CN104372070A - UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene promoter region A (TA) nTAA polymorphism fluorescence detection kit - Google Patents

UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene promoter region A (TA) nTAA polymorphism fluorescence detection kit Download PDF

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CN104372070A
CN104372070A CN201310353481.XA CN201310353481A CN104372070A CN 104372070 A CN104372070 A CN 104372070A CN 201310353481 A CN201310353481 A CN 201310353481A CN 104372070 A CN104372070 A CN 104372070A
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mgb
kit
polymorphism
probes
gene promoter
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孙顺昌
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

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Abstract

The invention discloses a human UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene promoter region A (TA) nTAA polymorphism sequence detection kit belonging to the technical field of molecular detection. The kit comprises a primer, fluorescence probes, a PCR (polymerase chain reaction) buffer, MgCl2, dNTPs, Taq enzyme and ultra pure water. The kit upstream primer sequence is 5'-CATTAACTTGGTGTATCGATTGGT-3', the downstream primer sequence is 5'-AGCAGGCCCAGGACAAGT-3', the fluorescent probe is 5'-FAM (or VIC)-TTGCCATATATATATATATAAGTAGGAMGB-3', and the other fluorescent probe is 5 '-VIC (or FAM)-TGCCATATATATATATATATAAGTAGGA-MGB-3'. The kit adopts the double probes for simultaneous detection of a UGT1A1 gene promoter region A (TA) nTAA polymorphism sequence, is simple in operation, reduces the environmental and reaction solution cross pollution. The probe 3 'ends are modified by use of MGB, the probes are stronger in hybridization ability and more stable, Tm value difference between the probes and paired and non paired templates can be improved, and the kit is especially suitable for rich-AT target sequence detection.

Description

UGT1A1 gene promoter area A (TA) ntAA polymorphism fluorescence detection reagent kit
Technical field
Molecular Detection.
Background technology
UDP-Glucuronosyltransferase 1A1(uridine diphosphate glucuronosyltransferase 1A1, UGT1A1) being that bilirubin is converted into the key enzyme of conjugative bilirubin at liver metabolism, there is A (TA) in research display UGT1A1 gene promoter area ntAA polymorphism.A (TA) ntAA polymorphism is not only relevant to individual abnormal level of serum total bilirubin, also exists relevant to the toxicity, mammary cancer susceptibility etc. of some medicines, therefore A (TA) ntAA polymorphic detection has important clinical meaning.A (TA) ntAA polymorphism comprises A (TA) 5tAA, A (TA) 6tAA, A (TA) 7tAA and A (TA) 8tAA tetra-kinds of polymorphisms, wherein A (TA) 6tAA is the most common, A (TA) 7tAA takes second place, A (TA) 8tAA is accidental in the crowd of Caucasia, A (TA) 5tAA is accidental in African crowd, only finds A (TA) in Chinese population 6tAA and A (TA) 7tAA two kinds of polymorphic sequences.A (TA) ntAA polymorphism belongs to microsatellite polymorphism, and traditional detection method has single strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, capillary electrophoresis, denaturing high-performance chromatography etc.These detection techniques respectively have self feature, application is to a certain degree obtained in some fields, but also there are some defects in them, as recall rate is not high, false positive rate is high, detect that flux is low, experimental technique require high, experimental period also longer, testing cost is high, therefore these technology are difficult to use in clinical detection.Microsatellite polymorphism new detecting method has intrusion probe analysis method and high resolving power melting curve analysis method.Invading probe analysis method is the qualitative or quantitative measurement technology of a kind of DNA and RNA of isothermal, simple to operate, but detection sensitivity is lower.High resolving power melting curve analysis is a kind of PCR detection technique of efficient stable, without the need to sequence-specific probes, directly high resolution melting is carried out after pcr amplification terminates, complete paired samples polymorphic detection, fast easy and simple to handle, cost is low, has been widely used in detecting single nucleotide polymorphism, detects microsatellite polymorphism resolving power not high.A common ground of all these detection techniques is that they can not share trace routine with the pathogenic microbes fluorescent PCR detection technique having entered clinical application at present.TaqMan probe technology, since the nineties in last century is born, has become a kind of quantitative PCR technique of classics, and it is strong that it has detection specificity, highly sensitive.TaqMan probe technology both can be used for quantitative analysis, can be used for qualitative detection again.TaqMan probe technology for detection key is synthesising probing needle and primer, and this brings difficulty to detection microsatellite polymorphism sequence variations.Key of the present invention is successful design amplification UGT1A1 gene promoter area A (TA) nthe probe of TAA polymorphic sequence and primer, and hold use MGB to modify at probe 3 ', make probe especially be applicable to detection and be rich in AT tumor-necrosis factor glycoproteins.The gene amplification of current clinical detection pathogenic microbes substantially all have employed Fluorescence PCR assay, and most of test kit shares a trace routine, greatly simplify clinical detection.The present invention uses Fluorescence PCR assay to detect A (TA) ntAA polymorphism, synchronously can carry out with pathogenic microbes gene test, conveniently detects, and has clinical generalization value.
Summary of the invention
UGT1A1 gene promoter area A (TA) ntAA polymorphism fluorescence detection reagent kit composition comprises two Auele Specific Primers, two the fluorescently-labeled probes of difference, PCR damping fluid, MgCl 2, dNTPs, Taq DNA polymerase and ultrapure water.Upstream primer sequence is 5 '-CATTAACTTGGTGTATCGATTGGT-3 ', and downstream primer sequence is 5 '-AGCAGGCCCAGGACAAGT-3 '.Article one, fluorescent probe is 5 '-FAM(or VIC)-TTGCCATATATATATATATAAGTAGGA-MGB – 3 ', another fluorescent probe is 5 '-VIC(or FAM)-TGCCATATATATATATATATAAGTAGGA-MGB-3 ', wherein FAM and VIC is fluorescent mark, two kinds of fluorescence arbitrarily can mark a wherein probe, but two probes can not mark identical fluorescence.MGB is the modification group that probe 3 ' is held.
Embodiment
human gene group DNA's extracting: use phenol chloroform method extracting human gene group DNA. reaction solution is prepared: by each reacted constituent preparation PCR reaction solution. fluorescent PCR amplification instrument requires: have 465-580 nm excitation wavelength, and detecting pattern is containing the fluorescent PCR amplification instrument of hydrolysis probes. establishment PCR response procedures: first 95 DEG C of sex change 5 minutes, then enter 40 cyclic amplifications, each circulation comprises 95 DEG C of sex change 15 seconds, 60 DEG C of extensions 1 minute.Fluorescent collecting is set as 60 DEG C of single-point acquirings, and detecting pattern is double-colored hydrolysis probes, and analytical model is terminal gene type, arranges contrast. analytical results: after having increased, according to contrast judged result.
Nucleotide sequence
Upstream primer sequence: 5 '-CATTAACTTGGTGTATCGATTGGT-3 '
Downstream primer sequence: 5 '-AGCAGGCCCAGGACAAGT-3 '
, a fluorescent probe sequence: 5 '-TTGCCATATATATATATATAAGTAGGA-3 '
Another fluorescent probe sequence: 5 '-TGCCATATATATATATATATAAGTAGGA-3 '
 

Claims (3)

1.UGT1A1 gene promoter area A (TA) nthe feature of TAA polymorphism fluorescence detection reagent kit is that use a pair special primer and 2 novel MGB modify the A (TA) that hydrolysis probes detects Chinese population UGT1A1 gene promoter area 6tAA and A (TA) 7tAA two kinds of polymorphic sequences, test kit can use on fluorescent PCR amplification instrument.
2. according to described in right 1, a pair special primer comprises upstream primer and downstream primer, and upstream primer sequence is 5 '-CATTAACTTGGTGTATCGATTGGT-3 ', and downstream primer sequence is 5 '-AGCAGGCCCAGGACAAGT-3 '.
3. according to described in right 1, the novel MGB of test kit modifies hydrolysis probes 2, article one, fluorescent probe is 5 '-FAM(or VIC)-TTGCCATATATATATATATAAGTAGGA-MGB – 3 ', another fluorescent probe is 5 '-VIC(or FAM)-TGCCATATATATATATATATAAGTAGGA-MGB-3 ', wherein FAM and VIC is fluorescent mark, two kinds of fluorescence arbitrarily can mark a wherein probe, but two probes can not mark identical fluorescence, and MGB is modification group.
CN201310353481.XA 2013-08-14 2013-08-14 UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene promoter region A (TA) nTAA polymorphism fluorescence detection kit Pending CN104372070A (en)

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CN201310353481.XA CN104372070A (en) 2013-08-14 2013-08-14 UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) gene promoter region A (TA) nTAA polymorphism fluorescence detection kit

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520950A (en) * 2016-11-16 2017-03-22 武汉海吉力生物科技有限公司 UGT1A1 gene polymorphism detection primer and probe and kit

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006027182A1 (en) * 2004-09-06 2006-03-16 Medizinische Hochschule Hannover Methods and its kits based on ugt1a7 promoter polymorphism

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006027182A1 (en) * 2004-09-06 2006-03-16 Medizinische Hochschule Hannover Methods and its kits based on ugt1a7 promoter polymorphism

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
URSULA EHMER等: "Gilbert Syndrome Redefined: A Complex Genetic Haplotype Influences the Regulation of Glucuronidation", 《HEPATOLOGY》 *
URSULA EHMER等: "Rapid Allelic Discrimination by TaqMan PCR for the Detection of the Gilbert’s Syndrome Marker UGT1A1*28", 《JOURNAL OF MOLECULAR DIAGNOSTICS》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520950A (en) * 2016-11-16 2017-03-22 武汉海吉力生物科技有限公司 UGT1A1 gene polymorphism detection primer and probe and kit

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Application publication date: 20150225