kit of claim 17, the PCR primer of the control site that wherein is used to increase is to being GAGTTTATTG GTACAGGTGC AG
ATGGACAGTA?GCCTATATGC?C。
19., wherein also comprise primer that detects control site of pcr amplification according to the kit of one of claim 12-14.
20., wherein be used to detect the PCR primer of control site to being according to the kit of claim 19
TGTATGCCTT?GGCAATTTAA
CATGTTTCAC?AGGAAAGTTC。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1836049B (en) * | 2003-07-04 | 2010-10-06 | 杰纳罗生物系统有限责任公司 | Method for detecting aneuploidy using microparticle multiplex detection |
| CN101137761B (en) * | 2005-03-18 | 2010-12-08 | 香港中文大学 | Markers for prenatal diagnosis and monitoring |
| CN101137760B (en) * | 2005-03-18 | 2011-01-26 | 香港中文大学 | Methods for detecting chromosomal aneuploidy |
| CN101871002B (en) * | 2009-04-22 | 2013-04-17 | 中山大学达安基因股份有限公司 | Kit for antenatally screening trisomy 21 syndrome noninvasively |
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| CN101323877B (en) * | 2007-06-15 | 2011-05-11 | 中山大学达安基因股份有限公司 | Reagent box for detecting No 21 chromosome and idiochromosome number abnormality |
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| CN1836049B (en) * | 2003-07-04 | 2010-10-06 | 杰纳罗生物系统有限责任公司 | Method for detecting aneuploidy using microparticle multiplex detection |
| CN101137761B (en) * | 2005-03-18 | 2010-12-08 | 香港中文大学 | Markers for prenatal diagnosis and monitoring |
| CN101137760B (en) * | 2005-03-18 | 2011-01-26 | 香港中文大学 | Methods for detecting chromosomal aneuploidy |
| CN101871002B (en) * | 2009-04-22 | 2013-04-17 | 中山大学达安基因股份有限公司 | Kit for antenatally screening trisomy 21 syndrome noninvasively |
| CN105132572A (en) * | 2015-09-25 | 2015-12-09 | 邯郸市康业生物科技有限公司 | Noninvasive prenatal screening 2, 1-trisomy syndrome kit |
| CN105132572B (en) * | 2015-09-25 | 2018-03-02 | 邯郸市康业生物科技有限公司 | A kind of patau syndrome kit of noninvasive Prenatal Screening 21 |
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| CN1110573C (en) | 2003-06-04 |
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