CN1207137A - Double-stranded conformational polymorphism analysis - Google Patents
Double-stranded conformational polymorphism analysis Download PDFInfo
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Abstract
Double-stranded conformational polymorphism analysis is performed by combining a probe comprising a cross-linking agent and optionally a label with a sample having a target sequence, which may be complementary or have one or a few mismatches with respect to the probe sequence. After sufficient time for hybridization under mild or lesser stringency conditions, hybridized pairs are irradiated to induce cross-link formation by the cross-linking agent. The sample is then analyzed by denaturing gel electrophoresis where the rate of migration depends upon the degree of complementarity between the probe and the target sequences. For more corroboration, in a second experiment, the probe may be combined with the sample under high stringency conditions, where it is found that the formation of cross-linked probe/target is substantially lower for pairs having mismatches than for fully matched pairs. After cross-linking, the sample may be separated by gel electrophoresis, and the amount of cross-linked nucleic acid determined.
Description
The present invention is used to detect dna mutation
Relevant genetic information amount human and other species has had great increase, particularly since the Human Genome Project progress.The discriminating of all odc genes makes us can have the ability to differentiate the sudden change relevant with some special phenotypes.But existing a considerable amount of gene pool let us go to differentiate the sudden change relevant with various diseases.We only need to consider such as Cysticfibrosis, enjoy court of a feudal ruler Dun Shi disease, β-thalassemia, sicklemia disease and other similar disease.In some instances, as sickle cell disease, exist a total point mutation relevant with disease.And in other examples,, then exist a large amount of point mutation relevant running through on the relevant gene with disease as Cysticfibrosis.
Under many situations, what people wished to know is whether to exist point mutation or interested special polymorphism in a patient or other species.We are not only interested in disease itself, and what cherish a special interest is in other species, also are interested in whether have a special allelotrope in the host.
Existing a large amount of technology is used for detecting the difference of known array and target sequence.
Allele specific oligonucleotide oligonucleotide (ASO) detection method is used to differentiate the mononucleotide mispairing that exists between short probe and target DNA or minute differences between the two.Target DNA is transferred on nylon membrane or the nitrocellulose filter at last by gel electrophoresis.With the probe of a mark under hybridization conditions with the incubation of this film; Whether exist to detect complementary.Detection depends on strictness and follows required hybridization and wash conditions, so that distinguish mispairing and complementary situation.
Polymerase chain reaction (PCR) has been applied to directly detect the difference of sequence.One is called technology that amplification do not answer abruptly-changing system (ARMS) and does not promptly play the PCR primer and act as the basis to observe oligonucleotide, and this Oligonucleolide primers except that 3 '-other parts and target complement sequence take place the mispairing in end with target sequence.Thereby, by select suitable primer to the PCR reaction conditions, can detect sudden change.In other words, selected primer causes normal or sudden change amplified production when forming, and will produce restriction site in one or other sequence.
It seems that single strand conformation polymorphism (SSCP) be to utilize non-denaturing polyacrylamide gel (PAGE) mobility difference to detect single base difference.After the target DNA sex change, available native gel detects the variation of single stranded DNA secondary structure.
DNA-DNA complementary and the generation mispairing is hybridized product sex change under the condition that differs from one another.This has been applied to denatured gradient and has coagulated in the arteries and veins electrophoresis (DGGE).Contain the denaturing agent that increases gradually in the DGGE gel, thereby can make double-stranded DNA (ds DNA) molecule of complementation and generation mispairing move and sex change at the different sites of gel.
Except that electrophoretic technique, some chemical technologies can be used for detecting the difference between sequence in addition, chemical modifier, and as osmic acid (Osmium tetroxide), the position that mispairing can take place in DNA azanol (hydroxylamine) etc. produces breach; The site that on the rnase cleavable DNA:RNA hybridization chain mispairing takes place; With passing through the PAGE methods analyst after their processing.Other technology also has heteroduple analysis and nucleotide sequence analysis.All these technology have certain limitation,, compare operational version complexity, the limitation on general method is learned etc. as the condition of need strictness.
The every technical article that detects mispairing comprises: Dowton and Slangh, clinical chemistry (Clin.Chem) 41:785-794 (1995); Newton etc., Nucl.Acids Res.17:2503-2516 (1989); 1989 the 17 volumes of nucleic acids research 2503-2516 page or leaf Haliassos Nacl.Acids Res.17:3606 (1989); Nucleic acids research was rolled up the 3606th page in 1989 the 17.Orita, Proc.Natl.Acad Sci.USA 86:2766-2770 (1989); Institute of American Academy of Sciences newspaper 1989, the 86th volume 2766-2770 page or leaf Sarkar, Nucl.Acids Res.20:871-878 (1992); Nucleic acids research 1992, the 20 volume 871-878 page or leaf Fischer and Lerman, Proc.Natl.Acad Sci.USA 80:1579-1583 (1983); Institute of American Academy of Sciences reports nineteen eighty-three, the 80 volume 1579-1583 page or leaf Cotton, Proc.Natl.Acad Sci.USA 85:4397-4401 (1988); Institute of American Academy of Sciences newspaper 1988, the 85 volume 4397-4401 page or leaf Myers, Science 230:1242-1246 (1985); Science 1985, the 230 volume 1242-1246 page or leaf White, Genomics 12:301-306 (1992).1992 the 12 volumes of genome 301-306 page or leaf
The method and composition that is used to detect the one or more mispairing of generation between a target sequence and a known array is provided.This method is included in and is no more than the target sequence that hybridization probe and length under the gentle condition are less than 300 bases.Wherein used probe comprises known array, an any detectable label and a linking agent.Through enough for a long time hybridization with the double-strandednucleic acid that produces detectable amount after, the condition of change medium with inducement crossbreeding to crosslinked.Sample separates by PAGE under the sex change condition then, can determine to be linked to the mobility of the label probe on the target nucleic acid from a known standard.The right mobility of probe/target pair that mispairing takes place and complementary probe/target is different.Be further conclusive evidence, select strict hybridization conditions, the right hybridization amount of the sequence of mispairing and complementary hybridization amount take place be suitable different.Method as described above heats this sample, and the quantity that crosslinked probe takes place is relevant with the mispairing degree between probe and target sequence, and under the mispairing situation, crosslinked number of probes is less significantly.
By the present invention, about detecting the obvious handiness that increases of condition tool that the target sequence sudden change exists.
By the present invention, the probe that is provided is used for whether existing between detection probes and target sequence the method for mispairing.The reason that mispairing takes place may be because sudden change, allelic variation, transmutation of species and alternately factor such as montage cause that the mispairing of unit point or multidigit point also may be by insertion, delete that the mispairing equity causes usually.
In general, this method has known array with one, and the optional detectable label and the probe of linking agent combine with target sequence as a target DNA major portion or a complex mixture composition.Target sequence exists with single stranded form.Probe and target sequence are hybridized under the condition that is no more than gentleness.After through the double-strandednucleic acid of sufficiently long time with the formation q.s, the change condition is so that take place crosslinked.After the crosslinked generation, sample separates by gel electrophoresis, and the mobility of double-strandednucleic acid that this moment, mispairing took place is different with the mobility of complementary double-strandednucleic acid.The generation that can detect mispairing by the mobility of double-stranded mixture of probe-target and standard substance relatively whether.
Target DNA can be to originate arbitrarily, and the about 25-300 Nucleotide of its mean size (nt) is more typically 50-250 Nucleotide, is preferably the Nucleotide with 50-200.DNA can come from prokaryotic organism or eukaryote, normally eukaryote.DNA can come from host genome, plasmid DNA, viral DNA (this virus natural or as the carrier of different sources DNA), pcr amplification product, or analogue.Target DNA can be the special allelotrope of a mammalian hosts, or MHC allelotrope, or the sequence of a kind of isozyme of a coding, or a special gene of unicellular organism body or be or other analogue.Target sequence also can be genomic dna, cDNA, RNA, or analogue.
The nucleic acid of desired length especially can pass through restriction enzyme digestion DNA, or utilizes methods such as primer and PCR to obtain.Preferably, the 80mol% that has an appointment at least, the target sequence at least about 90mol% has identical size usually.During with the Restriction Enzyme blanking method, adopt high frequency restriction endonuclease (frequently cuttingenzyme) (they normally have the enzyme of four base consensus sequences), or carry out the method (this moment, DNA can thoroughly be degraded) of multiple restriction enzyme digestion.Mispairing generally betides the inside of target sequence and can not be positioned on the restriction enzyme site of restriction enzyme of degraded sample DNA.Typical interesting sequence has the sickle-cell anaemia sudden change, the MHC relevant with IDDM reaches and cystic fibrosis, enjoy court of a feudal ruler Dun Shi disease, β-thalassemia, (as: those are by oncogene for senile dementia disease, all kinds of cancer, as ras, src, myc (etc.) activation and (or) tumor suppressor gene, as p53, the inactivation of RB etc. causes.) relevant sudden change.In some cases,, a single base mutation is arranged as sickle cell disease.In some other case,, then can detect a plurality of site mutation as cystic fibrosis.By selecting suitable Restriction Enzyme, can know to predict in the big or small fragment for one to have the zone of the one or more sudden changes that are suspected to have concealment, thereby can be by the existence that detects one or more sudden changes in the gene easily of uniting of probe.
Used DNA need pass through such as purifying such as isolated protein, removal Restriction Enzyme inhibitor.
Generally about 15-50 the Nucleotide of probe is more typically 20-35 Nucleotide.Have 1-5 linking agent on the probe, 1-3 linking agent more generally arranged.Used linking agent can not disturb hybridization, and they generally are positioned at thymus pyrimidine (T), on the relative position of cell pyrimidine (C) or uridylic (U) so that crosslinked.Many functionality are to have photochemical activity and can form covalent linkage with nearly all organic moiety.These groups comprise Cabbeen (Carbenes), nitrene (Nitrenes), ketene (Ketenes), free radical (freeradicals) etc.Can add in bulk solution and remove molecule (scavenging molecule), promptly non-target sequence nucleic acid so that those are not with target sequence bonded probe with remove molecular reaction, thereby has been avoided the non-specific crosslinked generation between probe and target sequence.Cabbeen (Carbenes) can pass through diazo compound, as diazo-ketones salt (diazonium salts), and acquisitions such as alkylsulfonyl hydrazone salt (Sulfonylhydrazone salts) or diaziranes.Ketene (Ketenes) can or go from diazo-ketones (diazoketones) to obtain the nitrine quinone (quinonediazides).Nitrene (Nitrenes) derives from aromatic yl azide (aryl azides); acyl azide (acyl azides) and triazo-compound (azido compounds); more relevant photodestruciton produces the non-information of sharing electrode pair; see also organic photochemistry preparation (PreparativeOrganic Photochemistry) (Springer-Verlag, NY 1968) of A.Schonberg.
In most cases, be used for crosslinked compound and be photoactivation compound and can form covalent linkage with base, particularly pyrimidine.These compounds comprise funtion part (functional moieties), as tonka bean camphor and substitute thereof, furans a pair of horses going side by side tonka bean camphor (furocoumarin), Isocoumarin 〉97 (isocoumarin), temparin (bis-coumarin), psoralen etc., quinones substance (quinones), pyrone (pyrones), α, beta-unsaturated acid (α, β-unsaturated acids), acid derivative (acid derivatives) (as: ester, ketone, and nitrile; Trinitride etc.).
Another kind of photoactivation reactant is the organometallic compound based on d district or f-district transition metal.Photoactivation can induce the disappearance of aglucon of metal to allow its substituent combination.Suitable aglucon comprises Nucleotide.The information of the light substituent of detailed relevant organometallic compound sees also " organometallic photochemistry (Organometallic Photochemistry) " book that G.F.Geoffrey and M.S.Wrighton collaborate.(Academic Press, San francisco, CA, 1979) (Science Press).
The probe homologous sequence that is attached on the target DNA has natural Nucleotide usually.Yet, in certain embodiments, phosphoric acid-sugar chain is used non-natural sugar and is modified, perhaps the Sauerstoffatom in its phosphate group is replaced by sulphur, carbon, nitrogen etc., or modify with some other modifying method, its objective is for comprehensive advantage is provided, the stability under the testing conditions, and the short degraded of antienzyme etc.
Probe can obtain with any method easily, and method is the Nucleotide synthesis method that appropriate location in building-up process progressively adds crosslinked modification the most easily.Connect various molecules to the existing lot of documents report of the method on the Nucleotide, need not to describe in detail herein.As consulting " oligonucleotide and analysis, hands-on approach (oligonueleotides and Analogues, A practial Approach) " book (Oxford University Press, 1991) (Oxford University Press) that EsksteinF etc. compiles.
Similarly, mark (if words are arranged) can be connected on the Nucleotide on any probe chain of being convenient to connect, but can not disturb the hybridization of probe and target sequence.In general, mark is little, is generally 100～1000Da.Mark can be any detectable material, and it can directly detect or be attached on the acceptor, and it needs molecule that easily detects on the mark equally.The molecule that detects by electrophoresis method comprises radio-labeling, (as
32P
35S etc.), fluorescent substance (as: rhodamine (rhodamine), fluorescein fluorescein etc.), the aglucon of acceptor (as: vitamin H that is used for streptavidin is used for the digoxigenin of anti-digoxin etc.), chemiluminescent substance and analogue.Perhaps,, DNA used such as ethidium bromide when separating in front and back or the sepn process, second ingot dimer, and thiazole orange (thiazole orange), tetrazolium bromide (thiazoleblue) and their dyeing such as dimer just needn't have been used above-mentioned mark.If use PCR method, then come the surrogate markers probe with labeled primer, detect with primer.If utilize part, acceptor can come mark with any mark that can directly detect.
This method is carried out with combining of target DNA by probe.The consumption of the target DNA of measuring general about 10
-20～10
-8Mole (moles) is more typically 10
17～10
-10Mole.The target nucleic acid amount of the amount of used probe and mensuration quite or excessive but generally is no more than 10 of target DNA consumption
9Doubly, more generally think and to surpass 10
7Doubly.Hybridization medium uses gentle to low severity, and is crosslinked to guarantee that all target nucleic acids all take place.It is 25-70 ℃ (using 40-70 ℃ usually, more through 30-50 ℃ commonly used) that this severity generally is equivalent to temperature, and sodium ions content is 0.05-1.5M (being more typically tool 0.25-1M sodium ion or 0-20% methane amide).When being RNA, the amount of guanidinium isothiocyanate should add to 0.1-6M.Except that methane amide, other denaturing agent also has urea, dimethyl sulfoxide (DMSO) (DMSO).Used hybridization conditions is the hybridization that mispairing or pairing the time all reach maximum quantity for providing between probe and target sequence.Hybridizing the used time answers sufficiently long, so that form the double-strandednucleic acid of detectable amount, the susceptibility when this depends on hybridization conditions and used marker detection.Time generally was at least five minutes, but can not surpass six hours, was more typically between ten minutes to one hour.
After hybridization takes place, the double-strandednucleic acid that contains probe will take place crosslinked.Use light wavelength should be equal to or greater than 300nm, natural-crosslinked to avoid nucleic acid to take place.In general, optical wavelength should be in the 300-400nm scope, and this can obtain by a light source that is connected with the Pyrex film.When using the chemokinesis method, owing to photodestruciton activates the more convenient alternative methods that becomes.General about 1 minute to two hours of irradiation time, more generally about five minutes to one hour, this depended on the size of sample, the amount of radiation source intensity and required product etc.In case of necessity, a desirable duplicate samples is carried out electrophoresis to determine whether forming the crosslinked of q.s.
After the irradiation, sample promptly passes through heating or adds indicating dye (front-runingdye), glycerine, and sucrose, methane amide etc. are handled so that go up sample.These technology are well-known in this area, do not need to add detailed description here again.
Carry out electrophoretic analysis with polyacrylamide gel electrophoresis, be generally the acrylamide of 5-23%, acrylamide with two-monomeric ratio is 10-30/1.Adopt the sex change condition to remove all non-crosslinked nucleic acid in the crosslinked nucleic acid region.Available other denaturing agent is to replace urea.Typical electrophoretic buffer can be selected boric acid-EDTA as Tris-for use.Electrophoresis can carry out under the condition that helps separating mismatch and matched sequence.Swimming lane adds the standard sequence of a suitable pairing or mispairing therein, just the band of sample and they can be compared.Standard sequence demonstrates target sequence with the difference of sample sequence and whether standard sequence is identical.Utilize suitable mark or gel is dyeed, just can check out whether mispairing has taken place between target sequence and probe sequence.
Further prove conclusively as need, can detect the double-stranded degree that forms in conjunction with the ASO technology.Yet the crosslinked meeting that supplies significantly reduces the severity of the condition of using.In this process, temperature is generally 50-70 ℃, and Na ion concentration is about 50-500mM, more generally about 100-400mM.For each target sequence and probe, can optimize used condition and make that forming double-stranded degree between mismatch and matched sequence reaches maximum difference.Best, have about twice ratio (being generally) between crosslinked paired sequence amount and crosslinked non-matching sequence amount at least at least about five multiple proportions rates.In this process, use strict condition.Otherwise, this condition in fact will with the conditional likelihood of mobility difference.The amount of crosslinked DNA can be determined easily by measuring with the signal of pairing standard sequence, probe sequence and the relevant band of target sequence.If signal obviously a little less than, illustrate that mispairing has taken place sequence.If signal is identical with the standard sequence signal, illustrate that sequence is a paired.
Convenience for the user provides test kit.The probe that comprises one or more (being generally two or more) in the test kit, especially a pair of probe, one of them probe and sequence complementation are referred to as " wild-type " sequence, and another probe is referred to as " sudden change " sequence.Yet, what should know is that these appellations are at random, because under many situations, people only wonder whether target sequence is identical with probe sequence, and should not form such notion: promptly a sequence is common or wild-type, another sequence right and wrong common or the sudden change.Give an example, wonder that as people which exists the difference of one or two mispairing in two MHC allelotrope.Probe between generally be no more than five, more generally be no more than three difference.Based on target sequence, may exist most probe, especially probe has many potential sudden changes to (being no more than about 12 pairs usually) in the target sequence, and they are distributed on the whole gene.Also have some subsidiaries in the test kit, as dyestuff, the antibody of mark (part wherein serves as a mark), the primer of the mark of using in the PCR method etc.
The following examples provide for the purpose of demonstration rather than restriction.
EXAMPLE Example 1: utilize (A) allele specific oligonucleotide hybridization and crosslinked and (B) the DSCP analytical procedure detect single base mispairing.
The oligonucleotide (Oligo#1) of the E6 gene 374-403 nucleotide sequence of tool HPV 16 is synthetic with the phosphoramidite method in the DNA synthetic method, its 5 '-end uses
32The P mark.Preparation simultaneously substitutes the G with A except that the 388th with Oligo#1, the oligonucleotide that all the other sequences are identical (Oligo#2), same using
32The P mark.
Oligo#1：5′-CAATACAACAAACCGTTGTGTGATTTGTTA-3′
Oligo#2：5′-CAATACAACAAACCATTGTGTGATTTGTTA-3′
Prepare one and have the photoactivation crosslinked group, the dna probe (Oligo#3) of the long 20-chain link (mer) of 3-oxo (7-tonka bean camphor base) glycerine (in the sequence be mark with X).The dna sequence dna of this probe and Oligo#1 sequence are complementary fully, and it also can form double-strandedly with Oligo#2 hybridization, but it is right to have an A/C mispairing in the two strands that forms.
Oligo#3??3′-TTGTTTGGCAACACACTAXA-5′
The Oligo#1/#3 two strands:
5′-CAATACAACAAACCGTTGTGTGATTTGTTA-3′
3′-TTGTTTGGCAACACACTAXA-5′
The Oligo#2/#3 two strands:
5′-CAATACAACAAACCATTGTGRGATTTGTTA-3′
3′-TTGTTTGGCAACACACTAXA-5′
The Oligo#3 of 20pmole is in the usefulness with 2pmole amount
32P carries out 5 '-Oligo#1 of end mark or the 0.15mL sample of Oligo#2 in incubation, temperature and NaCl concentration are summarized as follows:
| Sample | Oligonucleotide | Temperature, ℃ | NaCl concentration, mM |
| ????1 ????3 ????2 ????4 ????5 ????7 ????6 ????8 ????9 ???11 ???10 ???12 | ?????1+3 ?????1+3 ?????2+3 ?????2+3 ?????1+3 ?????1+3 ?????2+3 ?????2+3 ?????1+3 ?????1+3 ?????2+3 ?????2+3 | ?????45 ?????45 ?????45 ?????45 ?????50 ?????50 ?????50 ?????50 ?????55 ?????55 ?????55 ?????55 | ?????150 ?????300 ?????150 ?????300 ?????150 ?????300 ?????150 ?????300 ?????150 ?????300 ?????150 ?????300 |
Behind the incubation 20 minutes, solution places the light of UV-A wavelength to shine 45 minutes down.Before radiation treatment finishes, get 1/10th volumes (0.015ml) sample and isopyknic methane amide-tetrabromophenol sulfonphthalein dyestuff and mix, 70 ℃ were heated three minutes.In cooled on ice, application of sample is in 15% polyacrylamide gel that contains 7M urea (acrylamide/bisacrylamides of 19: 1) then with sample, and electrophoresis arrives the bottom of gel under the 300V to tetrabromophenol sulfonphthalein.Take off gel, cover x-ray film, spend the night in-80 ℃.
Method 1: allele specific oligonucleotide hybridization and crosslinked
By the experiment in the NaCl concentration range that under 45-55 ℃ hybridization temperature, reaches 15-300mM, can determine to form between complementary oligonucleotide #/1 and #3 the condition of significant cross linking, significant cross linking does not then take place in this condition in oligonucleotide #2 and #3 that mispairing takes place.For the top condition of determining that mispairing is differentiated, the radioactivity band is excised from glue, also determine quantitatively that with scintillation counter the percentage yield of cross-linking products is (with respect to nonreactive
32The oligonucleotide of P mark).The result shows below:
| Sample | Oligonucleotide | Temperature, ℃ | NaCl concentration, mM | Crosslinked, % |
| ??1 ??2 ??3 ??4 ??5 ??6 ??7 ??8 ??9 ??10 ??11 ??12 | ????1+3 ????2+3 ????1+3 ????2+3 ????1+3 ????2+3 ????1+3 ????2+3 ????1+3 ????2+3 ????1+3 ????2+3 | ????45 ????45 ????45 ????45 ????50 ????50 ????50 ????50 ????55 ????55 ????55 ????55 | ????150 ????150 ????300 ????300 ????150 ????150 ????300 ????300 ????150 ????150 ????300 ????300 | ????46 ????44 ????49 ????51 ????40 ????22 ????48 ????44 ????39 ?????3 ????43 ????18 |
The top condition that data from last table can determine to distinguish complementary and mispairing two strands is sample 9 and sample 10 (55 ℃, 150mM NaCl); With this understanding, complementary oligonucleotide is to producing the cross-linking products (39% to 3%) than many ten triplications of mispairing oligonucleotide.
Method 2:DSCP analyzes (double-stranded conformational polymorphism analysis)
Analysis is (45 ℃ of the conditions of minimum strictness, 300mM NaCl) sample 3 under and the radioactive intensity of sample 4 can clearly show the cross-linking products that forms between oligonucleotide #2 that mispairing takes place and #3 is slower than the cross-linking products that forms between complementary oligonucleotide #1 and #3 in the rate of migration on the gel rate of migration (DSCP effect).
From this result of experiment, the method for utilizing hybridization conditions to detect single base mispairing that the DSCP method is more traditional has two advantages:
1.DSCP method is simple, need not carefully to optimize hybridization conditions and distinguishes mismatch and matched sequence.The DSCP method is used the low stringency hybridization condition.
2. by nonstringent condition, crosslinked yield and detection signal thereof show the hybridization product that is better than under the stringent condition; (45 ℃ of the conditions that is used for the DSCP analytical procedure, 300mM NaCl) under, the cross-linking products yield that reaction forms between complementary oligonucleotide 1# and #3 is 49%, and the cross-linking efficiency when stringent hybridization condition (55 ℃, 150mM NaCl) produces best mispairing difference down is 39%.Thereby the DSCP method has strengthened 26% signal.
Embodiment 2: utilize the DSCP analytical procedure to detect normal (β
A) and sickle cell (β
S) betaglobulin allelotrope
Two long be 56 bases have normal people's betaglobulin gene (β
A-target) or sickle cell's betaglobulin gene (β
SPhosphoramidite method during the oligonucleotide of a part of sequence-target) synthesizes with DNA is synthetic, and in their 5 '-end usefulness
32The P mark.β
S-sphaeroprotein target sequence and β
AThe difference of target sequence is: replace A with a T, thereby replaced L-glutamic acid with Xie Ansuan in drepanocytic betaglobulin.
β
A-target sequence: 5 '-TGACTCCTGAGGAGAAGTCTGCCGTTACTG
CCCTGTGGGGCAAGGTGAACGTGGAT-3′
β
S-target sequence: 5 '-TGACTCCTGTGGAGAAGTCTGCCGTTACTG
CCCTGTGGGGCAAGGTGAACGTGGAT-3′
Synthetic two and β
A-target sequence or β
SProbe (the β of-target complement sequence
A-probe and β
S-probe).These two probes are all used the photoactivation crosslinked group: 3-oxo-(7-tonka bean camphor base)-glycerine (in the sequence be mark with X) is modified.
β
A-probe: 3 '-TGAGGACTCCTCTTCAXA-5 '
β
S-probe: 3 '-TGAGGACACCTCTTCAXA-5 '
Hybridization and crosslinked experiment show that the DSCP of available these two betaglobulin probes analyzes and detect and distinguish the target sequence of the β-ball egg that exists with independent form or have (as in the allos individuality) with the mixing of 1: 1 form.Experimental result is summarized as follows:
| Sample | The betaglobulin probe | The betaglobulin target | The UV-A irradiation |
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 | ?????β A?????β S?????β A?????4β A?????β S?????β S?????β A?????β S | ??????β A??????β S??????β A??????β S??????β S??????β A????β A/β S????β A/β S | ?????- ?????- ?????+ ?????+ ?????+ ?????+ ?????+ ?????+ |
Contain corresponding probe of 10pmole and 0.2pmole in each 0.05ml sample
32The target sequence of P mark (the every kind of target molecule that contains 0.2pmole in sample 7 and the sample 8).NaCl concentration in the solution is 0.75M.
Hybridized 20 minutes down at 35 ℃, sample is used UV-A light source irradiation 60 minutes.Take out 1/10th volumes (0.010ml) sample, after the equal-volume methane amide-the tetrabromophenol sulfonphthalein dye mixture mixes, 70 ℃ were heated three minutes, place cooled on ice then, this is handled sample be added on 10% polyacrylamide gel (acrylamide/bisacrylamide=19: 1) of tool 7M urea, the 300V electrophoresis arrives the bottom of gel to tetrabromophenol sulfonphthalein.Take off blob of viscose, cover x-ray film, spend the night under-80 ℃.
This experimental data shows: DSCP analyzes can be by the arbitrary loci that detects and distinguish these two betaglobulins with the probe of two crosslinked modifications.By complete complementary β
A-probe and β
A-target sequence (sample 3) and β
S-probe and β
SThe mobility of main crosslinked product structure on gel that reaction forms between-target sequence (sample 5) is obviously faster than the mobility of the cross-linking products that forms between mismatch probe and target sequence in sample 4 and the sample 6.Further analyze β
A-target sequence and β
SThere is the reaction result of (sample 7 and sample 8) down simultaneously in-target sequence, shows that these two probes can detect and distinguish this two allelotrope simultaneously.This result is clinical relevant, because have β in the DNA of individual of tool sickle cell disease simultaneously
A-and β
S-allelotrope.
Prove from above result, the easy accurate method that detects the mispairing of single base generation easily is provided.Its methodology is easy, and method is quick, also can not produce error result because of the trickle change of condition control.
Publication and patent application mentioned in all these specification sheetss are all incorporated the present invention into reference, are all commented particularly and respectively and incorporate the present invention into reference as each mentioned publication and patent application.
The present invention is set forth comprehensively, is apparent that the variation and the modification of the scope and spirit that can not break away from additional claim to those skilled in the art.
Claims (19)
1. existence whether method that detects at least one mispairing between nucleic acid probe and target nucleic acid, between the sequence of wherein said probe and target the difference that is no more than five mispairing is arranged, this probe comprises known array and photoactivation linking agent, wherein said probe sequence is with the hybridization of this target sequence the time, form covalent linkage by the photoactivation effect between this probe sequence and target sequence, said method comprises:
In a hybridization medium, in the hybridization conditions of gentleness with hybridization time enough bonding probes sequence with comprise that the nucleic acid samples of target sequence is so that this probe and target sequence hybridization;
Shine wherein said hybridization medium so that form crosslinked between the target sequence of this probe and its hybridization to form crosslinked double-strandednucleic acid;
By the nucleic acid in the sex change electrophoretic separation hybridization medium and the mobility of this crosslinked double-strandednucleic acid and known generation mispairing or the crosslinked double-strandednucleic acid standard substance of complementary relatively, with whether existing of definite at least one mispairing.
2. according to the process of claim 1 wherein that said probe carries out mark with a detectable mark.
3. according to the process of claim 1 wherein that said sample prepares with the polymerase chain reaction, this sample nucleic acid carries out mark with a detectable mark.
4. according to the process of claim 1 wherein that said electrophoresis is a polyacrylamide gel electrophoresis.
5. one kind is detected the method that whether at least one mispairing exists between nucleic acid probe and target nucleic acid, the difference that the mispairing that is no more than five is arranged between wherein said probe and target sequence, wherein said target sequence comprises the nucleic acid molecule that is about 25-300 Nucleotide, probe sequence comprises the sequence and the photoactivation linking agent of known long 15-50 Nucleotide, when this probe sequence and target sequence are hybridized, act on formation one covalent linkage between this target sequence and probe sequence by photoactivation.Said method comprises:
In hybridization medium, this target sequence of chien shih and probe sequence hybridization when making probe under the hybridization conditions of gentleness, combine sufficiently long with the nucleic acid samples that contains said target sequence;
At the said hybridization medium of wavelength region internal radiation of about 300-400nm, make form between the target sequence of this probe and its hybridization crosslinked to form crosslinked double-strandednucleic acid;
By the nucleic acid in the said impurity of sex change electrophoretic separation and the mobility of this crosslinked double-strandednucleic acid and known generation mispairing or the crosslinked double-strandednucleic acid standard substance of complementary relatively, the existence that can determine at least one mispairing whether.
6. according to the method for claim 5, wherein said sample obtains with restriction enzyme digestion degrading genes group DNA.
7. according to the method for claim 5, this sample nucleic acid carries out mark with a detectable mark to wherein said sample by the polymerase chain reaction preparation.
8. according to the method for claim 5, wherein said probe carries out mark with detectable mark.
9. according to the method for claim 5, wherein said electrophoresis is a polyacrylamide gel electrophoresis.
10. one kind is detected the method that whether at least one mispairing exists between nucleic acid probe and target nucleic acid, the difference that the mispairing that is no more than five is arranged between wherein said probe and target sequence, wherein said target sequence comprises the nucleic acid molecule that is about 25-300 Nucleotide, said probe sequence is the sequence and the photoactivation linking agent of known long 15-50 Nucleotide, when this probe sequence and target sequence are hybridized, act on formation one covalent linkage between this probe sequence and target sequence by photoactivation.Said method comprises:
In a hybridization medium, the nucleic acid samples that makes probe and contain this target sequence under the hybridization conditions of gentleness in conjunction with the sufficiently long time so that this target sequence and probe are hybridized, this condition is equivalent under 25-70 ℃, 0.1-1.5M NaCl;
With this hybridization medium of rayed of about 300-400nm wavelength, make form between the target sequence of this probe and its hybridization crosslinked to form the double-strandednucleic acid of interlinkage;
Utilize the sex change gel electrophoresis to separate the nucleic acid and the mobility of this crosslinked double-strandednucleic acid and known mispairing or the crosslinked double-strandednucleic acid standard of complementary relatively in this hybridization medium, with whether existing of definite at least one mispairing.
11. according to the method for claim 10, wherein said linking agent includes a tonka bean camphor group.
12. one kind is detected the method that whether at least one mispairing exists between nucleic acid probe and target nucleic acid, the mispairing difference that is no more than five is arranged between wherein said probe sequence and target sequence, this target sequence comprises the nucleic acid molecule that is about 25-300 Nucleotide, probe sequence is the sequence of known long 15-50 Nucleotide, and include the photoactivation linking agent, when this probe sequence and target sequence are hybridized, act on formation one covalent linkage between probe sequence and target sequence by photoactivation, said method comprises:
In a hybridization medium, the nucleic acid samples that makes probe and contain this target sequence hybridization sufficiently long time under highly strict hybridization conditions will produce at least about greater than the hybridization amount of doubling dose in the probe of a mispairing of generation with the probe of target complement sequence so that target sequence and probe sequence are hybridized;
With this hybridization medium of rayed of 300-400nm wavelength, make form between the target sequence of probe and its hybridization crosslinked to form crosslinked double-strandednucleic acid;
Utilize the sex change gel electrophoresis to separate the nucleic acid in the hybridization medium and determine the amount of this crosslinked double-strandednucleic acid, and between the amount of this crosslinked double-strandednucleic acid and probe and target sequence thereof mispairing have or not relevant.
13. according to the method for claim 12, the hybridization conditions of wherein said height strictness is equivalent to the temperature of 40-70 ℃ of scope at least, the Na ion concentration of 0.05-0.5M.
14. a test kit that contains two kinds of probes, feature is made up of 15-50 Nucleotide, and this each probe links to each other with a photoactivation linking agent, has between each probe and other probe to be no more than three mispairing, and is the sequence of natural generation.
15. according to the test kit of claim 14, wherein said photoactivation linking agent includes the tonka bean camphor group.
16. according to the test kit of claim 14, wherein said native sequences is relative to each other, one of them is another sudden change.
17. according to the test kit of claim 14, wherein said native sequences is relative to each other, one of them is another allelotrope.
18. according to the test kit of claim 14, wherein said probe carries out mark with a detectable label.
19. according to the test kit of claim 14, wherein said each probe has many linking agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 96199509 CN1207137A (en) | 1995-11-03 | 1996-11-01 | Double-stranded conformational polymorphism analysis |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US60/007,239 | 1995-11-03 | ||
| CN 96199509 CN1207137A (en) | 1995-11-03 | 1996-11-01 | Double-stranded conformational polymorphism analysis |
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| Publication Number | Publication Date |
|---|---|
| CN1207137A true CN1207137A (en) | 1999-02-03 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 96199509 Pending CN1207137A (en) | 1995-11-03 | 1996-11-01 | Double-stranded conformational polymorphism analysis |
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| CN (1) | CN1207137A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023116408A1 (en) * | 2021-12-21 | 2023-06-29 | 广东菲鹏生物有限公司 | Nucleic acid probe and method for using same |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023116408A1 (en) * | 2021-12-21 | 2023-06-29 | 广东菲鹏生物有限公司 | Nucleic acid probe and method for using same |
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