CN100535643C - Double color fluorescent quantitative co-amplified polymerase chain reaction detection kit - Google Patents
Double color fluorescent quantitative co-amplified polymerase chain reaction detection kit Download PDFInfo
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- CN100535643C CN100535643C CNB2007100143908A CN200710014390A CN100535643C CN 100535643 C CN100535643 C CN 100535643C CN B2007100143908 A CNB2007100143908 A CN B2007100143908A CN 200710014390 A CN200710014390 A CN 200710014390A CN 100535643 C CN100535643 C CN 100535643C
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Abstract
The invention discloses a reaction reagent box of double color fluorescence batching polymerase chain. Firstly, according to the distinguished DSCR sector sequence of the 21 chromosome and the GAPDH gene sequence of the 16 chromosome, the distinguished primer and the double color Taqman probe are separately synthesized, and then, the synthesis of the new chains are guided separately and simultaneously by the two pairs of primers through 45 PCR circulations in the identical expanded cushioning reacting system, and the altogether expanding of the two pairs of primers is realized. The signal changing situation of the fluorescence obtained because of the degrading of the Taqman probe in the expanding process is inspected when expanding the PCR, and the curve diagrams are obtained, and the Ct value of each gene is obtained. And therefore, the copy number ratio can be calculated, and the ploidy of the 21 chromosomes can be judged according to the ratio of the two gene copy number.
Description
Technical field
The present invention relates to a kind of double color fluorescent quantitative co-amplified polymerase chain reaction detection kit, specifically relate to the external coamplification PCR of the double color fluorescent quantitative polymerase chain reaction kit that a kind of No. 21 ploidies detect, belong to the molecular Biological Detection diagnostic field.
Background technology
(Down Syndrome DS), is called mongolism again to Down syndrome, is one of modal chromosomal abnormality syndrome in the life birth fetus, and its incidence of disease is about 1/600-1/800.The sign of DS is very various, be usually directed to the unusual of multiple histoorgan, but its outstanding, the most serious clinical manifestation is a mental retardation.This sick main clinical characteristics is: serious feeblemindedness, and the phenotype of feeblemindedness is obvious gradually with age growth, and action growth and sexual development are slow, do not have self care ability substantially.Have unique face and physical abnormality, congenital heart disease, alimentary canal deformity etc., the leukemic incidence of disease also than the high 10-30 of crowd average attack rate doubly.Patient's mean lifetime is about 30 years old.
The DS overwhelming majority is accidental, and each pregnant woman has the possibility of living infant, and fall ill no tangible racial difference and family build up phenomenon.The result of a large amount of mass surveies shows that the incidence of disease much at one between the race all over the world, No. 21 chromosomes are 3 in each body cell of Down syndrome, and other autosome is 2, it is the conventional method that its laboratory is detected that lymphoblast or amniotic fluid cast-off cells in vitro culture and Chromosome Preparation are observed, though this method accuracy height, but technical sophistication length consuming time can not realize robotization and high throughput testing.Because the single copy gene dosage on No. 21 chromosomes of Down syndrome patient is 1.5 times of single copy gene dosage on other autosome, and DSCR is crucial single copy section of Down syndrome morbidity, therefore can select No. 21 single copy gene such as single copy gene on DSCR1 and the autosome such as the GAPDH gene on the chromosome, the ratio that detects their relative copy number by fluorescent quantitative PCR technique is judged chromosomal ploidy No. 21, thereby can detect Down syndrome from the minim DNA sample.
The real-time fluorescence quantitative PCR technology is the quantitative new technology of state-of-the-art gene dosage that can carry out accurate quantification to specific DNA (gene) copy number that development in recent years is got up.Its ultimate principle is to add TaqMan probe (special oligonucleotide probe) in the PCR reaction system simultaneously, this probe 5 ' the end mark fluorescence report group (R), 3 ' end mark a fluorescent quenching group (Q).When this probe is in good working condition or does not react, a part of fluorescence that R produced will be absorbed or cancellation by Q.In the PCR course of reaction, this probe can with PCR target gene specific hybrid, by 5 ' the circumscribed active degradation of TaqDNA polymerase, R is separated with Q then, it is detected that free R sends fluorescence signal.Along with the carrying out of PCR reaction, free R and the corresponding increase of PCR product.Therefore, according to the fluorescence signal intensity that each circulation back R produces, can measure the quantity of PCR reaction product in real time.If use the known DNA standard items of copy number to do typical curve simultaneously, can do accurate quantification to the target DNA of the sample that detected.
Summary of the invention
The object of the invention is to set up a kind of easy and simple to handle, can quick and precisely detect Down syndrome patient's double color fluorescent quantitative co-amplified polymerase chain reaction kit.
Technical scheme of the present invention is: this kind double color fluorescent quantitative co-amplified polymerase chain reaction kit, the quantitative fluorescent PCR reagent that contains special single copy section DSCR of No. 21 chromosomes of amplification and No. 16 special single copy section GAPDH amplification in vitros of chromosome, it is characterized in that, at first according to synthetic respectively its special primer of GAPDH gene order and double-colored Taqman fluorescence probe on No. 21 special DSCR sector sequences of chromosome and No. 16 chromosome, then with 0.1-0.5umol/L DSCR and the special primer of GAPDH, Two Colour Fluorescence Taqman probe adds in the same pcr amplification buffering reaction system, after carrying out 96 ℃ of pre-sex change 2min on the polychrome real-time fluorescence quantitative PCR instrument, 94 ℃ of sex change 10sec again, 52-56 ℃ of renaturation 30sec, 60-70 ℃ is extended 40sec, totally 45 circulations, and when extending fluorescence intensity, make two pairs of primers guide new chain synthetic respectively simultaneously, realize the detection by quantitative of coamplification and its copy number of two pairs of primers.
Above-mentioned DSCR and GAPDH special primer and double-colored Taqman fluorescence probe is characterized in that its sequence is respectively: DSCR upstream primer 5 '-CAG AAA TCC CAG TTC ATG TTG CT-3 '; DSCR downstream primer: 5 '-CAT TCC CGGGTG CCA TGA ACA GT-3 '; DSCR TaqMan probe: 5 '-[FAM] CAA GGC CGT GTC CCC TTGTTC[TAMRA]-3 '; GAPDH upstream primer: 5 '-ctc cct ctt tct ttg cag caa t-3 '; GAPDH downstream primer: 5 '-cag ctc tca tac cat gag tcc t-3 '; GAPDH TaqMan probe: 5 '-[HEX] ctg caccac caa ctg ctt agc[TAMRA]-3 '.
Used amplification buffering reaction system, it is characterized in that, be by primer, probe, hot resistant DNA polymerase, dNTPs, template DNA, Mg++, the PCR reaction buffer, ultrapure waters etc. are formed, the concentration content of each composition is as follows respectively: the primer that DSCR and GAPDH are special, each 0.1-0.5umol/L of Two Colour Fluorescence Taqman probe, Taq archaeal dna polymerase 1-3U/50ul, dNTPs substrate 0.1-0.5mmol/L, template DNA is some, Mg++1.5-3.5mmol/L, reaction buffer 0.5-1.5XPCR, wherein the 1XPCR reaction buffer comprises 50mmol/L KCl, 10mmol/L Tris.HCl PH8.3,0.01% gelatin.
When this kind double color fluorescent quantitative co-amplified polymerase chain reaction kit is realized the coamplification of two pairs of primers by detecting in the amplification procedure because the power of the fluorescence signal that degraded Taqman probe obtains, obtain amplification curve diagram separately, and then draw the Ct value of each gene.Per sample Ct value and typical curve utilize following formula to calculate to calculate respectively the GAPDH gene order content on DSCR sector sequence and No. 16 chromosome: Nx=No*Y
Cto-CtxWherein: Nx is the initial copy number of the target gene of sample x, and Y is an amplification efficiency, is equivalent to the multiple that the Ct value reduces the increase of 1 corresponding template DNA that circulates, by formula Y=10
1/ Δ Ct(10 times of the every dilutions of Δ Ct=target DNA, the average Ct value number that increases) calculates and obtains; Ctx is the Ct value of the target gene of amplification pipe x; Cto is the Ct value when the target gene copy number is No on the typical curve; No is initial copy number.Calculate the ratio R of the two: R=Nd/Ng according to following formula, wherein Nd is the DSCR copy number, and Ng is the GAPDH copy number; Judge No. 21 chromosomal ploidies according to the ratio R of the two at last.
This kind double color fluorescent quantitative co-amplified polymerase chain reaction kit is compared with the method that other detects No. 21 ploidies, has the following advantages: 1, easy and simple to handle quick, can in 2-3 hour, obtain testing result accurately; 2, stopped pipe detects, and can not cause amplified production to pollute; 3, automaticity height, cost of labor is low, and all right high throughput testing can detect 90 above samples at every turn; 4, the detection sample material trace of Xu Yaoing, each reaction only needs DNA 50-100ng to be checked, is convenient to draw materials.
Embodiment
Embodiment 1:
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
Amplified production 96bp, the position in gene is as follows:
cacgcgacga?ggacgcattc?caaatcatac?tcacgggagg?aatcttttac 34812197
tgtggaggtg?gctggtcacg?acttcttcgg?aggtggcagc?cgagatcggg 34812147
gtggc
cag?aagagaat
34812097
t?aatgctgcac?accagtt
34812047
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Amplified production 112bp, the position in gene is as follows:
gggtgtgaac?catgagaagt?atgacaacag?cctcaagatc?atcaggtgag 6516648
gaaggcaggg?cccgtggaga?agcggccagc?ctggcaccct?atggacacgc 6516698
tcccctgact?tgcgccccg
gcctc
6516748
tatcgtgga
gggaatggg?actgaggctc?6516848
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 10pmol of two primers, each probe 10pmol, patient or normal person DNA 50-100ng, 1X PCR damping fluid, Taq archaeal dna polymerase 2u, Mg++ final concentration 2mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 52 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.85 ± 0.27, and the difference of normal person's GAPDH and DSCR1 is 1.06 ± 0.176, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.58～1.43, and difference between the two has statistical significance.
Embodiment 2:
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Primer probe and the amplified production position in gene is with embodiment 1.
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 15pmol of two primers, each probe 15pmol, patient or normal person DNA 50-100ng, 1X PCR damping fluid, Taq archaeal dna polymerase 2u, Mg++ final concentration 3mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 56 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.88 ± 0.29, and the difference of normal person's GAPDH and DSCR1 is 1.16 ± 0.17, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.61～1.53, and difference between the two has statistical significance.
Embodiment 3
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Primer probe and the amplified production position in gene is with embodiment 1.
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 15pmol of two primers, each probe 15pmol, patient or normal person DNA 50-100ng, 1X PCR damping fluid, Taq archaeal dna polymerase 2u; Mg++ final concentration 3mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 53 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.78 ± 0.27, and the difference of normal person's GAPDH and DSCR1 is 1.05 ± 0.25, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.51～1.43, and difference between the two has statistical significance.
Embodiment 4
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Primer probe and the amplified production position in gene is with embodiment 1.
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 15pmol of two primers, each probe 15pmol, patient or normal person DNA 50-100ng, 1.5X the PCR damping fluid, Taq archaeal dna polymerase 2u, Mg++ final concentration 3mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 53 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.69 ± 0.15, and the difference of normal person's GAPDH and DSCR1 is 1.02 ± 0.12, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.53 :～1.46, and difference between the two has statistical significance.
Embodiment 5
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Primer probe and the amplified production position in gene is with embodiment 1.
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 25pmol of two primers, each probe 25pmol, patient or normal person DNA 50-100ng, 1X PCR damping fluid, Taq archaeal dna polymerase 2u, Mg++ final concentration 2.5mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 53 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.85 ± 0.22, and the difference of normal person's GAPDH and DSCR1 is 1.24 ± 0.32, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.55 :～1.48, and difference between the two has statistical significance.
Embodiment 6
1. the design of primer and probe
According to DSCR1[gi:7768679 among the genebank] the primer of sequences Design DSCR as follows:
Upstream primer DSCRF:5 '-CAGAAATCCCAGTTCATGTTGCT-3 ';
Downstream primer DSCRR:5 '-CATTCCCGGGTGCCATGAACAGT-3 ';
TaqMan probe DSCRTM:5 '-[FAM] CAAGGCCGTGTCCCCTTGTTC[TAMRA]-3 ';
According to [gi:32891804] design internal reference GAPDH[glyceraldehyde 3-phosphatedehydrogenase among the genebank, glyceraldehyde 3-phosphate dehydrogenase] primer as follows:
Upstream primer GAPDF:5 '-ctccctctttctttgcagcaat-3 ';
Downstream primer GAPDR:5 '-cagctctcataccatgagtcct-3 ';
TaqMan probe GAPDTM:5 '-[HEX] ctgcaccaccaactgcttagc[TAMRA]-3 ';
Primer probe and the amplified production position in gene is with embodiment 1.
2. double color fluorescent quantitative PCR reaction
Mix the 50ul reaction system, include each 20pmol of two primers, each probe 20pmol, patient or normal person DNA 50-100ng, 1X PCR damping fluid, Taq archaeal dna polymerase 1.5u, Mg++ final concentration 2.5mmol/L, 96 ℃ of following pre-sex change 2min on FTC2000 real-time fluorescence quantitative PCR instrument then, 94 ℃ of sex change 10sec then, 56 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when extending for 60 ℃, take a picture.Each sample triplicate experiment.
3. the typical curve of quantitative fluorescent PCR
10 times of dna profilings, 100 times, 1000 times, 10000 times of proportional diluted are done the typical curve of DSCR1 and GAPDH gene, with relatively and draw the amplification efficiency of two genes.
4. result
Experiment shows that Down syndrome patient's GAPDH and the difference of DSCR1 are 1.75 ± 0.28, and the difference of normal person's GAPDH and DSCR1 is 1.15 ± 0.12, with 2
-Δ Δ CTMethod is calculated the difference that changes copy number into and is about: 1/1.65 :～1.55, and difference between the two has statistical significance.
The nucleotides sequence tabulation
＜110〉Shandong Yada Pharmaceutical Co., Ltd.
＜120〉a kind of double color fluorescent quantitative co-amplified polymerase chain reaction kit
<140>2007100143908
<141>2007-04-29
<160>8
<210>1
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for the upstream primer of the special single copy section DSCR of No. 21 chromosomes of amplifying human (Homo sapiens)
<400>1
cagaaatccc?agttcatgtt?gct 23
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for the downstream primer of the special single copy section DSCR of No. 21 chromosomes of amplifying human (Homo sapiens)
<400>2
cattcccggg?tgccatgaac?agt?23
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for detecting the TaqMan probe of the special single copy section DSCR amplified production of human (Homo sapiens) No. 21 chromosomes
<400>3
caaggccgtg?tccccttgtt?c 21
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for the upstream primer of the special single copy section GAPDH of No. 16 chromosomes of amplifying human (Homo sapiens)
<400>4
ctccctcttt?ctttgcagca?at?22
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for the downstream primer of the special single copy section GAPDH of No. 16 chromosomes of amplifying human (Homo sapiens)
<400>5
cagctctcat?accatgagtc?ct 22
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for detecting the TaqMan probe of the special single copy section GAPDH amplified production of human (Homo sapiens) No. 16 chromosomes
<400>6
ctgcaccacc?aactgcttag?c 21
<210>7
<211>200
<212>DNA
＜213〉special single copy section [gi:7768679] DSCR of human (Homo sapiens) No. 21 chromosomes
<220>
<221>repeat_unit
<222>(105)...(200)
<400>7
cacgcgacga?ggacgcattc?caaatcatac?tcacgggagg?aatcttttac?tgtggaggtg?60
gctggtcacg?acttcttcgg?aggtggcagc?cgagatcggg?gtggacgaaa?tcccagttca?120
tgttgctcag?aagagaatca?aggccgtgtc?cccttgttct?aatgctgcac?accagttact?180
gttcatggca?cccgggaatg 200
<210>8
<211>250
<212>DNA
＜213〉special single copy section [gi:32891804] GAPDH of human (Homo sapiens) No. 16 chromosomes
<220>
<221>repeat_unit
<222>(120)...(231)
<400>8
gggtgtgaac?catgagaagt?atgacaacag?cctcaagatc?atcaggtgag?gaaggcaggg?60
cccgtggaga?agcggccagc?ctggcaccct?atggacacgc?tcccctgact?tgcgccccgc?120
tccctctttc?tttgcagcaa?tgcctcctgc?accaccaact?gcttagcacc?cctggccaag?180
gtcatccatg?acaactttgg?tatcgtggaa?ggactcatgg?tatgagagct?ggggaatggg?240
actgaggctc 250
Claims (2)
1, a kind of double color fluorescent quantitative co-amplified polymerase chain reaction kit, the quantitative fluorescent PCR reagent that contains special single copy section DSCR1 of No. 21 chromosomes of amplification and No. 16 special single copy section GAPDH amplification in vitros of chromosome, it is characterized in that, at first according to synthetic respectively its special primer of GAPDH gene order and double-colored Taqman fluorescence probe on the special sequence D SCR1 sector sequence of No. 21 chromosome and No. 16 chromosome, then with 0.2umol/L DSCR1 and the special primer of GAPDH, Two Colour Fluorescence Taqman probe adds in the same pcr amplification buffering reaction system, after carrying out 96 ℃ of pre-sex change 2min on the multicolor fluorescence quantitative PCR instrument, 94 ℃ of sex change 10sec again, 52 ℃ of renaturation 30sec, 60 ℃ are extended 40sec, totally 45 circulations, and when 60 ℃ are extended fluorescence intensity, make two pairs of primers guide new chain synthetic respectively simultaneously, realize the detection by quantitative of coamplification and its copy number of two pairs of primers, the sequence of above-mentioned DSCR1 and GAPDH special primer and double-colored Taqman fluorescence probe is respectively: DSCR1 upstream primer 5 '-CAG AAA TCC CAG TTC ATG TTG CT-3 '; DSCR1 downstream primer: 5 '-CAT TCCCGG GTG CCA TGA ACA GT-3 '; DSCR TaqMan probe: 5 '-[FAM] CAA GGC CGT GTCCCC TTG TTC[TAMRA]-3 '; GAPDH upstream primer: 5 '-ctc cct ctt tct ttg cag caat-3 '; GAPDH downstream primer: 5 '-cag ctc tca tac cat gag tcc t-3 '; GAPDHTaqMan probe: 5 '-[HEX] ctg cac cac caa ctg ctt agc[TAMRA]-3 '.
2, a kind of double color fluorescent quantitative co-amplified polymerase chain reaction kit according to claim 1 is characterized in that, in the 50ul amplification buffering reaction system, the concentration ratio of described DSCR1 and GAPDH special primer and double-colored Taqman fluorescence probe is 1: 1.
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| CNB2007100143908A CN100535643C (en) | 2007-04-29 | 2007-04-29 | Double color fluorescent quantitative co-amplified polymerase chain reaction detection kit |
| PCT/CN2008/000871 WO2008134933A1 (en) | 2007-04-29 | 2008-04-29 | A double-color fluorescent kit useful for quantitative detecting co-amplification polymerase chain reaction |
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| CNB2007100143908A CN100535643C (en) | 2007-04-29 | 2007-04-29 | Double color fluorescent quantitative co-amplified polymerase chain reaction detection kit |
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| CN101105454A CN101105454A (en) | 2008-01-16 |
| CN100535643C true CN100535643C (en) | 2009-09-02 |
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| CN104087672A (en) * | 2014-07-14 | 2014-10-08 | 钦州市妇幼保健院 | Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique |
| CN106755443B (en) * | 2016-12-30 | 2018-11-30 | 人和未来生物科技（长沙）有限公司 | A kind of method and reagent of the super sensitivity detection trace amount DNA from a variety of hybrid dnas |
| CN109628559B (en) * | 2019-01-29 | 2023-05-02 | 阅尔基因技术（苏州）有限公司 | Method and kit for detecting Y chromosome copy number variation |
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| 双色实时荧光定量PCR及荧光原位杂交检测唐式综合征. 王玲.中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑，第01期. 2006 |
| 双色实时荧光定量PCR及荧光原位杂交检测唐式综合征. 王玲.中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑，第01期. 2006 * |
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| CN101105454A (en) | 2008-01-16 |
| WO2008134933A1 (en) | 2008-11-13 |
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