CN101760516A - Method for detecting genotypes of two bit points of G34A and C421A in BCRP gene - Google Patents
Method for detecting genotypes of two bit points of G34A and C421A in BCRP gene Download PDFInfo
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- CN101760516A CN101760516A CN200810188315A CN200810188315A CN101760516A CN 101760516 A CN101760516 A CN 101760516A CN 200810188315 A CN200810188315 A CN 200810188315A CN 200810188315 A CN200810188315 A CN 200810188315A CN 101760516 A CN101760516 A CN 101760516A
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Abstract
The invention relates to a method for utilizing the fluorescent quantitative PCR method for determining the genotypes of two bit points of G34A and C421A in human breast cancer resistance protein (BCRP) gene.
Description
Invention field
The present invention relates to utilize the method for quantitative fluorescent PCR to measure the method for G34A and two loci gene types of C421A in human breast cancer drug-resistant protein (BCRP) gene.
Background of invention
Single nucleotide polymorphism mark SNP (singlenucleotide Poly=rphisms as " third generation DNA genetic marker ", SNP) have that the site is abundant, representative, genetic stability and characteristics such as analysis automated, be the molecular basis of research pharmacogenomics.The variation great majority of Human genome sequence are SNP, but the distribution of SNP there are differences in different crowds, these differences can be represented the heritable variation between a certain race or certain crowd. therefore, research SNP helps to explain individual phenotypic difference, different groups and individual to disease, particularly to the susceptibility of complex disease and to the tolerance of various medicines and the difference that environmental factors is reacted.If determined the genotype of certain SNP and the strong correlation relation of certain tumor susceptibility, just can this SNP as molecular genetic marker, carry out the molecule marker diagnosis, in epidemiology survey, determine to suffer from the high risk population of certain tumour, and carry out ill hazard level evaluation, reach effective primary prevention purpose.
BC RP＜A BCG2 gene is positioned at chromosome 22, is made of the transmembrane protein of coding 72-kDa 655 amino acid.BCRP antibody is located to express at placenta epithelium, enteric epithelium, breast duct etc.On structure, it has only an ATP-binding domain, one to stride the film district, and is different with the MDR gene.Constructional unique its drug transport mechanism of prompting may be different with other abc transport albumen.BCRP genetic expression increase can be observed in the drug-resistant tumor of number of different types.
Because there are upper frequency and occurred frequently in most of ethnic populations in BCRP gene G34A (Val12Met) and C421A (Gln141Lys) polymorphism, discover that its expression is relevant with the BCRP protein-active.The expressed BCRP protein-active of different genotype also is not quite similar, in human placenta, the homozygous protein level of 421A allelotrope is more much lower than 421C allelotrope homozygote, then between by-level and the contrast of wild allelotype, 34A and 42A allelic variation can reduce BCRP translocator activity to heterozygote simultaneously.The apparent BCRP gene G34A of many results of study is not relevant with existence with the DLBCL susceptible with the C421A polymorphism.
Nucleic acid primer and the probe polymorphism that detect BCRP gene G34A and C421A site of the present invention by designing special quantitative fluorescent PCR avoided the complicated processes of order-checking, thereby can have been realized genotypic detection more accurately, quickly and easily.
Summary of the invention
The invention provides a kind of method that is used for measuring human breast cancer drug-resistant protein gene (BCRP) G34A and two loci gene types of C421A, this method is by one group of probe special, that be used for the quantitative fluorescent PCR reaction, the multiple sample that comprises blood sample is handled, analyzed its result and judge genotype.
The accompanying drawing summary
Fig. 1. the figure of the quantitative fluorescent PCR of the genotypic two kinds of probes of BCRP gene G34A site G/A in the sample.
Fig. 2. the figure of the quantitative fluorescent PCR of the genotypic two kinds of probes of BCRP gene G34A site G/G in the sample.
Fig. 3. the figure of the quantitative fluorescent PCR of the genotypic two kinds of probes of BCRP gene G34A site A/A in the sample.
Fig. 4. the figure of the quantitative fluorescent PCR of the genotypic two kinds of probes of BCRP gene C 421A site C/A in the sample.
Fig. 5. the figure of the quantitative fluorescent PCR of the genotypic two kinds of probes of BCRP gene C 421A site C/C in the sample.
Detailed Description Of The Invention
The invention provides the human cell, organize and comprise the method that detects G34A and two locus gene types of C421A in the BCRP gene in the body fluid of serum and saliva.
In embodiments, the invention provides a kind of method of measuring in the blood sample G34A and two loci gene types of C421A in the BCRP gene, this method is by using quantitative fluorescent PCR, utilize two groups of special probes that sample is handled, wherein every kind of probe all only can combine with a kind of special genes type nucleic acid, by this group probe is judged the genotype in G34A and two sites of C421A to the result of same sample process.Confirm that by order-checking the judgement based on this method is accurately.
Embodiment
Following embodiment is used to those skilled in the art to provide about how implementing and use complete disclosure of the present invention and description, and these examples are not that the invention scope that is intended to the present inventor is thought limits, and the also non-experiment that means hereinafter is effective whole experiment and is only enforceable experiment.
Embodiment 1: BCRP gene G34A site G/A is genotypic two kinds in the drop of blood sample
The figure of the quantitative fluorescent PCR of probe.
We use the nucleic acid primer of SEQ ID NO.2 and SEQ ID NO.3 and the nucleic acid probe of SEQID NO.6 and SEQ ID NO.7 respectively, use quantitative fluorescent PCR that same sample is handled, obtain the set of diagrams shape among Fig. 1, judge that the genotype in this sample G34A site is G/A.This result obtains conclusive evidence by order-checking.
Embodiment 2: BCRP gene G34A site G/G is genotypic two kinds in the blood sample
The figure of the quantitative fluorescent PCR of probe.
We use the nucleic acid primer of SEQ ID NO.2 and SEQ ID NO.3 and the nucleic acid probe of SEQID NO.6 and SEQ ID NO.7 respectively, use quantitative fluorescent PCR that same sample is handled, obtain the set of diagrams shape among Fig. 1, judge that the genotype in this sample G34A site is G/G.This result obtains conclusive evidence by order-checking.
Embodiment 3: BCRP gene G34A site A/A is genotypic two kinds in the blood sample
The figure of the quantitative fluorescent PCR of probe.
We use the nucleic acid primer of SEQ ID NO.2 and SEQ ID NO.3 and the nucleic acid probe of SEQID NO.6 and SEQ ID NO.7 respectively, use quantitative fluorescent PCR that same sample is handled, obtain the set of diagrams shape among Fig. 1, judge that the genotype in this sample G34A site is A/A.This result obtains conclusive evidence by order-checking.
Embodiment 4: BCRP gene C 421A site C/A is genotypic two kinds in the blood sample
The figure of the quantitative fluorescent PCR of probe.
We use the nucleic acid primer of SEQ ID NO.4 and SEQ ID NO.5 and the nucleic acid probe of SEQID NO.8 and SEQ ID NO.9 respectively, use quantitative fluorescent PCR that same sample is handled, obtain the set of diagrams shape among Fig. 1, judge that the genotype in this sample C421A site is C/A.This result obtains conclusive evidence by order-checking.
Embodiment 5: BCRP gene C 421A site C/C is genotypic two kinds in the blood sample
The figure of the quantitative fluorescent PCR of probe.
We use the nucleic acid primer of SEQ ID NO.4 and SEQ ID NO.5 and the nucleic acid probe of SEQIDNO.8 and SEQ ID NO.9 respectively, use quantitative fluorescent PCR that same sample is handled, obtain the set of diagrams shape among Fig. 1, judge that the genotype in this sample C421A site is C/C.This result obtains conclusive evidence by order-checking.
Sequence table
＜110〉Tianjin tumour hospital
＜120〉method of G34A and two loci gene types of C421A in the detection BCRP gene
<130>
<150>
<151>2008-12-19
<160>9
<210>1
<211>2418
<212>DNA
＜213〉people BCRP sequence
<400>1
gggaggaggc?agcctgtgga?ggaactgggt?aggatttagg?aacgcaccgt?gcacatgctt?60
ggtggtcttg?ttaagtggaa?actgctgctt?tagagtttgt?ttggaaggtc?cgggtgactc?120
atcccaacat?ttacatcctt?aattgttaaa?gcgctgcctc?cgagcgcacg?catcctgaga?180
tcctgagcct?ttggttaaga?ccgagctcta?ttaagctgaa?aagataaaaa?ctctccagat?240
gtcttccagt?aatgtcgaag?tttttatccc?agtgtcacaa?ggaaacacca?atggcttccc?300
cgcgacagct?tccaatgacc?tgaaggcatt?tactgaagga?gctgtgttaa?gttttcataa?360
catctgctat?cgagtaaaac?tgaagagtgg?ctttctacct?tgtcgaaaac?cagttgagaa?420
agaaatatta?tcgaatatca?atgggatcat?gaaacctggt?ctcaacgcca?tcctgggacc?480
cacaggtgga?ggcaaatctt?cgttattaga?tgtcttagct?gcaaggaaag?atccaagtgg?540
attatctgga?gatgttctga?taaatggagc?accgcgacct?gccaatttca?aatgtaattc?600
aggttacgtg?gtacaagatg?atgttgtgat?gggcactctg?acggtgagag?aaaacttaca?660
gttctcagca?gctcttcggc?ttgcaacaac?tatgacgaat?catgaaaaaa?acgaacggat?720
taacagggtc?attcaagagt?taggtctgga?taaagtggca?gactccaagg?ttggaactca?780
gtttatccgt?ggtgtgtctg?gaggagaaag?aaaaaggact?agtataggaa?tggagcttat?840
cactgatcct?tccatcttgt?tcttggatga?gcctacaact?ggcttagact?caagcacagc?900
aaatgctgtc?cttttgctcc?tgaaaaggat?gtctaagcag?ggacgaacaa?tcatcttctc?960
cattcatcag?cctcgatatt?ccatcttcaa?gttgtttgat?agcctcacct?tattggcctc?1020
aggaagactt?atgttccacg?ggcctgctca?ggaggccttg?ggatactttg?aatcagctgg?1080
ttatcactgt?gaggcctata?ataaccctgc?agacttcttc?ttggacatca?ttaatggaga?1140
ttccactgct?gtggcattaa?acagagaaga?agactttaaa?gccacagaga?tcatagagcc 1200
ttccaagcag?gataagccac?tcatagaaaa?attagcggag?atttatgtca?actcctcctt 1260
ctacaaagag?acaaaagctg?aattacatca?actttccggg?ggtgagaaga?agaagaagat 1320
cacagtcttc?aaggagatca?gctacaccac?ctccttctgt?catcaactca?gatgggtttc 1380
caagcgttca?ttcaaaaact?tgctgggtaa?tccccaggcc?tctatagctc?agatcattgt 1440
cacagtcgta?ctgggactgg?ttataggtgc?catttacttt?gggctaaaaa?atgattctac 1500
tggaatccag?aacagagctg?gggttctctt?cttcctgacg?accaaccagt?gtttcagcag 1560
tgtttcagcc?gtggaactct?ttgtggtaga?gaagaagctc?ttcatacatg?aatacatcag 1620
cggatactac?agagtgtcat?cttatttcct?tggaaaactg?ttatctgatt?tattacccat 1680
gacgatgtta?ccaagtatta?tatttacctg?tatagtgtac?ttcatgttag?gattgaagcc 1740
aaaggcagat?gccttcttcg?ttatgatgtt?tacccttatg?atggtggctt?attcagccag 1800
ttccatggca?ctggccatag?cagcaggtca?gagtgtggtt?tctgtagcaa?cacttctcat 1860
gaccatctgt?tttgtgttta?tgatgatttt?ttcaggtctg?ttggtcaatc?tcacaaccat 1920
tgcatcttgg?ctgtcatggc?ttcagtactt?cagcattcca?cgatatggat?ttacggcttt 1980
gcagcataat?gaatttttgg?gacaaaactt?ctgcccagga?ctcaatgcaa?caggaaacaa 2040
tccttgtaac?tatgcaacat?gtactggcga?agaatatttg?gtaaagcagg?gcatcgatct 2100
ctcaccctgg?ggcttgtgga?agaatcacgt?ggccttggct?tgtatgattg?ttattttcct 2160
cacaattgcc?tacctgaaat?tgttatttct?taaaaaatat?tcttaaattt?ccccttaatt 2220
cagtatgatt?tatcctcaca?taaaaaagaa?gcactttgat?tgaagtattc?aatcaagttt 2280
ttttgttgtt?ttctgttccc?ttgccatcac?actgttgcac?agcagcaatt?gttttaaaga 2340
gatacatttt?tagaaatcac?aacaaactga?attaaacatg?aaagaaccca?aaaaaaaaga 2400
tatcactcag?cataatga 2418
<210>2
<211>21
<212>DNA
＜213〉people BCRP primer
<400>2
5’-tcaggtcattggaagctgtcg-3’
<210>3
<211>23
<212>DNA
＜213〉people BCRP primer
<400>3
5’-gtcacctagtgtttgcaatctca-3’
<210>4
<211>19
<212>DNA
＜213〉people BCRP primer
<400>4
5’-agttgttgcaagccgaaga-3’
<210>5
<211>20
<212>DNA
＜213〉people BCRP primer
<400>5
5’-tgttgtgatgggcactctga-3’
<210>6
<211>15
<212>DNA
＜213〉people BCRP probe
<400>6
5’-tccttgtgacactgg-3’
<210>7
<211>17
<212>DNA
＜213〉people BCRP probe
<400>7
5’-tttccttgtgacattgg-3’
<210>8
<211>16
<212>DNA
＜213〉people BCRP probe
<400>8
5’-ctgctgagaactggaa-3’
<210>9
<211>17
<212>DNA
＜213〉people BCRP probe
<400>9
5’-ctgctgagaactgtaag-3’
Claims (8)
1. method that is used for measuring human breast cancer drug-resistant protein gene (BCRP) G34A and two loci gene types of C421A, this method comprises: thus (a) contact with the human cell with two kinds of human BCRP specific compoundss and form many mixtures of two kinds of compound-BCRP; And (b) existence by detecting this mixture and ratio thereof and two loci gene types of G34A and C421A among this cell BCRP are measured; Wherein the BCRP specific compounds is selected from: in conjunction with nucleic acid primer and the probe of BCRP, described nucleic acid primer and probe comprise antisense scant polymer, siRNA and shRNA, these probes should be hybridized with BCRP DNA, wherein the nucleotide sequence of BCRP is SEQ ID No:1, and perhaps any have the sequence of at least 80% homogeny with Seq ID No:1.
2. the method in the claim 1, wherein cell is selected from adrenal cells, brain cell, mammary gland cell, colon cell, epithelial cell, endotheliocyte, heart cell, immunocyte, kidney cell, liver cell, pneumonocyte, gonad cell, pancreatic cell, prostatic cell, skin cells, splenocyte, gastric cells, testicular cell, thyroid cell, uterine cell and vascular cell, cancer cells or is in cell in the tumor microenvironment.
3. the method for claim 2, wherein epithelial cell is selected from endotheliocyte, non-colloid neurocyte, colon cell, mammary gland cell, the near little tube cell of kidney, prostatic smooth muscle cell, the smooth muscle cell in uterus and the smooth muscle cell of testis.
4. the method for claim 2, wherein immunocyte is selected from polymorphonuclear leukocyte, monocyte, scavenger cell, epithelioid cell, giant cell, microgliacyte, Kupffer cell and pulmonary alveolar macrophage.
5. the method for claim 2, wherein this cancer cells is selected from lung cancer, mammary cancer, carcinoma of the colon and rectum, carcinoid tumor, cancer of the stomach, neurospongioma, hepatoma, leiomyosarcoma, liver cancer, kidney, bladder cancer, uterus carcinoma, head and neck cancer, vagina or carcinoma of testis, brain tumor, cervical cancer, esophagus cancer, lymphoma, malignant melanoma, mesothelioma, myelomatosis, ovarian cancer, carcinoma of the pancreas, prostate cancer, thyroid carcinoma, renal cell carcinoma, cancer eye, rhabdosarcoma, sarcoma, does not break up knurl and leukemia.
6. the method for claim 2, wherein this cell that is in the tumor microenvironment is selected from gelatinous fiber archeocyte, colloid monocyte and myofibroblast.
7. the method for claim 2, the nucleic acid primer sequence that wherein is used for measuring BCRP gene G34A loci gene type is SEQ ID NO.2 and SEQ ID NO.3, and the nucleic acid primer sequence that is used for measuring BCRP gene C421A loci gene type is SEQ ID NO.4 and SEQ ID NO.5.
8. the method for claim 2, the nucleic acid probe sequence that wherein is used for measuring BCRP gene G34A loci gene type is SEQ ID NO.6 and SEQ ID NO.7, and the nucleic acid probe sequence that is used for measuring BCRP gene C421A loci gene type is SEQ ID NO.8 and SEQ ID NO.9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102912008A (en) * | 2011-08-05 | 2013-02-06 | 广州益善生物技术有限公司 | ABCG2 gene polymorphism detection specific primer and liquid phase chip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102912008A (en) * | 2011-08-05 | 2013-02-06 | 广州益善生物技术有限公司 | ABCG2 gene polymorphism detection specific primer and liquid phase chip |
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Application publication date: 20100630 |