CN100396792C - Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia - Google Patents
Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia Download PDFInfo
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Abstract
The present invention provides a nucleic acid hybridization film strip for clinically diagnosing beta-Mediterranean anemia and a reagent box thereof. The nucleic acid hybridization film strip comprises a base and specific probes which ar fixed to the base, wherein each specific probe respectively aims at a mutational beta-globin gene site to be called as a mutational probe; the mutational beta-globin gene site is selected form one or the combination of 27-28M, 41-42M, 654M,-28M, 71-72M, 17M, beta EM, 31M, IVS1-1M, 43M,-32M,-29M,-30M, 14-15M, CAP, IntM and IVS1-5M; each mutational probe comprises a sequence of 14 to 20 basic groups, and the middle position of the basic group sequence comprises a corresponding mutational beta-globin gene mutational basic group. The nucleic acid hybridization film strip for diagnosing beta-Mediterranean anemia diseases has the advantages of easy judgment of normal and mutational heterozygote and mutational homozygote, good signal specificity, high diagnosing efficiency, high diagnosing accuracy, low cost, and easy popularization in hospitals.
Description
Technical field
The present invention relates to be used to diagnose thalassemic nucleic acid hybridization film bar, relate in particular to and be used for diagnosing beta-thalassemic nucleic acid hybridization film bar.
The invention still further relates to the PCR primer of the sample DNA to be checked that is used for increasing.
The invention still further relates to and be used for diagnosing beta-thalassemic test kit.
Background technology
Thalassemia (be called for short ground poor) is a kind of common heredopathia, and generation is all arranged all over the world, sees the west at most from the mediterranean region, in through Turkey, various countries, the Middle East, to the east of South East Asia and south China.The clinical phenotypes of thalassemia is various, can be from normally transfusing blood repeatedly, even threat to life to needs, and cause the patient at teenage day before yesterday folding even stillborn foetus.Because this disease at the southern high incidence of China, produces huge spirit and economic pressures to society and family.Yet, still lack therapy measure at present, therefore carry out genetic counseling and antenatal diagnosis, stop the birth of such seriously ill disease infant, to this type of disease of effective monitoring, it is significant to improve the health of the people.
Thalassemia is divided into α-Di Zhonghaipinxue and β-thalassemia (it is poor to be called for short β-ground).β-thalassemia (be called for short β-ground poor) is a kind ofly to cause the peptide chain imbalance of expression and the monogenic inheritance hemopathy that produces by the beta-globin gene unconventionality,, be one of inherited disease that each province, China south is the most common, harm is maximum how by due to the beta-globin point mutation.
Because thalassemic clinical symptom has bigger variability, light-duty patient and carrier's detection is prone to omission.Along with the progress of thalassemia molecule genetics research, make gene diagnosis become possibility.β-thalassemic genetic flaw overwhelming majority belongs to non-deletion type, promptly because point mutation causes the beta-globin resulting anomaly.β-thalassemia has kind of a group specificity, show as many sudden changes and occur over just among certain indivedual race, and almost each race has several frequently seen sudden change.By 1998, in Chinese β-thalassemia colony, found 23 kinds of different mutation types, the each province, continent is found 22 kinds (wherein 3 kinds are detected in ethnic minority) altogether, Taiwan is found 18 kinds altogether.In Chinese population common mutational site have 41-42M, 654M ,-28M, 71-72M etc., wherein 5 kinds of sudden changes show higher frequency of disease development in Chinese population, account for more than 90% of total incidence.Table 1 is the statistics of the various mutation type occurrence frequencies of β-thalassemia.
The statistics of the various mutation type occurrence frequencies of table 1 β-thalassemia
β-thalassemia mutation type | Mutation rate (%) |
CD41-42 | 36.22 |
IVS-2-654 | 24.80 |
-28 | 16.54 |
βE(CD26) | 5.12 |
CD17 | 3.94 |
CD71-72 | 3.54 |
CD43 | 3.54 |
-29 | 1.97 |
CD27-28 | 1.97 |
Other | 2.36 |
Amount to | 100 |
The diagnostic techniques commonly used of β-thalassemia has: nucleic acid hybridization film bar method, PCR-SSCP, PCR-heteroduple analysis, PCR-DGGE, PCR-ARMS, PCR-ASO, PCR-RFLP, PCR-mispairing chemical cracking and DNA chip (fluorescent mark glass-chip) method etc.
Application number is that 02117287.0 Chinese patent application discloses and is used to diagnose thalassemic DNA chip and preparation method thereof, a kind of poor DNA chip of clinical diagnosis α-type and β-type ground that is used for is provided, chip is by a substrate and be fixed in suprabasil probe and form, at hitherto known α-and the sequence of beta-globin mutator gene, according to the oligonucleotide probe of these sudden changes of diagnosis of the principle design of dna sequence dna specific recognition.
Aforementioned DNA chip is realized all being different from present patent application on the carrier in probe design principle and product.In this patent application, adopt sheet glass as substrate (being carrier), promptly adopt the glass-chip method to detect dna mutation, and when the designing probe sequence, the base of suddenling change is designed in the end that is positioned at probe.The weak point of this method is that mutant probe and normal probe in the same position of slide, just lean on the color difference to distinguish, and the sample of heterozygous mutation is exactly a mixture colors like this, bad judgement; The unicity of adding this method signal is relatively poor, and often with spurious response, being difficult to sometimes distinguish when mixed signal occurring is with spurious response or heterozygous mutation.And detect needs with this method and buy expensive laser scanner, the general hospital laboratory of China does not also have such economic condition.This has significant limitation with regard to causing this method product in the popularization of China.
What nucleic acid hybridization film bar method detected β-thalassemia application is that PCR adds reversal point hybridization (RDB) technology, and this technology is on the basis of PCR-ASO technology, is proposed by people such as Saiki in 1989.This method is that a series of known detection probes are fixed on the carrier, is hybridized with it by the PCR product of test sample, and again by a series of color reactions, whether test sample PCR product has and known probe complementary series.This method has that to detect flux big, characteristics such as signal specificity height.From know-why, nucleic acid hybridization film bar also is a kind of in the DNA chip (generally being middle density chip) in itself, but it is different from general said glass DNA chip, its detected result with the naked eye just can directly be judged, and need not to buy expensive laser scanner, be easier to carry out clinical expansion in the hospital of China.
With the nylon membrane is the nucleic acid hybridization film bar technology of substrate, and its main ultimate principle is as follows: oligonucleotide probe is the oligonucleotide fragment that contains specific dna sequence, and its end has amino labeled.The nylon membrane of activated processing has carboxyl, can form covalent attachment with amino.Utilize this method can be with oligonucleotide probe by the firm surface that is fixed on nylon membrane of certain arrangement regulation.In conjunction with round pcr patient's genome DNA sample is carried out a large amount of rapid amplifyings on this basis, the PCR product after the amplification contains biotin labeling (being marked in advance on the PCR primer).After probe on PCR product and the film bar carries out specific hybrid, by series reaction such as vitamin H, Streptavidin-peroxidase, tetramethyl benzidines, there is the correspondent probe site in the hybridization of PCR product just to have blue hybridization signal demonstration, just can judges having or not of clinical sample corresponding positions point mutation in view of the above.Because corresponding normal control has mostly been designed in the mutational site on the film bar, have or not in conjunction with normal probe signals and just can judge that the corresponding positions point mutation is heterozygote or homozygote.Its main advantage is that signal specificity is strong, several specific signals nothing but.Remove in addition, its advantage is mainly simple and easy to do in addition, and technical requirements is low, and plant and instrument drops into little, is easy to do clinical expansion etc. in developing country.Hospital only need buy a common PCR instrument, a hybridization instrument and other laboratory common equipment just can finish whole experiment, can accomplish at the hospital laboratory that China is general.
Up to now, still not having bibliographical information is the nucleic acid hybridization film bar that substrate or mutator gene are positioned at oligonucleotide probe middle part with the nylon membrane at β-thalassemia.
Summary of the invention
One object of the present invention is to provide a kind of poor nucleic acid hybridization film bar in clinical diagnosis β-ground that is used for.
Another object of the present invention be to be provided for the to increase PCR primer of DNA in the sample to be checked.
A further object of the present invention is to be provided for diagnosing beta-thalassemic test kit.
According to an aspect of the present invention, the invention provides a kind of diagnosing beta-thalassemic nucleic acid hybridization film bar that is used for, comprise a substrate; And
Be fixed in described suprabasil specific probe; It is characterized in that:
The described specific probe of each bar at the beta-globin gene locus of a sudden change, is called mutant probe respectively;
The beta-globin gene locus of described sudden change be selected from 27-28M, 41-42M, 654M ,-28M, 71-72M, 17M, β EM, 31M, IVS1-1M, 43M ,-32M ,-29M ,-the one or more combination of 30M, 14-15M, CAP, IntM and IVS1-5M;
Wherein each bar mutant probe comprises the sequence of 14 to 20 bases, comprises the mutating alkali yl of the beta-globin gene of its corresponding sudden change at the medium position of this base sequence.
Compare in the end of oligonucleotide probe with the mutational site, the mutational site can obviously reduce the non-specific hybridization signal in the centre of oligonucleotide probe, improves the specificity that detects greatly.
In the present invention, N represents normal type, and M represents mutant, and the implication normal or mutational site that relates in the present invention is listed as follows:
Table 2:
Letter | Implication |
27-28M | The base mutation of beta-globin the 27/28th amino acids correspondence |
41-42M | The base mutation of beta-globin the 41/42nd |
654M | The 654th bit base sudden change of second intron of beta-globin |
-28M | The 28th bit base sudden change of beta-globin gene promoter upstream |
71-72M | The base mutation of beta-globin the 71/72nd |
17M | The base mutation of beta-globin the 17th amino acids correspondence |
βEM | The base mutation of beta-globin the 26th amino acids correspondence (being 26M) |
31M | The base mutation of beta-globin the 31st amino acids correspondence |
IVS1-1M | The 1st bit base sudden change of first intron of beta-globin |
43M | The 43rd bit base sudden change of beta-globin gene |
-32M | The 32nd bit base sudden change of beta-globin gene promoter upstream |
-29M | The 29th bit base sudden change of beta-globin gene promoter upstream |
-30M | The 30th bit base sudden change of beta-globin gene promoter upstream |
14-15M | The base mutation of beta-globin the 14/15th amino acids correspondence |
CAP | Beta-globin transcription initiation is undergone mutation |
IntM | The sudden change of beta-globin translation initiation site |
IVS1-5M | The 5th bit base sudden change of first intron of beta-globin |
654N | The 654th bit base of second intron is normal beta-globin gene, and other back mark N's can the rest may be inferred |
The substrate that is used for this nucleic acid hybridization film bar can be any substrate that is suitable for the immobilized oligonucleotide probe, for example nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, silica gel wafer, micro magnetic bead etc.
In a preferred embodiment of the present invention, this substrate is a nylon membrane.Use the activation solution pre-treatment after 30 minutes nylon membrane, the activated surface carboxyl, the activatory carboxyl reacts with the terminal amino that has the oligonucleotide probe of amino labeled and generates stable covalent linkage then, thereby can be with probe stationary on the nylon membrane surface.
Being fixed in suprabasil probe then is the sequence that detects gene at aforesaid beta-globin sudden change, oligonucleotide probe according to these sudden changes of diagnosis of the principle design of dna sequence dna specific recognition, by spot sample device probe is arranged in substrate according to certain rule, as on the nylon membrane etc., through just having made nucleic acid hybridization film bar after fixing and the relevant treatment.
According to the probe of about 14 to 20 bases of the different designs of GC content, but each probe all must include corresponding mutational site during mutant probe in design, generally in the centre.
Described mutant probe is selected from least one sequence among SEQ ID NO.1~SEQ ID NO.17 or the DNA of its reverse complementary sequence, has covered 17 mutational sites of beta-globin.When selecting probe, can choose one or more groups as required wantonly, from every group, choose one wantonly then.The designed probe sequence sees Table 3:
The dna sequence dna of table 3 mutant probe
Sequence numbering | The probe title | Base length (bp) | Sequence |
SEQ ID NO.1 | 27-28M | 14 | 5’GGTGAGGCCCCTGG 3’ |
SEQ ID NO.2 | 41/42M | 19 | 5’CAAAGGACTCAACCTCTGG 3’ |
SEQ ID NO.3 | 654M | 19 | 5’ATTGCTATTACCTTAACCC 3’ |
SEQ ID NO.4 | -28M | 17 | 5’CCCTGACTTCTATGCCC 3’ |
SEQ ID NO.5 | 71/72M | 17 | 5’GGTGCCTTTAAGTGATG 3’ |
SEQ ID NO.6 | 17M | 17 | 5’TGTGGGGCTAGGTGAAC 3’ |
SEQ ID NO.7 | βEM | 16 | 5’TTGGTGGTAAGGCCCT 3’ |
SEQ ID NO.8 | 31M: | 16 | 5’CTTAGGTGCTGGTGGT 3’ |
SEQ ID NO.9 | IVS1-1M | 16 | 5’CTGGGCAGWTTGGTAT 3’ |
SEQ ID NO.10 | 43M | 19 | 5’GGTTCTTTTAGTCCTTTGG 3’ |
SEQ ID NO.11 | -32M: | 17 | 5’CCTGACTTTTATTCCCA 3’ |
SEQ ID NO.12 | -29M | 17 | 5’CCCTGACTTTCATGCCC 3’ |
SEQ ID NO.13 | -30M: | 16 | 5’CCTGACTTTTGTGCCC 3’ |
SEQ ID NO.14 | 14/15M | 16 | 5’CCCTGGTGGGGCAAGG 3’ |
SEQ ID NO.15 | CAP | 17 | 5’CATGGTGTCTGAGGTTG 3’ |
SEQ ID NO.16 | IntM: | 17 | 5’GGTGCACCCTGGTGTCT 3’ |
SEQ ID NO.17 | IVS 1-5M: | 16 | 5’GCAGGTTGCTATCAAG 3’ |
Annotate: the W=A/T in the SEQ ID NO.9 probe I V S1-1M sequence.
Further, described nucleic acid hybridization film bar also comprises and is fixed in described suprabasil specific probe at the beta-globin normal gene, is called normal probe, and itself and mutant probe are individually fixed on the different positions of substrate.
Described normal probe is in contrast, is beneficial to judge more accurately mutation type.Above probe also can be the sequence of reverse complemental.Normal can also be different sections with mutant probe.
Described normal probe is selected from least one sequence among SEQ ID NO.18~SEQ ID NO.24 or the DNA of its reverse complementary sequence; Each normal probe all corresponds respectively to the beta-globin gene locus of one or several sudden change; Specifically see Table 4 and table 5:
Table 4: normal probe at the mutational site
Normal probe sequence numbering | Normal probe title | At the mutational site |
SEQ ID NO.18 | 41/42N | 41-42M、43M |
SEQ ID NO.19 | 654N | 654M |
SEQ ID NO.20 | -28N | -28M、-29M、-30M、-32M |
SEQ ID NO.21 | 71/72N | 71-72M |
SEQ ID NO.22 | |
17M、14-15M |
SEQ ID NO.23 | BEN | BEM |
SEQ ID NO.24 | 31N | 31M |
The dna sequence dna of the normal probe of table 5
Sequence numbering | The probe title | Base length (bp) | Sequence |
SEQ ID NO.18 | 41/42N | 20 | 5’CAGAGGTTCTTTGAGTCCTT 3’ |
SEQ ID NO.19 | 654N | 19 | 5’GGGTTAAGGCAATAGCAAT 3’ |
SEQ ID NO.20 | -28N | 18 | 5’TGGGCATAAAAGTCAGGG 3’ |
SEQ ID NO.21 | 71/72N | 17 | 5’TCGGTGCCTTTAGTGAT 3’ |
SEQ ID NO.22 | 17N | 17 | 5’TGTGGGGCAAGGTGAAC 3’ |
SEQ ID NO.23 | BEN | 16 | 5’CAGGGCCTCACCACCA 3’ |
SEQ ID NO.24 | 31N | 16 | 5’CTTAGGCTGCTGGTGG 3’ |
Further, said mutation probe and/or normal probe can further carry out amino or oligo mode marks such as (T) at 5 ' end or 3 ' end, so that probe can combine better with substrate.Mark and join method with ultraviolet not also can stationary probe.
The above-mentioned preparation method who is used for the poor nucleic acid hybridization film bar in diagnosing beta-ground comprises synthesising probing needle, with synthetic probe and corresponding control group DNA point sample and being arranged in the substrate in sequence, the dna probe behind the point sample is fixed in prepares nucleic acid hybridization film bar in the substrate.
According to another aspect of the present invention, the invention provides the PCR primer of the sample DNA to be checked that is used for increasing.The amplified production of described primer contains and can the dna sequence dna of specific hybrid take place with the beta-globin detection probes (part wherein normal and/or mutant probe) of suddenling change, described primer comprises two pairs of primers, is respectively SEQ ID NO.25 and SEQ IDNO.26, SEQ ID NO.27 and SEQ ID NO.28;
Wherein the amplified production of SEQ ID NO.27 and SEQ ID NO.28 can be hybridized with 654N and/or 654M, is used to detect 654 site mutations; The amplified production of SEQ ID NO.25 and SEQ ID NO.26 is used to detect other site mutation except that 654 sites.
The beta-globin gene PCR primed DNA sequence of table 6 different lengths
Sequence numbering | The primer title | Base length (bp) | Sequence |
SEQ ID NO.25 | B1 | 19 | 5’CACTTAGACCTCACCCTGT 3’ |
SEQ ID NO.26 | B2 | 20 | 5’TGACATGAACTTAACCATAG 3’ |
SEQ ID NO.27 | B3 | 20 | 5’TAACAGTGATAATTTCTGGG 3’ |
SEQ ID NO.28 | B4 | 20 | 5’CCAGTTTAGTAGTTGGACTT 3’ |
To partly or entirely carrying out vitamin H or radioactivity, fluorescence mode marks such as (as CY5, CY3, TAMRA) in the above-mentioned primer, make and contain corresponding mark in the product of amplification, so that can after hybridization, analyze to results of hybridization.
According to a further aspect of the invention, provide the test kit that is used to detect β-thalassemia, wherein it comprises
Above-mentioned diagnosing beta-thalassemic nucleic acid hybridization film the bar that is used for; And
The PCR reaction solution of the above-mentioned sample DNA to be checked that is used for increasing;
The temperature of reaction of nucleic acid hybridization is 40 ℃~46 ℃, and its optimal reaction temperature is 42 ℃~44 ℃.
When this test kit is used to detect β-thalassemia, may further comprise the steps:
1) will carry out pcr amplification with Auele Specific Primer of the present invention from the DNA of sample to be checked and obtain amplified production;
2) product and the diagnosing beta-thalassemic nucleic acid hybridization film bar that is used for of the present invention that obtain are hybridized;
3) detected result.
In a preferred embodiment of the present invention, prepare nucleic acid hybridization film bar of the present invention and utilize the method for its test sample to be summarized as follows:
1, the making of film bar
The activated liquid pre-treatment of nylon membrane is after 30 minutes, the activated surface carboxyl; Simultaneously, after will being diluted to suitable concentration with damping fluid at the probe that 5 ' or 3 ' end has an amino labeled, be added on the nylon membrane corresponding position, at this moment by array order point, amino reacting with the activatory carboxyl generates stable covalent linkage, thus with probe stationary on the nylon membrane surface; At last, handle nylon membrane with the liquid of blockading, seal the not surperficial carboxyl of bonding probes, the film bar completes.
2, the preparation of sample DNA to be checked
Adopt boiling lysis or whole blood DNA rapid extraction test kit extraction method from blood, to extract the human gene group DNA.
3, pcr amplification sample to be checked
The amplification PCR fragment comprises the testing gene fragment, and has biotin labeling at PCR product 5 ' end.
4, hybridization
Product to be measured behind the mark and the probe that is fixed on the film bar are hybridized, and its hybridization principle is identical with common nucleic acid hybridization.The length of the testing sample behind hybridization temperature and probe and the mark, GC content, salt concn, methane amide etc. are relevant.In system of the present invention, hatch the results of hybridization that can obtain satisfaction after 1.5~4 hours for 42 ℃.
5, wash-out non-specific binding thing
Utilize different elution requirements, remove non-specific hybridization to greatest extent as temperature, salt concn etc.
6, colour developing
The Streptavidin (streptavidin) that is marked with peroxidase (POD) is held the vitamin H specific combination that has with PCR product 5 ', and issuing biochemical color reaction in the existence of hydrogen peroxide and brown substrate TMB (tetramethyl benzidine), the correspondent probe position manifests blue spot on the film bar.
7, detected result
Can judge detected result through visual inspection.Also can after reading apparatus is read, carry out Computer Analysis, obtain final results of hybridization by the corresponding software system analysis, and automatically the result is preserved and adds up signal and background.
Diagnosing beta-thalassemic nucleic acid hybridization film the bar that is used for of the present invention, its probe that is used to detect sample that is fixed in substrate has comprised the main mutational site relevant with the poor generation in β-ground, in the probe sequence base of sudden change is included in the roughly mid-way of whole sequence, and mutant probe and normal probe are arranged in respectively on the different positions of substrate, to normally, heterozygous mutation, the sudden change homozygote all is easy to judge, and signal specificity is very good, can improve diagnosis efficiency and diagnostic accuracy, reduce cost, shorten Diagnostic Time, reduce cost, be easy to promote in hospital.
In order to understand essence of the present invention better,,, describe in detail but do not limit the present invention by description to better embodiment of the present invention below in conjunction with accompanying drawing.
Description of drawings
Fig. 1 is the detected result figure of the nucleic acid hybridization film bar of substrate with the nylon membrane for the present invention, wherein, be followed successively by three of nucleic acid hybridization film bar A~C figure as a result from top to bottom, among each figure, first row be followed successively by from left to right 41-42N, 654N ,-28N, 71-72N, 17N, β EN, 31N, 27-28M, second row be followed successively by from left to right 41-42M, 654M ,-28M, 71-72M, 17M, β EM, 31M, IVS1-1M, second row be followed successively by from left to right 43M ,-32M ,-29M ,-30M, 14-15M, CAP, IntM, IVS1-5M;
Fig. 2 is for being the detected result figure of the DNA chip of substrate with the sheet glass, and wherein, figure (A) is normal people's a sample, the sample of figure (B) for undergoing mutation, and among two figure ,-28 sites are all played second on first a row left side, and the 41-42 site is all played the 4th on second a row left side;
Fig. 3 is the detected result figure of the nucleic acid hybridization film bar of substrate with the nylon membrane for the present invention, wherein, is followed successively by the figure as a result of nucleic acid hybridization film bar D and E from top to bottom, among each figure, and the same Fig. 1 in the position of probe.
Embodiment
The used experiment material of the present invention if no special instructions, is the commercially available prod.
[embodiment 1] is used to detect the preparation of the poor nucleic acid hybridization film bar in β-ground
1, the preparation of probe
According to the sequence synthesising probing needle in the table 3, the probe of reverse complementary sequence have with the table in the identical effect of sequence, this table in list no longer one by one.5 ' end or 3 ' end to probe carry out amino labeled and have identical effect, and the synthetic method be the DNA synthesis method of routine.
The dna sequence dna of table 3 mutant probe
Sequence numbering | The probe title | Base length (bp) | Sequence |
SEQ ID NO.1 | 27-28M | 14 | 5’GGTGAGGCCCCTGG 3’ |
SEQ ID NO.2 | 41/42M | 19 | 5’CAAAGGACTCAACCTCTGG 3’ |
SEQ ID NO.3 | 654M | 19 | 5’ATTGCTATTACCTTAACCC 3’ |
SEQ ID NO.4 | -28M | 17 | 5’CCCTGACTTCTATGCCC 3’ |
SEQ ID NO.5 | 71/72M | 17 | 5’GGTGCCTTTAAGTGATG 3’ |
SEQ ID NO.6 | 17M | 17 | 5’TGTGGGGCTAGGTGAAC 3’ |
SEQ ID NO.7 | BEM | 16 | 5’TTGGTGGTAAGGCCCT 3’ |
SEQ ID NO.8 | 31M: | 16 | 5’CTTAGGTGCTGGTGGT 3’ |
SEQ ID NO.9 | IVS1-1M | 16 | 5’CTGGGCAGWTTGGTAT 3’ |
SEQ ID NO.10 | 43M | 19 | 5’GGTTCTTTTAGTCCTTTGG 3’ |
SEQ ID NO.11 | -32M: | 17 | 5’CCTGACTTTTATTCCCA 3’ |
SEQ ID NO.12 | -29M | 17 | 5’CCCTGACTTTCATGCCC 3’ |
SEQ ID NO.13 | -30M: | 16 | 5’CCTGACTTTTGTGCCC 3’ |
SEQ ID NO.14 | 14/15M | 16 | 5’CCCTGGTGGGGCAAGG 3’ |
SEQ ID NO.15 | CAP | 17 | 5’CATGGTGTCTGAGGTTG 3’ |
SEQ ID NO.16 | IntM: | 17 | 5’GGTGCACCCTGGTGTCT 3’ |
SEQ ID NO.17 | IVS1-5M: | 16 | 5’GCAGGTTGCTATCAAG 3’ |
Annotate: the W=A/T in the SEQ IDNO.9 probe I V S1-1M sequence.
The dna sequence dna of the normal probe of table 5
Sequence numbering | The probe title | Base length (bp) | Sequence |
SEQ ID NO.18 | 41/42N | 20 | 5’CAGAGGTTCTTTGAGTCCTT 3’ |
SEQ ID NO.19 | 654N | 19 | 5’GGGTTAAGGCAATAGCAAT 3’ |
SEQ ID NO.20 | -28N | 18 | 5’TGGGCATAAAAGTCAGGG 3’ |
SEQ ID NO.21 | 71/72N | 17 | 5’TCGGTGCCTTTAGTGAT 3’ |
SEQ ID NO.22 | 17N | 17 | 5’TGTGGGGCAAGGTGAAC 3’ |
SEQ ID NO.23 | BEN | 16 | 5’CAGGGCCTCACCACCA 3’ |
SEQ ID NO.24 | 31N | 16 | 5’CTTAGGCTGCTGGTGG 3’ |
2, the making of nucleic acid hybridization film bar
Finish after oligonucleotide probe synthetic, need carry out the fixing of direct oligonucleotide probe.Earlier nylon membrane is cut into certain size, and the probe title of printed grid and corresponding grid thereon, activated liquid pre-treatment is after 30 minutes, the activated surface carboxyl; Simultaneously, will be diluted to suitable concentration with probe dilution liquid at the probe that 5 ' or 3 ' end has an amino labeled after, with spot sample device each probe points is added on the corresponding grid.The carboxyl of the amino of probe end and film surface active reacts and generates stable covalent linkage, thus with probe stationary on nylon membrane.At last, handle nylon membrane with the liquid of blockading, seal the not surperficial carboxyl of bonding probes, nucleic acid hybridization film bar completes.
The array order of table 7 nucleic acid hybridization film bar point sample
41-42N | 654N | -28N | 71-72N | 17N | B EN | 31N | 27-28M |
41- |
654M | -28M | 71- |
17M | B EM | 31M | IVS1-1M |
43M | -32M | -29M | -30M | 14-15M | CAP | IntM | IVS1-5M |
Annotate: 43M and 41-42M can be reference with 41-42N simultaneously ,-32M ,-30M ,-29M and-28M all can-28N is a normal control, 14-15M and 17M can do reference by 17N.
[embodiment 2] be used for increasing primer design of sample DNA to be checked
According to the sequence data of β-thalassemia transgenation, with two pairs specific, have different lengths and Tm value and contain two fragments of biotin labeled primer amplification beta-globin gene at 5 ' end.The nucleotide sequence of these two pairs of primers sees Table 6.In this table, the amplified production of SEQ ID NO.27 and SEQ ID NO.28 can be hybridized with 654N and/or 654M, is used to detect 654 site mutations; The amplified production of SEQ ID NO.25 and SEQ ID NO.26 is used to detect other site mutation except that 654 sites.
The beta-globin gene PCR primed DNA sequence of table 6 different lengths
Sequence numbering | The primer title | Base length (bp) | Sequence |
SEQ ID NO.25 | B1 | 19 | 5’CACTTAGACCTCACCCTGT 3’ |
SEQ ID NO.26 | B2 | 20 | 5’TGACATGAACTTAACCATAG 3’ |
SEQ ID NO.27 | B3 | 20 | 5’TAACAGTGATAATTTCTGGG 3’ |
SEQ ID NO.28 | B4 | 20 | 5’CCAGTTTAGTAGTTGGACTT 3’ |
The detection of [embodiment 3] sample DNA to be checked
One, tests with instrument and reagent
1, test instrument
PCR instrument, DNA electrophoresis apparatus, molecular hybridization case, shaking table
2, test reagent
(1)3%H
2O
2
(2) 20 * SSC (pH7.0): get NaCl 175.3g, Trisodium Citrate 88.2g adds H
2O is molten to 1000mL, transfers pH to 7.0, autoclaving with pH meter.
(3) 10%SDS (pH 7.0): 20g SDS is added H2O 180mL dissolving, transfer pH to 7.0, be settled to 200mL at last with HCl.
(4) A liquid (pH 7.4 for 2 * SSC, 0.1%SDS): get 20 * SSC 100mL, 10%SDS 10mL, add H
2O is molten to 1000mL, transfers pH to 7.4.
(5) B liquid (pH 7.4 for 0.5 * SSC, 0.1%SDS): get 20 * SSC 25mL, 10%SDS 10mL, add H
2O is molten to 1000mL, transfers pH to 7.4.
(6) C liquid (0.1M Trisodium Citrate, pH 5.4): get Trisodium Citrate 14.7g, be dissolved in 450mL water, transfer pH to 5.0, be settled to 500mL at last with dense HCl.
(7) strepto-affinity element-peroxidase (streptavidin-POD)
(8) (TMB) of tetramethyl benzidine
Two, the detection of sample DNA to be checked
1, the preparation of sample DNA to be checked
Get three parts of blood samples (being designated as A, B, C respectively), any that select following two kinds of methods for use extracts.
(1) boiling lysis
Get 100 μ L anticoagulated whole bloods, add the 1mL sterilized water, abundant mixing, room temperature is placed 5 minutes with splitting erythrocyte, and centrifugal 1 minute of 13000rpm abandons supernatant and as seen manages the end one white precipitate is arranged; Repeat this operation 1~2 time, in the white precipitate piece, almost can not see blood red.Blot supernatant liquor, add lysate 50 μ L, fully mixing boiled 10 minutes in 100 ℃ of water-baths; Centrifugal 5 minutes of 13000rpm gets supernatant liquor and promptly can be used as the poor pcr template in β-ground.
(2) whole blood DNA rapid extraction test kit extraction method
Use the Qiagen whole blood to extract whole blood DNA rapid extraction test kit extracting human gene group DNA from anticoagulated whole blood of test kit or Yaneng Biotechnology (Shenzhen) Co., Ltd..
2, the pcr amplification of goal gene
Get three of PCR-Mix reaction tubess, carry out mark on tube wall, and in 5000rpm centrifugal 2 seconds, (it is about 50~100ng) to contain genomic dna, and this moment, reaction totally was 25 μ L then to add the testing sample DNA2 μ L that extracted respectively.
β-PCR Mix-I 23μL
Total 25μL
Increase by following condition:
50℃ 15min
95℃ 10min
72℃ 5min
The amplification PCR fragment includes the testing gene fragment, and has biotin labeling at PCR product 5 ' end.
3, hybridization
Get the 15mL plastic centrifuge tube, indicate sample number into spectrum to be checked, put into the nucleic acid hybridization film bar (by embodiment 1 preparation) that indicates sample number into spectrum to be checked equally, add A liquid 5-8mL and PCR product, lid is screwed on.Plastics tubing is put into 10 minutes (guaranteeing that the hybridization solution liquid level is positioned under the water-bath liquid level fully) of boiling water heating, take out and tighten lid, put into 42 ℃ of hybridization casees hybridization 1.5~4 hours.Get a 50mL plastics tubing, add 40mLB liquid and in hybridization case or incubator, be preheated to 42 ℃.
4, wash film
Take out the film bar, move in the 50mL pipe that preheating B liquid is housed, in 42 ℃ of jog washings 15 minutes.By aforesaid operations, can remove non-specific hybridization to greatest extent.
5, colour developing
With strepto-affinity element-peroxidase (streptavidin-POD) solution of 1: 2000 of A liquid preparation.Earlier the film bar was soaked 30 minutes with this streptavidin-POD solution room temperature jog, discard streptavidin-POD solution; Wash film twice with A liquid chamber temperature jog again, each 5 minutes; Washed film 1~2 minute with C liquid chamber temperature then, (colour developing liquid needs fresh preparation to cofabrication colour developing liquid.Be sequentially added into 19mL0.1M Trisodium Citrate, 1mLTMB, 10 μ L 3%H
2O
2, formulated.)。The film bar is soaked in the colour developing liquid, and the lucifuge colour developing was that visible spot manifests in 10~15 minutes.The Streptavidin (streptavidin) that is marked with peroxidase (POD) is held the vitamin H specific combination that has with PCR product 5 ', and issues biochemical color reaction in the existence of hydrogen peroxide and brown substrate TMB (tetramethyl benzidine).Like this, there is the film bar correspondent probe position of hybridization product will manifest blue spot.
The detected result of three duplicate samples is seen Fig. 1.As shown in the figure, in film bar A, blue spot appears in 71-72N and M and 654N, M simultaneously, shows that sample A to be checked is 71-72 and 654 double heterozygotes; In film bar B, β EN immaculate, and blue spot appears in β EM, shows that sample B to be checked is a β EM homozygote; In film bar C, blue spot appears simultaneously at 41-42N and M, show that sample C to be checked is the 41-42 heterozygote.
6, the result judges
Can judge the type of transgenation by the colour developing result of film bar, also can further after reading apparatus is read, carry out Computer Analysis, directly provide results of hybridization, and the result is preserved and statistical study signal and background.
The contrast experiment of [embodiment 4] nucleic acid hybridization film bar method and glass-chip method
1, glass-chip method
The glass-chip method is the method that the DNA chip of substrate detects for adopting with the sheet glass.What be used for the poor transgenation in diagnosing beta-ground is that the DNA chip production method of substrate and detection method are 02117287.0 Chinese patent application referring to application number with the sheet glass.
That utilizes that this DNA chip detects the results are shown in Figure 2.In Fig. 2, A figure is normal people's sample (being called for short No. 1 sample), and B figure is the sample (being called for short No. 2 sample) of undergoing mutation.The corresponding site of normal people's sample is red, and sudden change is green.
As shown in the figure ,-28 sites of No. 1 sample and 41-42 site all are red; The 41-42 site of No. 2 samples is a green, and sudden change has taken place, and-28 is yellow signal (red and green mixed signal), can't judge whether this sample-28 site suddenlys change, and then, detects this sample through sequencing, does not undergo mutation in confirmation-28 sites.
The mutant probe of glass-chip method and normal probe are just distinguished by the color difference in same site, and the sample of heterozygous mutation is exactly a mixture colors like this, bad judgement; The unicity of adding this method signal is relatively poor, and often with spurious response, being difficult to sometimes distinguish when mixed signal occurring is with spurious response or heterozygous mutation.
2, nucleic acid hybridization film bar method
Nucleic acid hybridization film bar method is the method that the film bar of substrate detects for adopting with the nylon membrane.What be used for the poor transgenation in diagnosing beta-ground is that the preparation method of nucleic acid hybridization film bar of substrate and detection method are referring to the foregoing description 1~3 with the nylon membrane.
That utilizes that this Hybond membrane bar detects the results are shown in Figure 3.In Fig. 3, D figure is normal people's sample (being called for short No. 4 sample), and E figure is the sample (being called for short No. 5 sample) of undergoing mutation.As shown in the figure, blue spot does not all appear in the mutant probe position of No. 4 sample film bars, shows that No. 4 samples do not undergo mutation; No. 5 sample film bars-28M and N occur blue spot simultaneously, show that No. 5 samples are-28 heterozygotes.
In nucleic acid hybridization film bar method and since the principle of design of its probe different with as above glass-chip method, mutant probe and normal probe on different positions, normal, heterozygous mutation, sudden change homozygote all are easy to judge, and signal specificity are very good respectively.
More than the description of embodiments of the invention is not limited the present invention, those skilled in the art can make various changes and distortion according to the present invention, these changes and distortion all should belong to the defined scope of accompanying Claim of the present invention.
Sequence table (SEQUENCE LISTING)
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<120〉be used for diagnosing beta-thalassemic nucleic acid hybridization film bar and test kit
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Claims (8)
1. one kind is used for diagnosing beta-thalassemic nucleic acid hybridization film bar, comprises
One substrate; And
Be fixed in described suprabasil specific probe; It is characterized in that:
The described specific probe of each bar at the beta-globin gene locus of a sudden change, is called mutant probe respectively;
The beta-globin gene locus of described sudden change be selected from 27-28M, 41-42M, 654M ,-28M, 71-72M, 17M, β EM, 31M, IVS1-1M, 43M ,-32M ,-29M ,-the one or more combination of 30M, 14-15M, CAP, IntM and IVS1-5M;
Described mutant probe is selected from least one sequence among SEQ ID NO.1~SEQ ID NO.17 or the DNA of its reverse complementary sequence, and the medium position of the base sequence of mutant probe comprises the mutating alkali yl of the beta-globin gene of its corresponding sudden change.
2. nucleic acid hybridization film bar according to claim 1 is characterized in that: comprise also and be fixed in described suprabasil specific probe at the beta-globin normal gene that be called normal probe, itself and mutant probe are individually fixed on the different positions of substrate.
3. nucleic acid hybridization film bar according to claim 2 is characterized in that: described normal probe is selected from least one sequence among SEQ IDNO.18~SEQ ID NO.24 or the DNA of its reverse complementary sequence;
Each normal probe all corresponds respectively to the beta-globin gene locus of one or several sudden change, wherein, SEQ IDNO.18 at the mutational site be 41-42M and 43M; SEQ ID NO.19 at mutational site 654M; SEQ IDNO.20 at the mutational site be-28M ,-29M ,-30M and-32M; SEQ ID NO.21 at the mutational site be 71-72M; SEQ ID NO.22 at the mutational site be 17M and 14-15M; SEQ ID NO.23 at the mutational site be β EM; SEQ ID NO.24 at the mutational site be 31M.
4. according to the described nucleic acid hybridization film of one of claim 1 to 3 bar, it is characterized in that: described substrate is a nylon membrane.
5. nucleic acid hybridization film bar according to claim 1 is characterized in that: described mutant probe carries out amino labeled at 5 ' end or 3 ' end.
6. according to claim 2 or 3 described nucleic acid hybridization film bars, it is characterized in that: described normal probe carries out amino labeled at 5 ' end or 3 ' end.
7. one kind is used for diagnosing beta-thalassemic test kit. and it is characterized in that: it comprises
Described diagnosing beta-thalassemic nucleic acid hybridization film the bar that is used for of claim 1; And
The PCR primer of sample DNA to be checked is used for increasing; The amplified production of this primer contains the dna sequence dna with beta-globin mutant probe generation specific hybrid, and described primer comprises two pairs of primers, is respectively SEQ ID NO.25 and SEQ IDNO.26, SEQ ID NO.27 and SEQ ID NO.28;
Wherein the amplified production of SEQ ID NO.27 and SEQ ID NO.28 can be hybridized with 654N and/or 654M, is used to detect 654 site mutations; The amplified production of SEQ ID NO.25 and SEQ ID NO.26 is used to detect the sudden change of 27-28,41-42 ,-28,71-72,17, β E, 31, IVS1-1,43 ,-32 ,-29 ,-30,14-15, CAP, Int and IVS1-5 gene locus;
The temperature of reaction of nucleic acid hybridization is 40 ℃~46 ℃.
8. diagnosing beta-thalassemic the test kit that is used for according to claim 7 is characterized in that 5 ' end of described primer carries out biotin labeling.
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CN102115781B (en) * | 2009-12-30 | 2013-04-03 | 中山大学达安基因股份有限公司 | Beta-thalassemia mutation detection kit |
CN111593112A (en) * | 2020-05-12 | 2020-08-28 | 深圳市星蝶科技有限公司 | PCR reagent and kit for detecting beta-thalassemia |
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