CN101684492A - Kit for detecting genes of leukemia of children - Google Patents
Kit for detecting genes of leukemia of children Download PDFInfo
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- CN101684492A CN101684492A CN200810200586A CN200810200586A CN101684492A CN 101684492 A CN101684492 A CN 101684492A CN 200810200586 A CN200810200586 A CN 200810200586A CN 200810200586 A CN200810200586 A CN 200810200586A CN 101684492 A CN101684492 A CN 101684492A
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Abstract
The invention discloses a kit used for detecting genes of leukemia of children, which can detect susceptibility genes of leukemia of children conveniently and quickly before the disease possibly occurs. The kit comprises a DNA extraction reagent, premixed PCR reaction liquid, a standard positive control substance and a standard negative control substance. When the kit is used, the DNA samples of the children to be detected are taken as the detecting objects, after the sample DNA is extracted, the kit is used for reaction on a fluorescence quantitative PCR instrument and diagnosis is carried out in combination with the reaction results. The kit can detect the genes of leukemia of children to realize early discovery and early diagnosis and adopt preventive measures in advance so as to avoidtragedies from happening to the greatest extent.
Description
Technical field
The present invention relates to a kind of diseases predisposing gene detection technique, specifically, relate to a kind of test kit of relevant leukemia of children gene test.
Background technology
Leukemia is the malignant tumour of the common blood system of children's.Be a kind of malignant proliferation disease that causes owing to the hemopoietic stem cell proliferation prosoplasia, it not only influences marrow and whole hemopoietic system, and immerses other organs of health.According to statistics, the sickness rate of leukemia of children below 15 years old is 3,/10 ten thousand～4/,100,000.Childhood lymphoblastic leukemia occupies the first place of the various malignant tumours of children's, and any age all can fall ill, and wherein sees with pre-school age and school age children more.According to slow, clinical, the blood of urgency of morbidity and marrow performance etc., generally be divided into acute and chronic.Wherein, based on acute leukemia, account for 97% of childhood lymphoblastic leukemia sum.Acute leukemia can be divided into acute lymphoblastic leukemia and acute nonlymphocytic leukemia again.The former accounts for 70-85%.
Leukemic clinical manifestation is by normal plasma cell minimizing and some organ of leukemiacell infiltration and organizes caused.Patient usually goes sick because of symptoms such as fever, pale, abnormal sweating, ecchymosis and pain.Morbidity may be very hidden, also may be very unexpected, and symptom can gently can weigh.Great majority had a couple of days medical history, minority can reach several weeks to the several months before making a definite diagnosis.That utmost point discrete case shows as fulminant high heat, weakness, anaemia, no fix or localized pain and large stretch of ecchymosis, progress is exceedingly fast.
When having a member that leukemia takes place in the family, leukemic probability takes place its close relative can be higher 4 times than common people.If suffer from acute leukemia for one in the monozygotic twins tire, another incidence is 20%～25%.The above fact all points out the leukemic cause of disease may be relevant with heredity.In recent years studies confirm that numerical abnormalities such as the increase of karyomit(e) quantity or minimizing, and textural anomaly such as transposition, inversion, disappearance make structure, the abnormal expression of gene.The inactivation of genetic expression or gene is one of basis of malignant change of cell.
Leukemia of children has two characteristics: the one, and grade malignancy height, PD are acute rapidly, mostly; The 2nd, very sensitive to chemotherapy, cancer cells is killed easily.General 1 to 5 years old child the easiest be acute lymphoblastic leukemia, this age bracket patient effect after treating is best.Generally before 9 years old, can both better cure, and heal than refractory with regard to the same later in 14 years old with the adult.Therefore, the leukemia in infancy if can in time be found, adopts suitable treatment means, often can both obtain satisfactory effect.
People wish to have a kind of easy and objective method can be carried out the leukemia of children gene test, accomplish early to find, diagnosis early, take preventive measures ahead of time, avoid the generation of tragedy as far as possible.
Summary of the invention
For can be easy and objectively leukemia of children is tested, the invention provides a kind of kit for detecting genes of leukemia of children, can be test sample book with children's dna sample, and the gene of leukemia of children is detected, thereby, provide important reference information for the prevention leukemia of children.
This test kit is by the DNA extraction agent, pre-mixed PCR reaction solution, and standard positive reference substance, standard negative reference substance is formed.Dna sample with tested children when using is detected object, after the sample DNA extracting, uses test kit to react on quantitative real time PCR Instrument, and association reaction is the result diagnose.
Described pre-mixed PCR reaction solution contains SYBR green I, and the relevant GSTP1 of leukemia of children and the amplimer of XRCC1 gene mutation site; Amplimer is upstream primer and the normal downstream primer that comprises the mutational site, and sequence is as follows respectively:
The primer sequence of amplification GSTP1 gene is as follows:
Upstream primer F1A (C) 5-GAC CTC CGC TGC AAA TCC A-3 19bp,
Upstream primer F1G (C) 5-AC CTC CGC TGC AAA TCC G-3 18bp,
Downstream primer R1 5-GCA CTG GGG GCT GAA CAAA-3 19bp;
The primer sequence of amplification XRCC1 gene is as follows:
Upstream primer F2A (C) 5-GCA CAC TCC GGA ATG GCGA-3 19bp,
Upstream primer F2G (C) 5-GCA CAC TCC GGA ATG GCG G-3 19bp,
Downstream primer R2 5-CCT CCG GCG TAG CAC GCA-3 18bp.
Above-mentioned kit for detecting genes of leukemia of children, described positive reference substance have 4, comprise following sequence respectively:
SEQ1：
GGACCAGCAG?GAGGCAGCCC?TGGTGGACAT?GGTGAATGAC
GGCGTGGAGG?ACCTCCGCTG?CAAATACATC?TCCCTCATCT
ACACCAACTA?TGTGAGCATC?TGCACCAGGG?TTGGGCACTG
GGGGCTGAAC?AAAGAAAGGG?GCTTCTTGTG?CCCTCACCCC
CCTTACCCCT?CAGGTGGCTT?GGGCTGACCC?CTTCTTGGG；
SEQ2：
GGACCAGCAG?GAGGCAGCCC?TGGTGGACAT?GGTGAATGAC
GGCGTGGAGG?ACCTCCGCTG?CAAATACGTC?TCCCTCATCT
ACACCAACTA?TGTGAGCATC?TGCACCAGGG?TTGGGCACTG
GGGGCTGAAC?AAAGAAAGGG?GCTTCTTGTG?CCCTCACCCC
CCTTACCCCT?CAGGTGGCTT?GGGCTGACCC?CTTCTTGGG；
SEQ3：
TGCCCCTCAG?ATCACACCTA?ACTGGCATCT?TCACTTCTGC
CCCCCACCAG?CTGTGCCTTT?GCCAACACCC?CCAAGTACAG
CCAGGTCCTA?GGCCTGGGAG?GCCACATCGT?GCGTAAGGAG
TGGGTGCTGG?ACTGTCACCG?CATGCGTCGG?CGGCTGCCCT
CCCAGAGGTA?AGGCCTCACA?CGCCAACCCT?GCTC；
SEQ4：
GAGCAGGGTT?GGCGTGTGAG?GCCTTACCTC?CGGGAGGGCA
GCCGCCGACG?CATGCGGTGA?CAGTCCAGCA?CCCACTCCTT
ACGCACGATG?CGGCCTCCCA?GGCCCAGGACC?TGGCTGTACT
TGGGGGTGTT?GGCAAAGGCA?CAGCTGGTGG?GGGGCAGAAG
TGAAGATGGC?CAGTTAGGTG?TGATCTGAGG?GGCA。
Above-mentioned kit for detecting genes of leukemia of children, the sequence that described negative control product comprise is the arabidopsis gene sequence, following ``:
SEQ5：
TTCGTTATTG?AATCTTGTTT?TGGTTCTCTT?GTGTGGAATG
CATTGTTCTT?GCTTCTCTGG?AACTTGGGAT?ATGATGACAA
AAATACGATA?TCTCTAAGGC?TAATTGCTGT?GCGGATGAAA
TATATTTGCA?CGTCAGTCTG?ATCCCATTAT?GTATATAACA
TTAGGAGTTG?GTGCATGCTC。
Use of the present invention does not need children to show up, and only need get its dna sample, and is such as the oral cavity sample, more timely and convenient.Can accomplish early to find, early diagnose to leukemia of children, take preventive measures ahead of time, avoid the generation of tragedy as far as possible.
Description of drawings
Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 are the amplification curve diagram on the quantitative real time PCR Instrument.The Delta Rn vs Cycle of each figure top is meant the logarithmic amplification collection of illustrative plates of Δ Rn with circulation change; Δ Rn (Delta Rn) is meant the logarithmic value at one group of given following fluorescent signal that generates of PCR condition.The X-coordinate of this figure is represented cycle number (Cycle Number), and ordinate zou is represented fluorescent value (Delta Rn).Horizontal line is represented fluorescence thresholding, and the pairing cycle number in the point of crossing of curve and fluorescence thresholding curve is the Ct value.
Fig. 1 represents the product amplification curve diagram of the homozygous sample of GSTP1 gene A A: the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C1, and the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C2.
Fig. 2 represents the product amplification curve diagram of GSTP1 gene GA heterozygous sample: the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C3, and the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C4.
Fig. 3 represents the product amplification curve diagram of the homozygous sample of GSTP1 gene GG: the Ct cycling numerical value that thresholding is represented to reach in curve 2 and the point of crossing of fluorescence thresholding curve is C5, and the Ct cycling numerical value that thresholding is represented to reach in curve 1 and the point of crossing of fluorescence thresholding curve is C6.
Fig. 4 represents the product amplification curve diagram of the homozygous sample of XRCC1 gene A A: the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C7, and the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C8.
Fig. 5 represents the product amplification curve diagram of XRCC1 Gene A heterozygous sample: the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C9, and the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C10.
Fig. 6 represents the product amplification curve diagram of the homozygous sample of XRCC1 gene GG: the Ct cycling numerical value that thresholding is represented to reach in curve 6 and the point of crossing of fluorescence thresholding curve is C11, and the Ct cycling numerical value that thresholding is represented to reach in curve 5 and the point of crossing of fluorescence thresholding curve is C12.
Specific embodiment:
Below in conjunction with specific embodiment, further set forth the present invention.
Be to be understood that, unaccounted normal condition and method among the following embodiment, usually according to the conventional employing method of affiliated field experimenter: as " molecular cloning experiment guide " third edition of Sa nurse Brooker and Russell chief editor, or the step and the condition of advising according to manufacturer.
Embodiment 1:
The preparation of test kit.Comprise the preparation of (1) probe; (2) DNA extraction agent; (3) premix reaction solution; (4) standard positive control; (5) standard negative control.
(1) preparation of probe;
Synthetic by common nucleotide primer synthesizer.Sequence is as follows:
The amplimer of GSTP1 gene:
Upstream primer F1A (C) 5-GAC CTC CGC TGC AAA TCC A-3 19bp
Upstream primer F1G (C) 5-AC CTC CGC TGC AAA TCC G-3 18bp
Downstream primer R1 5-GCA CTG GGG GCT GAA CAAA-3 19bp
The amplimer of XRCC1 gene:
Upstream primer F2A (C) 5-GCACAC TCC GGA ATG GCGA-3 19bp,
Upstream primer F2G (C) 5-GCA CAC TCC GGA ATG GCG G-3 19bp,
Downstream primer R2 5-CCT CCG GCG TAG CAC GCA-3 18bp,
Be the amount of water of 100 μ mol/L (being 100pmol/ μ l) concentration by every OD primer dilution before using, opened under the rotating speed that is preferably in 3000-4000 rev/min before the bottle cap dissolving centrifugal 1 minute that primer scatters and disappears when preventing to uncap.Before the use, experimentize after strong solution is diluted to working fluid 10pmol/ml.
(2) DNA extraction agent:
(3) pre-mixed PCR reaction solution
The premix reaction solution comprises 4 kinds, and every kind includes a pair of upstream and downstream amplimer, each corresponding genotype.Press the 1ml proportional quantity and calculate, the common composition is formulated as: the dNTP of 100 μ M, 20 μ l; 500U/ μ l TaqDNAPolymerase 20 μ l; 2 * Quant One Step SYBR, 1300 μ l; The upstream primer 50 μ l of 10 μ M; The downstream primer 50 μ l of 10 μ M; RNase-free ddH
2O 860 μ l.
1 pair of primer of pipe 1 is:
Upstream primer F1A (C) 5-GAC CTC CGC TGC AAA TCC A-3 19bp
Downstream primer R1 5-GCA CTG GGG GCT GAA CAAA-3 19bp
1 pair of primer of pipe 2 is:
Upstream primer F1G (C) 5-AC CTC CGC TGC AAA TCC G-3 18bp
Downstream primer R1 5-GCA CTG GGG GCT GAA CAAA-3 19bp
1 pair of primer of pipe 3 is:
Upstream primer F2A (C) 5-GCA CAC TCC GGAATG GCG A-3 19bp
Downstream primer R2 5-CCT CCG GCG TAG CAC GCA-3 18bp
1 pair of primer of pipe 4 is:
Upstream primer F2G (C) 5-GCA CAC TCC GGA ATG GCG G-3 19bp
Downstream primer R2 5-CCT CCG GCG TAG CAC GCA-3 18bp
(4) standard positive control:
Standard positive template is to contain to contain the Puc18-T recombinant plasmid that mutation nucleotide fragment constitutes in two genes of GSTP1 and XRCC1, this reorganization material transformed into escherichia coli DH5 α propagation back alkaline lysis method of extracting, through DNA purification kit purifying, quantitatively and be diluted to 10 with spectrophotometric instrumentation A260
9Copy/ μ l ,-20 ℃ of preservations.Storage saves as 10
9Copy/ μ l, working concentration are 10
5Copy/ μ l.
Standard positive template provides 4 kinds, and every kind of concentration is 10
5Copy/ul.Sequence is as follows:
SEQ1：
GGACCAGCAG?GAGGCAGCCC?TGGTGGACAT?GGTGAATGAC
GGCGTGGAGG?ACCTCCGCTG?CAAATACATC?TCCCTCATCT
ACACCAACTA?TGTGAGCATC?TGCACCAGGG?TTGGGCACTG
GGGGCTGAAC?AAAGAAAGGG?GCTTCTTGTG?CCCTCACCCC
CCTTACCCCT?CAGGTGGCTT?GGGCTGACCC?CTTCTTGGG；
SEQ2：
GGACCAGCAG?GAGGCAGCCC?TGGTGGACAT?GGTGAATGAC
GGCGTGGAGG?ACCTCCGCTG?CAAATACGTC?TCCCTCATCT
ACACCAACTA?TGTGAGCATC?TGCACCAGGG?TTGGGCACTG
GGGGCTGAAC?AAAGAAAGGG?GCTTCTTGTG?CCCTCACCCC
CCTTACCCCT?CAGGTGGCTT?GGGCTGACCC?CTTCTTGGG；
SEQ3：
TGCCCCTCAG?ATCACACCTA?ACTGGCATCT?TCACTTCTGC
CCCCCACCAG?CTGTGCCTTT?GCCAACACCC?CCAAGTACAG
CCAGGTCCTA?GGCCTGGGAG?GCCACATCGT?GCGTAAGGAG
TGGGTGCTGG?ACTGTCACCG?CATGCGTCGG?CGGCTGCCCT
CCCAGAGGTA?AGGCCTCACA?CGCCAACCCT?GCTC；
SEQ4：
GAGCAGGGTT?GGCGTGTGAG?GCCTTACCTC?CGGGAGGGCA
GCCGCCGACG?CATGCGGTGA?CAGTCCAGCA?CCCACTCCTT
ACGCACGATG?CGGCCTCCCA?GGCCCAGGACC?TGGCTGTACT
TGGGGGTGTT?GGCAAAGGCA?CAGCTGGTGG?GGGGCAGAAG
TGAAGATGGC?CAGTTAGGTG?TGATCTGAGG?GGCA。
(5) standard negative control:
Standard negative template is to contain the Puc18-T recombinant plasmid that 180 Nucleotide of arabidopsis gene segment constitute.Alkaline lysis method of extracting use in this reorganization material transformed into escherichia coli DH5 α propagation back, and is through DNA purification kit purifying, quantitative and be diluted to 10 with spectrophotometric instrumentation AA260
9Copy/ μ l ,-20 ℃ of preservations.Storage saves as 10
9Copy/ μ l, working concentration are 10
5Copy/ μ l.Sequence is as follows:
SEQ5：
TTCGTTATTG?AATCTTGTTT?TGGTTCTCTT?GTGTGGAATG
CATTGTTCTT?GCTTCTCTGG?AACTTGGGAT?ATGATGACAA
AAATACGATA?TCTCTAAGGC?TAATTGCTGT?GCGGATGAAA
TATATTTGCA?CGTCAGTCTG?ATCCCATTAT?GTATATAACA
TTAGGAGTTG?GTGCATGCTC。
Embodiment 2: the use of test kit
(1) preparation of sample
Sampling mode: use cotton swab wiping 10 times in cheek.
Attention: do not polluted in order to guarantee sample, take a sample and to take food and to drink water in preceding 30 minutes by food or beverage.
A) handle material: cotton swab transposition that will wiping in cheek is cut the cotton swab part with scissors in the 2ml centrifuge tube from its bar, add 400 μ l damping fluid GA.
B) add 20 μ l Proteinase K solution, 10 seconds mixings of vortex were placed 60 minutes for 56 ℃, and per therebetween 15 minutes vortex mixings for several times.
C) add 400 μ l damping fluid GB, fully put upside down mixing, placed 10 minutes for 70 ℃, this moment, the solution strain was limpid, and is brief centrifugal to remove the drop of cap wall.
D) add 200 μ l dehydrated alcohols, fully put upside down mixing, brief centrifugal to remove the drop of cap wall.
E) previous step gained solution and flocks are all added (adsorption column CR puts into collection tube) among the adsorption column CR, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.
F) in adsorption column CR, add 500 μ l damping fluid GD, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.
G) in adsorption column CR, add 700 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back to collection tube with adsorption column.
H) in adsorption column CR, add 500 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.
I) adsorption column CR is put back in the collection tube, 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column CR room temperature was placed several minutes, thoroughly to dry rinsing remaining in the sorbing material.
J) adsorption column CR is changed in the clean centrifuge tube, to the unsettled dropping in adsorption film mid-way 20-50 μ l elution buffer TB, room temperature was placed 2-5 minute, and 12,000rpm (～13,400 * g) centrifugal 2 minutes.Repeat once.
(2) experimentation
A) getting the every pipe 23ul of pre-mixed PCR reaction solution by sample number n (sample number=number of awaiting test sample+1+positive control of negative control 1) is sub-packed in the reaction tubes.
B) the above-mentioned sample to be tested of handling well and feminine gender, positive control (must vibrate the mixing several seconds) are respectively got 2ul and added respectively in the reaction tubes, mixing, carries out pcr amplification reaction immediately at the low-speed centrifugal several seconds.
C) PCR condition
95℃10sec
(95 ℃ of 15sec, 60 ℃ of 60sec) * 40 circulations
(3) interpretation of result and judgement:
A) judging criterion of detection by quantitative SNP:
The absolute value of the difference of the Ct value of two curves is designated as | Δ Ct|, when | Δ Ct|≤1 is judged as heterozygous; When | Δ Ct| 〉=5 are judged as homozygous.
As figure, among Fig. 1 | Δ Ct| 〉=5, represent that this sample is the homozygous sample of GSTP1 gene A A.Among Fig. 2, | Δ Ct|≤1, represent that this sample is a GSTP1 gene GA heterozygous sample.Among Fig. 3, | Δ Ct| 〉=5, represent that this sample is the homozygous sample of GSTP1 gene GG.Among Fig. 4 | Δ Ct| 〉=5, represent that this sample is the homozygous sample of XRCC1 gene A A.Among Fig. 5, | Δ Ct|≤1, represent that this sample is an XRCC1 Gene A heterozygous sample.Among Fig. 6, | Δ Ct| 〉=5, represent that this sample is the homozygous sample of XRCC1 gene GG.
B) interpretation of result:
Sequence table
＜110〉Shanghai Yulong Biological Technology Co., Ltd
＜120〉a kind of kit for detecting genes of leukemia of children
<160>11
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of GSTP1 gene order in the box, called after upstream primer F1A (C).
<400>1
gacctccgct?gcaaatcca 19
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of GSTP1 gene order in the box, called after upstream primer F1G (C).
<400>2
acctccgctg?caaatccg 18
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of GSTP1 gene order in the box, called after upstream primer R1.
<400>3
gcactggggg?ctgaacaaa 19
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of XRCC1 gene order in the box, called after upstream primer F2A (C).
<400>4
gcacactccg?gaatggcga 19
<210>5
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of XRCC1 gene order in the box, called after upstream primer F2G (C).
<400>5
gcacactccg?gaatggcgg 19
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to be used as the leukemia of children gene detection reagent
Detect the dna primer of the relevant sudden change of XRCC1 gene order in the box, called after upstream primer R2.
<400>6
cctccggcgt?agcacgca 18
<210>7
<211>199
<212>DNA
＜213〉Genus Homo (Homo)
<400>7
ggaccagcag?gaggcagccc?tggtggacat?ggtgaatgac?ggcgtggagg?acctccgctg 60
caaatacatc?tccctcatct?acaccaacta?tgtgagcatc?tgcaccaggg?ttgggcactg 120
ggggctgaac?aaagaaaggg?gcttcttgtg?ccctcacccc?ccttacccct?caggtggctt 180
gggctgaccc?cttcttggg 199
<210>8
<211>199
<212>DNA
＜213〉Genus Homo (Homo)
<400>8
ggaccagcag?gaggcagccc?tggtggacat?ggtgaatgac?ggcgtggagg?acctccgctg 60
caaatacgtc?tccctcatct?acaccaacta?tgtgagcatc?tgcaccaggg?ttgggcactg 120
ggggctgaac?aaagaaaggg?gcttcttgtg?ccctcacccc?ccttacccct?caggtggctt 180
gggctgaccc?cttcttggg 199
<210>9
<211>194
<212>DNA
＜213〉Genus Homo (Homo)
<400>9
tgcccctcag?atcacaccta?actggcatct?tcacttctgc?cccccaccag?ctgtgccttt 60
gccaacaccc?ccaagtacag?ccaggtccta?ggcctgggag?gccacatcgt?gcgtaaggag 120
tgggtgctgg?actgtcaccg?catgcgtcgg?cggctgccct?cccagaggta?aggcctcaca 180
cgccaaccct?gctc 194
<210>10
<211>195
<212>DNA
＜213〉Genus Homo (Homo)
<400>10
gagcagggtt?ggcgtgtgag?gccttacctc?cgggagggca?gccgccgacg?catgcggtga 60
cagtccagca?cccactcctt?acgcacgatg?cggcctccca?ggcccaggac?ctggctgtac 120
ttgggggtgt?tggcaaaggc?acagctggtg?gggggcagaa?gtgaagatgg?ccagttaggt 180
gtgatctgag?gggca 195
<210>11
<211>180
<212>DNA
＜213〉Arabidopis thaliana (Arabidopsis thaliana)
<400>11
ttcgttattg?aatcttgttt?tggttctctt?gtgtggaatg?cattgttctt?gcttctctgg 60
aacttgggat?atgatgacaa?aaatacgata?tctctaaggc?taattgctgt?gcggatgaaa 120
tatatttgca?cgtcagtctg?atcccattat?gtatataaca?ttaggagttg?gtgcatgctc 180
Claims (3)
1. kit for detecting genes of leukemia of children, by the DNA extraction agent, pre-mixed PCR reaction solution, standard positive reference substance, standard negative reference substance is formed; Described pre-mixed PCR reaction solution contains SYBR greenI, and the amplimer of GSTP1 gene relevant with leukemia of children and XRCC1 gene mutation site; Amplimer is upstream primer and the normal downstream primer that comprises the mutational site, and sequence is as follows:
The primer sequence of amplification GSTP1 gene is as follows:
Upstream primer F1A (C) 5-GAC CTC CGC TGC AAA TCC A-3 19bp,
Upstream primer F1G (C) 5-AC CTC CGC TGC AAA TCC G-3 18bp,
Downstream primer R1 5-GCA CTG GGG GCT GAA CAAA-3 19bp;
The primer sequence of amplification XRCC1 gene is as follows:
Upstream primer F2A (C) 5-GCA CAC TCC GGA ATG GCG A-3 19bp,
Upstream primer F2G (C) 5-GCA CAC TCC GGA ATG GCG G-3 19bp,
Downstream primer R2 5-CCT CCG GCG TAG CAC GCA-3 18bp.
2. kit for detecting genes of leukemia of children as claimed in claim 1 is characterized in that: described positive reference substance has 4, comprises following sequence respectively:
SEQ1：
GGACCAGCAG GAGGCAGCCC TGGTGGACAT GGTGAATGAC
GGCGTGGAGG ACCTCCGCTG CAAATACATC TCCCTCATCT
ACACCAACTA TGTGAGCATC TGCACCAGGG TTGGGCACTG
GGGGCTGAAC AAAGAAAGGG GCTTCTTGTG CCCTCACCCC
CCTTACCCCT CAGGTGGCTT GGGCTGACCC CTTCTTGGG；
SEQ2：
GGACCAGCAG GAGGCAGCCC TGGTGGACAT GGTGAATGAC
GGCGTGGAGG ACCTCCGCTG CAAATACGTC TCCCTCATCT
ACACCAACTA TGTGAGCATC TGCACCAGGG TTGGGCACTG
GGGGCTGAAC AAAGAAAGGG GCTTCTTGTG CCCTCACCCC
CCTTACCCCT CAGGTGGCTT GGGCTGACCC CTTCTTGGG；
SEQ3：
TGCCCCTCAG ATCACACCTA ACTGGCATCT TCACTTCTGC
CCCCCACCAG CTGTGCCTTT GCCAACACCC CCAAGTACAG
CCAGGTCCTA GGCCTGGGAG GCCACATCGT GCGTAAGGAG
TGGGTGCTGG ACTGTCACCG CATGCGTCGG CGGCTGCCCT
CCCAGAGGTA AGGCCTCACA CGCCAACCCT GCTC；
SEQ4：
GAGCAGGGTT GGCGTGTGAG GCCTTACCTC CGGGAGGGCA
GCCGCCGACG CATGCGGTGA CAGTCCAGCA CCCACTCCTT
ACGCACGATG CGGCCTCCCA GGCCCAGGACC TGGCTGTACT
TGGGGGTGTT GGCAAAGGCA CAGCTGGTGG GGGGCAGAAG
TGAAGATGGC CAGTTAGGTG TGATCTGAGG GGCA。
3. kit for detecting genes of leukemia of children as claimed in claim 1 or 2 is characterized in that: the sequence that described negative control product comprise is:
SEQ5：
TTCGTTATTG AATCTTGTTT TGGTTCTCTT GTGTGGAATG
CATTGTTCTT GCTTCTCTGG AACTTGGGAT ATGATGACAA
AAATACGATA TCTCTAAGGC TAATTGCTGT GCGGATGAAA
TATATTTGCA CGTCAGTCTG ATCCCATTAT GTATATAACA
TTAGGAGTTG GTGCATGCTC。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810200586A CN101684492A (en) | 2008-09-26 | 2008-09-26 | Kit for detecting genes of leukemia of children |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1724692A (en) * | 2005-07-19 | 2006-01-25 | 北京大学 | Reagent and method for detecting leucocythemia susceptibility |
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| CN1724692A (en) * | 2005-07-19 | 2006-01-25 | 北京大学 | Reagent and method for detecting leucocythemia susceptibility |
Non-Patent Citations (4)
| Title |
|---|
| CHRISTINE F. SKIBOLA, JOHN D. CURRY, ALEXANDRA NIETERS: "Genetic susceptibility to lymphoma", 《THE HEMATOLOGY JOURNAL》 * |
| PASCUAL BOLUFER ET AL.: "Influence of genetic polymorphisms on the risk of developing leukemia and on disease progression", 《LEUKEMIA RESEARCH》 * |
| SAMART PAKAKASAMA ET AL.: "Genetic Polymorphisms and Haplotypes of DNA Repair Genes in Childhood Acute Lymphoblastic Leukemia", 《PEDIATR BLOOD CANCER》 * |
| SARA ROLLINSON ET AL.: "High-Throughput Association Testing on DNA Pools to Identify Genetic Variants that Confer Susceptibility to Acute Myeloid Leukemia", 《CANCER EPIDEMIOL BIOMARKERS PREV》 * |
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