CN1293204C - Method for determining alleles - Google Patents
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- CN1293204C CN1293204C CNB018183670A CN01818367A CN1293204C CN 1293204 C CN1293204 C CN 1293204C CN B018183670 A CNB018183670 A CN B018183670A CN 01818367 A CN01818367 A CN 01818367A CN 1293204 C CN1293204 C CN 1293204C
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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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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides methods and kits for separating and identifying alleles, and thereby the haplotype, in genomic DNA samples. The method generally involves hybridizing primers specific to polymorphic sites within the alleles to the DNA sample, elongating the primers by one or more nucleic acids, separating the elongated primers and identifying the alleles utilizing the elongated primer. The method also allows for a ligation of two primers, their separation and subsequent use in identifying the targeted allele. The method further provides that another primer can be used as a blocking site for elongation of the first primer such that a stretch of DNA that includes a polymorphic site is replicated and identified. The unextended or extended primers can be labeled so that the primer can be easily separated and/or identified.
Description
Technical field
The present invention relates to be tested and appraised one or more heterologous sequences site in the gene and separate and identify allelic method.More specifically, the present invention relates to use one or more primers to separate and identify allelic method.
Background of invention
The modal form of sequence difference is a single nucleotide polymorphism between the individuality, and common general knowledge is SNP.Along with finishing of human genome plan, estimate that the mean number that SNP exists is to have one in per 1000 Nucleotide, but the frequency that exists in some DNA district is higher.The focus of making great efforts is to utilize SNP to identify the target gene relevant with disease or drug reaction now.But because cognation is not strong, a lot of scientists and researchist suspect that with individual SNP be the viewpoint of basis to medicine and disease personalization, thus the importance that haplotype (Haplotype) is analyzed appear in one's mind out, become the key method of SNP medical use.
Haplotype is the organizational form of individual SNP along given dna fragmentation, and this is a common general knowledge.The classics definition of haplotype is the allelotrope combination that trends towards being genetic to from a generation jointly follow-on close linkage locus in the single karyomit(e) in given population.What the molecular cell type was measured is the drafting of linkage disequilibrium collection of illustrative plates on the other hand, and it is considered to the important tool in the positional cloning of disease gene now, analyses along with the genetics of complicated phenotype digs, and a variety of application have just become apparent.
Since 1989, scientists study various molecular cell type measuring methods, they utilize the unit molecule dilution (SMD) of genomic dna, with physical method for separation allelotrope, perhaps carry out allelotrope from heterozygosis template selective amplification hemizygote dna fragmentation and distinguish by allele-specific primers.Yet, just these methods have been developed at short-movie section (approximately 500bp), but the molecular cell type is measured the PCR (marker enlarges 10-20 doubly at interval again) that has been applied to long scope recently, and uses the prototype system of CD4 locus as this analytical procedure of exploitation.Also attempted measuring other method of dna sequence dna haplotype, but these methods are very unsuccessful, unreliable or cost is high.Therefore still exist needs to the economical molecular cell type measuring method reliable, that treatment capacity is big.
Summary of the invention
The present invention relates to be tested and appraised in the gene one or more heterologous sequences site and measure allelic method.This method can be used for measuring the haplotype of concrete gene, and has using value in a lot of fields, comprises the mensuration of human leucocyte antigen (HLA) (HLA) type.The invention still further relates to the test kit that is used for this type mensuration.
The present invention includes the method for separating the allele-specific nucleic acid molecule.In the single stranded nucleic acid molecule that comprises one or more heterologous sequences site, add in one or many middle heterologous sequence locus specificity nucleic acid primers, and make its hybridization.In one embodiment, 3 of each primer ' end is corresponding to the pleomorphism site in target heterologous sequence site.In such embodiments, can make this 3 ' end carry out single-basic extension, be connected in 5 ' end and second primer that 3 of this heterologous sequence locus specificity primer ' end is adjacent, perhaps can extend several bases.Separate the heterologous sequence locus specificity hybridized primer and the nucleic acid molecule that extend or connect then, and reclaim as required, be used for further genotype detection.In another embodiment, each primer comprises the one or more polymorphism bases that are positioned at primer, like this can selective removal and the primer that is less than the hybridization of 100% complementary base, and those primers of 100% complementary base hybridization are unaffected.
The invention still further relates to a plurality of allelic method that comprises in these allelic nucleic acid molecule of identifying.Selection contains the single stranded nucleic acid molecule in a plurality of heterologous sequences site, adds two kinds of primers, a kind of allos primer, a kind of homology primer in this nucleic acid molecule.Described allos primer can be hybridized with 3 ' end heterologous sequence site of the 3 ' end that is positioned at 5 ' heterologous sequence site on the same nucleic acid molecule.3 of described allos primer ' end base is so only just extended when 3 of allos primer ' end is hybridized with described single-chain nucleic acid corresponding to the polymorphism base in described heterologous sequence site.Described homology primer can be at 5 ' end position that is positioned at 5 ' heterologous sequence site and same making nucleic acid molecular hybridization.These primers and making nucleic acid molecular hybridization, and extend the allos primer, with regard to described 5 ' heterologous sequence site of reproducible between primer, nucleic acid molecule, when the allos primer that extends reached the homology primer, the homology primer played the effect that stops the allos primer extension like this.With the allos primer sex change of nucleic acid molecule and extension, and separate and analysis allos primer, to determine 5 ' heterologous sequence site.Utilize these information to determine that a new cover contains the nucleic acid primer of another kind of allos primer and another kind of homology primer, the allos primer of this new cover primer can be hybridized (3 of this allos primer ' end base is corresponding to the polymorphism base) with 5 ' heterologous sequence site, this 5 ' heterologous sequence site is positioned at 3 of another heterologous sequence site on the identical nucleic acid molecule ' end, and the homology primer of this new cover primer can be in 5 of another heterologous sequence site ' end and same making nucleic acid molecular hybridization.Repeat the step of front, in every next round hybridization/extension, use a new cover primer,, be used for identifying allelotrope up on this nucleic acid molecule, identifying enough heterologous sequence sites.Can measure the haplotype of nucleic acid molecule in this way.
The invention still further relates to a plurality of allelic methods in the nucleic acid molecule of identifying, comprise to one group of globule adding and contain a plurality of allelic nucleic acid samples, each globule is connected with two different primers, at least one primer is to the allelic primer of a kind of uniqueness on each globule, makes the part hybridization of the allelic at least a primer of a kind of uniqueness and this nucleic acid samples.With the primer amplification of hybridization, extend hybridized primer, produce the primer nucleic acid that extends.Remove nucleic acid samples then with the nucleic acid samples and the primer sex change of hybridizing, and from globule.The primer that extends then with globule on second kind of primer hybridization, and this second kind of primer that increase.Then the globule that contains dual amplimer is analyzed, determined the allelotrope that exists in the nucleic acid samples.
Brief Description Of Drawings
Fig. 1 is that explanation application allele-specific primers extension method of the present invention is identified allelic synoptic diagram.
Fig. 2 is the synoptic diagram that the single-basic extension multiple alleles authentication method of primer size marking method is used in explanation.
Fig. 2 A is the synoptic diagram that the single-basic extension multiple alleles authentication method of primer size marking method is used in explanation.
Fig. 3 is that allele-specific connection of the present invention is used in explanation and the big tick marks of primer is identified allelic synoptic diagram.
Fig. 4 is that hybridization of the present invention is used in explanation and the big tick marks of primer is identified allelic synoptic diagram.
Fig. 5 is the synoptic diagram that the multiple alleles authentication method of homology primer of the present invention and allos primer sets is used in explanation.
Fig. 6 A-6F is that the explanation application fluorescence globule that comprises multiple primer of the present invention is identified a plurality of allelic method synoptic diagram.
Detailed description of the present invention
The present invention is with U.S. Provisional Patent Application No.60/228, and 994 is basis (this full patent texts is incorporated by reference here), relates to the method for determining the allele mark.
The application uses following term from start to finish, and is defined as follows:
Allele: the variant form of given gene. Such variant comprises SNP, insertion, counter-rotating, transposition and disappearance.
Avidin: on the function with the protein families of the ability definition of its high affinity, specific binding biotin. Avidin is quite little oligomer albumen, is comprised of four identical subunits, and each subunit is with a single biotin binding site. Therefore every mole of avidin can be in conjunction with 4 moles biotin. Avidin comprises the protein that (a) amphibian, reptile and birds produce, described protein is present in their egg, be called avidin, (b) protein of streptomyces avidin streptomycete (Streptomyces avidinii) generation is called streptavidin. " avidin " used herein comprises all above-mentioned albumen.
Vitamin H: " vitamin H " used herein comprises vitamin H, wherein vitamin H is by adding the adorned commercial vitamin H product of alkyl and biotin derivative such as active ester, amine, hydrazides and sulfydryl, and described derivative has extra reactive group such as amine, acyl group and alkyl leavings group, carbonyl and alkyl halide or Mi Xieer receptor on polymkeric substance.
Detection molecules: the general molecule covalently bound that makes nucleic acid detect and/or to remove by extra power with nucleic acid.Such molecule can comprise dyestuff, changeable weight molecule (comprising poly A tail and poly-thymidylic acid tail), joint, vitamin H, avidin, digoxigenin, digoxigenin antibody and other analogous material well known in the art that can be connected with globule (comprising the magnetic globule).
Genotype: the specific allelotrope that on locus, carries.
Haplotype: represent collective's genotype of some close linkage locus, be allelic sufficient sequence on the same karyomit(e).
Allos primer: the primer of under stringent condition, hybridizing with a unique allelotrope.
Heterologous sequence site: have not homotactic two allelotrope in the sequence site of determining and be called as and have the heterologous sequence site.
Homology primer: the primer of all hybridizing with parental generation both sides' allelotrope.
Parental generation allelotrope: contain a cover from maternal side karyomit(e) and an allelotrope that overlaps from paternal line chromosomal Mammals diploid cell on one side on one side.
Primer: can with the oligonucleotide of dna profiling hybridization.
Here Yin Shu all patent documentations and reference are incorporated by reference here.
Method of the present invention has some important advantage.Method of the present invention can be fast, low-cost, accurately measure allelotrope, comprise that complete genotype and haplotype measure.The length nucleic acid that this method can be analyzed be standard amplification method well known in the art (for example polymerase chain reaction) the nucleic acid fragment length that can not increase fully.
The present invention relates to separate and identify the method for allele-specific nucleic acid molecule.Can use any nucleic acid molecule, preferred thymus nucleic acid.Can identify the allelotrope, gene fragment and the non-expression fragment that comprise multiple row gene (polyallelic genes) with isolating allele-specific nucleic acid molecule.
Therefore method of the present invention and test kit can be used for all to have two or more heterologous sequences site, has a diploid genetic material of multiple allelic gene type.Can use the example that has the gene of a plurality of allelotrope of the present invention is Mammals mhc gene such as human leucocyte antigen (HLA) (HLA) I class and II genoid, Mammals TXi Baoshouti gene, TAP, LMP, ras, atypia HLA I genoid, the gene of people's complement factor C4 and C2, Bf in the people HLA complex body and be arranged in the gene of Mitochondrial DNA, bacterial chromosome and viral DNA.
In a method of the present invention, obtain to contain a plurality of allelic nucleic acid samples, each allelotrope has a unique cover heterologous sequence site.By any method well known in the art this nucleic acid samples that increases, an embodiment is by polymerase chain reaction (PCR), as the laid-open U.S. Patents No.4 in 28 days July in 1988 of Mullis, described in 683,202.Nucleic acid samples sex change with amplification is a single-chain nucleic acid then.Can analyze this single-chain nucleic acid then, measure the heterologous sequence site that exists, determine the allelotrope that exists by the whole bag of tricks of the present invention.
Method of the present invention is used one or more primers.Primer of the present invention comprises the nucleotide sequence with aim sequence hybridization.In some cases, require primer under the condition of extending during the amplification, hybridizing.Under the other situation, the higher Tm of primer that mates less than 100% complementation when the primer of 100% complementary coupling had than hybridization when requirement was hybridized.Generally speaking, primer of the present invention can be any effective length, but generally comprises about 12-25 Nucleotide or at least 18 Nucleotide, and preferred length is about 18-22 Nucleotide.In the method for the invention, need to identify one or more primer sequences, so that identify the pleomorphism site of aim sequence for target DNA uniqueness in the sample.A lot of multiallelic this polymorphisms identify it is well known in the art.For example, HLA-A, HLA-B and HLA-C gene have about 222 kinds of known allelotrope, and these allelic sequences are well known in the art.Referring to Arnett and Parham, tissue antigen (Tissue Antigens) 45:217-257 page or leaf, 1995 and Baxter-Lowe etc., on December 30th, 1997 laid-open U.S. Patents No.5,702,885.
The phrase of the description making nucleic acid molecular hybridization that the present invention uses " in hybridization under the height stringent condition " refers to hybridize under low ionic strength and high wash temperature condition.Phrase " hybridize under low stringency condition " refers to hybridize under high ionic strength and cold condition.
The variable that influences severity comprises, for example, and temperature, salt concn, probe/sample homology and wash conditions.Along with the rising of hybridization temperature, when other was all identical, severity improved.Severity improves makes non-specific hybridization reduce, and promptly background interference is little." height stringent condition " of nucleic acid hybridization and " medium stringent condition " have explanation below in the document: Current Protocols inMolecular Biology, Ausubel etc., 1998, Green Publishing Associatesand Wiley Interscience, NY, instruction content wherein is incorporated by reference.Certainly, the technician understands that in order to comprise or get rid of complementary degree different between probe and the analyte, in order to meet the requirements of sensing range, the severity of hybridization conditions can change as required.
Can use various detection molecules in the present invention.These molecules can with one or more primer couplings, perhaps can be directly and the ddNTPs coupling that in extending step, is inserted in the nucleic acid.These molecules can comprise the instrument that detects described molecule, for example dyestuff, radio-labeling etc.; Perhaps they can comprise the instrument that separates described molecule, biological example element/avidin, magnetic and/or fluorescent bead etc.; Perhaps contain both.For example when using vitamin H/avidin, can use one or more primers of biotin labeling, when primer and single-chain nucleic acid were hybridized, a chain had biotin labeling in the double-stranded DNA of generation like this.This double-stranded DNA can combine with the solid phase carrier that is coated with avidin then.
The solid phase carrier that uses among the present invention can be any this carrier well known in the art, for example globule, affinity chromatographic column.Preferred carrier is the magnetic bead form.When carrier is the globule form, by washing globule, separate two chains of the nucleic acid of amplification with the attraction globule and in that double-strandednucleic acid is dissociated under the condition of single-chain nucleic acid.Usually under alkaline condition, generally be 0.1M or 0.15M NaOH, under the room temperature,, carry out dissociation by repeatedly the about 5-10 of incubation globule minute several times.Can reclaim each bar chain and further analysis then.
Use various analytical technologies and identify that isolating heterologous sequence site determines allelotrope.These technology are well known in the art, include but not limited to electrophoresis such as polyacrylamide gel electrophoresis, flow cytometry, high pressure liquid chromatography, laser scanning and mass spectroscopy.These technology can manual operationss or are operated by automatic system.Such automatic system is well known in the art and comprises the automatic sequencer or the capillary electrophoresis apparatus that can scan multiple color fluorescence.
First method of the present invention illustrates in Fig. 1 and 2, and this method depends on the extension of the heterologous sequence locus specificity primer of hybridization.This method is specially adapted to measure allelotrope or the haplotype specific gene type information in the height polymorphism chromosomal region.As shown in Figure 1, produce the single stranded DNA fragment after DNA sample amplification and the sex change, be added in 5 ' end one or more heterologous sequence locus specificity primers with the detection molecules mark.Add heterologous sequence locus specificity primer in the single stranded nucleic acid molecule and make its hybridization.In preferred embodiments, the polymorphism base complementrity in 3 of each primer ' end and heterologous sequence site.Therefore, if primer and wherein not complementary heterologous sequence site hybridization of 3 ' end base can not extend when making it to be in extension condition primer of following time.Preferably, use the enzyme that to distinguish single nucleotide difference.As shown in Figure 1, make the primer extension of hybridization then, have only the primer of hybridizing to extend with complementary 3 ' end base coupling.Removing primer by detection molecules then, is the example explanation with the vitamin H among Fig. 1.Use bag to be removed primer by the vitamin H on the primer by the magnetic bead of avidin.Then with those without the removed condition of primer bonded dna fragmentation of extending under the primer/dna fragmentation of washing hybridization.Make the double-strandednucleic acid sex change of extension then.Analyze no longer and globule bonded chain, determine the heterologous sequence site.
Perhaps, the primer that uses among the present invention can be not in 5 ' end and detection molecules coupling, but make primer hybridization as previously mentioned, use with detection molecules link coupled ddNTPs to make those primers carry out single-basic extension, as shown in Figure 2 with complementary 3 ' end hybridization.Utilize the detection molecules of extending on the primer to separate primer, make the primer sex change then and analyze definite heterologous sequence site that exists.
When using following method, the present invention is applicable to that also use can scan the automatic sequencer of four kinds of color fluorescence or the high-throughput single nucleotide polymorphism of capillary electrophoresis apparatus is measured.Also same method correct can be measured other heritable variation type outside the single nucleotide polymorphism, comprise polybase Quito state property, insertion, counter-rotating, transposition and disappearance.
Another kind of method of the present invention depends on allele-specific and connects.Fig. 3 has illustrated this method.As shown in Figure 3, the adding of heterologous sequence locus specificity primer is contained in the single stranded DNA fragment in one or more heterologous sequences site.The polymorphism base complementrity in 3 of each primer of a described heterologous sequence Auele Specific Primer ' end and a heterologous sequence site, and itself and described dna fragmentation are hybridized.Add then and connect primer, and itself and described dna fragmentation are hybridized.Each connects primer and has a part of complementary sequence with one of described dna fragmentation, makes to connect 5 of primer ' end directly in abutting connection with 3 of heterologous sequence locus specificity primer ' end.If this heterologous sequence locus specificity primer with the hybridization of described dna fragmentation, then when being in condition of contact following time, connecting primer can not be connected with heterologous sequence locus specificity primer.If possible, then primer is connected, make it then to be in be enough to make do not have the primer sex change that connects but not enough so that with the temperature condition of the primer sex change that is connected of dna fragmentation hybridization under.Generally speaking, when using the primer of 20 Nucleotide, such temperature is about 60 ℃.Can remove the connection primer of hybridization by any method well known in the art then.As shown in Figure 3, a cover primer can be connected with detection molecules, as vitamin H.Detection molecules can be connected on the heterologous sequence Auele Specific Primer or be connected and connect on the primer.In addition, described different methods combination as shown in Figure 3, by the polymorphism of a heterologous sequence site of a kind of method detection, can be measured the polymorphism of other site by other method described herein.Also as shown in Figure 3, one or more primers can have 5 ' end coupling of molecule and each primer of changeable weight, like this, do not have two primers to have identical molecular weight.The molecule of this changeable weight can be nonreactive any suitable material in hybridization/amplification step, comprises poly homotype nucleic acid tail, for example poly A tail (poly A).The length difference of such poly A tail is a 2-4 base, but can be any different lengths that is enough to separate tool poly A tail primer on standard separate apparatus example gel electrophoresis.
Fig. 4 illustrates another kind of method of the present invention.According to such method, a cover heterologous sequence Auele Specific Primer adding is comprised in the dna fragmentation in a plurality of heterologous sequences site.Each primer has at least one polymorphism base, and it is positioned within each primer, like this, after the hybridization of primer and dna fragmentation, has the Tm of those primers of base mismatch lower than the Tm of those primers that do not have base mismatch in the hybridization.Utilize this species diversity of Tm to remove those primers that are less than 100% complementary hybridization then.Such base mispairing generally betides near the primer sequence center.Removal is less than after the primer/dna fragmentation conjugate of 100% complementary hybridization, analyzes the conjugate that stays, and measures specificity heterologous sequence site, determines concrete allelotrope.This can carry out with various methods.According to shown in Figure 4, all primers can connect the molecule of changeable weight.All primers in each specificity heterologous sequence site can connect the molecule of specific changeable weight.Each primer in each polymorphism in one or more specificity heterologous sequences site connects different detection molecules.According to detection molecules the primer of hybridization is divided into each group, and respectively organizes by further mensuration and to have any changeable weight molecule in the primer, determine the allele specific identity.
Another kind of method of the present invention can be measured a plurality of heterologous sequences site on the nucleic acid long segment, and described nucleic acid long segment may be oversize, with ordinary method for example PCR can not increase fully.As shown in Figure 5, select to contain the single stranded nucleic acid molecule in a plurality of heterologous sequences site.In this nucleic acid molecule, add two kinds of primers, a kind of allos primer and a kind of homology primer.The allos primer can be hybridized with 3 ' heterologous sequence site of the 3 ' end that is positioned at the 5 ' heterologous sequence site on the identical nucleic acid molecule.3 of described allos primer ' end base is so only just extended when 3 of allos primer ' end is hybridized with described single-chain nucleic acid corresponding to the polymorphism base in described heterologous sequence site.Described homology primer can be hybridized at the 5 ' end position in 5 ' heterologous sequence site with same nucleic acid molecule.Primer and making nucleic acid molecular hybridization, and extend the allos primer, so that duplicate 5 ' heterologous sequence site of the nucleic acid molecule between primer.With the allos primer sex change of nucleic acid molecule and extension, and separate and analysis allos primer, determine 5 ' heterologous sequence site.Utilize this information to identify that a new cover contains the nucleic acid primer of allos primer and homology primer, the allos primer of the cover primer that this is new can be hybridized (3 of this allos primer ' end base is corresponding to the polymorphism base) with 5 ' heterologous sequence site, described 5 ' heterologous sequence site is positioned at 3 of another heterologous sequence site on the same nucleic acid molecule ' end, the homology primer of the cover primer that this is new can with the same nucleic acid molecule in position in 5 of another heterologous sequence site ' hybridize the position of end.Repeat the step of front, below each is taken turns in hybridization/extensions use one and overlaps new primer, is used for identifying heterologous sequence site on the allelic nucleic acid molecule up to what identify q.s.Can measure the haplotype of nucleic acid molecule in this way.
Shown in Fig. 6 A-6F, the invention still further relates to a plurality of allelic methods in the nucleic acid molecule of identifying.As shown in Figure 6A, this method comprises, adds to one group of globule and contains a plurality of allelic nucleic acid samples, and each globule is connected with two different primers, and at least one primer on each globule is to the allelic primer of a kind of uniqueness.Shown in Fig. 6 B, make this nucleic acid reaction then, so that make the part hybridization of unique allelic at least a primer and this nucleic acid samples.Make the primer amplification of hybridization,, produce the primer nucleic acid that extends, shown in Fig. 6 C to extend the primer of hybridization.Referring to Fig. 6 D, with the nucleic acid samples and the primer sex change of hybridization, remove nucleic acid samples then from globule.The primer that extends then with globule on second kind of primer hybridization (6E), and this second kind of primer (6F) that increase.Then the globule that contains dual amplimer is analyzed, determined the allelotrope that exists in the nucleic acid samples.In order to remove primer from globule easily, primer can have cracking site.
The invention still further relates to the test kit that is used to implement the method for the invention.In most of basic embodiments, test kit of the present invention comprises about implementing the explanation of aforesaid method.In addition, test kit can contain the reagent of necessity of using at least a or several the inventive method, for example a cover or an a few cover locus specificity amplimer, the polymerase chain reaction buffer reagent, dideoxy nucleotide (wherein one or more are labeled as required), nucleic acid amplification reagent, produce single-chain nucleic acid fragment agents useful for same, one or more heterologous sequence locus specificity primers (as required with at least a detection molecules coupling), one or more connect primer, be used to connect the reagent of contiguous hybridized primer, the globule and the one or more aseptic microtubule that comprise one or more detection molecules.
Will be better appreciated by the present invention from the following examples.But, one of ordinary skill in the art will readily recognize that the concrete grammar and the result that are discussed are in order to describe the present invention in detail rather than will to limit the present invention.
Embodiment
Present embodiment relates to be used three kinds of strategies and verifies not homoallelic catching, and polymorphism specific in described different allelotrope and the HLA gene is relevant: i) hybridize; Ii) single-basic extension; Iii) connect.
Each that use these conditions helps to identify that as setting up one thereby suitable allelotrope identifies the test of the testing method of the specific polymorphism relevant with this equipotential gene.The two kinds of methods in back are the tests based on enzyme, require to use Taq ligase enzyme and Thermus Sequenase, utilize the ability of the single nucleotide difference of specific site on these enzyme difference single stranded DNAs.Observe, these methods are enough sensitive to the single nucleotide polymorphism or the sudden change of differentiating in the specific allelotrope of being studied.
1.A. hybridization
A kind of detection method is the specific target thing and the hybridization of oligonucleotide link coupled microballoon of catching, and analyzes this complex body.The as described below reaction.The allelotrope that will catch is carried out two-wheeled hybridization.The special oligonucleotide coupling globule of different homozygous dnas and hybrid DNA and identification particular sequence is used in first round hybridization.Second takes turns the globule that distinguished sequence in the another set of identification target thing is used in hybridization, confirms the allelic existence of catching.But beginning is carried out single-wheel hybridization earlier as measuring oligonucleotide coupling microballoon to not homoallelic specific control experiment in the target thing.
The range gene group DNA sample (UCLA 210, UCLA 230 and UCLA 243) that use obtains from the UCLA register office uses the 158 bp dna fragmentations that adopted primer 5 ' A200A and antisense primer 3 ' A322-1 amplification HLA-A locus are arranged.For this embodiment, the application standard amplification method prepares this 158 bp fragment.The primer of homozygous dna and hybrid DNA of being used in this embodiment increasing is: 5 ' A200A 5 '-ACA GCG ACG CCG CGA GCC A-3 ', position 182-200, adopted primer 3 ' A322-1 5 '-CCTCGCTCTGGTTGTAGTA-3 ' is arranged, position 322-340, antisense primer
By asymmetric PCR preparation be used to connect, the single stranded DNA (ss) of single-basic extension or hybridization.Adopted primer concentration is arranged than low 50 times of the antisense primer except what add, the condition of asymmetric PCR as mentioned above.Antisense primer is biotinylated, produces the biotin labeled strand PCR fragment of 5 ' end.
Perhaps, use 5 '-3 ' excision enzyme, T7 gene 6 excision enzymes to prepare ssDNA.In this case, protect purpose chain by introducing 4 phosphorothioate bonds at oligonucleotide between synthesis phase at 5 of PCR primer ' end.The T7 excision enzyme does not comprise the chain degradation of thiophosphatephosphorothioate base with 5 of primer ' end
1.B. single base extension (SBER)
Single base extension among this embodiment (SBER) uses and extends primer, designs this primer and makes its 3 ' end in the place's annealing of contiguous polymorphism base.The extension method of this embodiment uses Thermo Sequenase or the big fragment polysaccharase of Klenow, introduces the polymorphism base respectively in circulation or acyclic reaction.
Use single base extension to attempt to catch special allelotrope; Use primer mixture (PM) H001 and H002 to carry out allele-specific PCR (ASPCR).Use these two primer mixtures to introduce particular bases at pleomorphism site.Two PM use common 5 ' primer (agcgacgccgcgagcca), but are to use allele-specific 3 ' primer.When using hybrid DNA, PM H001 introduces " C " (ccaagagcgcaggtcctcg) base in corresponding pleomorphism site place specificity, and PM H002 introduces " A " (ccaagagcgcaggtcctct) specifically.
Carry out extension as mentioned above.Product from extension is purified and combines with the streptavidin magnetic bead.Streptavidin makes that to the high binding affinity of vitamin H the separation of biotin labeled target thing molecule is quick and effective.With the complex body washing for several times, to reduce the possibility of any non-binding mark, this non-binding mark is the factor that possible influence next step reaction of experiment.
Multiple different sample is tested, with the allelotrope of catching by the ASPCR checking.Use PMH001 and H002 to carry out ASPCR 5 times.The experiment condition and the negative control of typical extension method are as follows: the laboratory sample that uses in the extension is biotinylated A or C.Suppose that Sequenase can correctly introduce particular bases, therefore will be detected on the basis of using primer mixture from the special allelic correct signal of catching.Laboratory sample is of identical composition among two negative controls and the SBER, has just cancelled ddNTPs A or C from reaction.Another negative control only uses does not have the ddNTPs of mark A, C, G and T.Use primer mixture H001 and H002, the supernatant liquor of each group reaction below the ASPCR checking.The supernatant liquor of test divides following 5 groups: after the extension, extension products with after magnetic bead combine, wash after the several and under the high temperature after the magnetic bead eluted product.
Circulating reaction
Strand (ss) DNA of 100ng HLA A locus is used in per 20 μ l reaction, as mentioned above to obtaining this single stranded DNA behind the genomic dna pcr amplification; Add the Sequenase that 2 μ M extend primer, 125nM and respectively do not have the ddNTP of vitamin H-mark of the dideoxy terminator (ddG, T, A or C) of mark and 500nM (A or C depend on the special base that will introduce at pleomorphism site), 10X enzyme reaction buffer reagent (being diluted to the final concentration of 1X) and 5 units to reaction mixture.Be reflected at and carried out under 94 ℃ 1 minute, then carry out 94 ℃ of 40 round-robin 10 seconds and 60 ℃ 30 seconds.Carried out final extension circulation in 10 minutes at 72 ℃, and keep down at 4 ℃, this is the extension in the present embodiment.
Acyclic reaction
When using the big fragment polymeric enzyme reaction of Klenow to extend, the first step requires to extend primer and single stranded DNA hybridization.The extension primer annealing of the ssDNA of 100ng and 20 μ M.Primer and DNA mixed 5 minutes down at 90 ℃, and slow then cool to room temperature generates hybrid like this.This process continues about 1 hour.Next step comprises that adding specificity does not have vitamin H ddNTPs mark and mark (1.5 μ M) and the big fragment of 5U Klenow, and 37 ℃ of following incubations 30 minutes.After extending end, in reaction mixture, add the 0.5M EDTA of 1.5 μ l.
Use QIAQUICK post (Qiagen) purifying extension products (circulation or acyclic), remove the vitamin H that is not impregnated in.The magnetic bead (in 2X binding buffer liquid 10mM Tris pH 7.5,1mM EDTA is among the 2.0mM NaCl) of 10 μ l streptavidin bag quilts was at room temperature mixed 20 minutes with the extension products of 20 μ l purifying.Magnetic bead is applied magnetic field, and reject does not have the bonded extension products.With the identical binding buffer liquid of 1ml with magnetic bead washing at least twice, by 95 ℃ down heating 2 minutes the purpose chain is eluted from magnetic bead.
Use special primer that the chain of wash-out is carried out the polymorphism that allele-specific PCR (ASPCR) confirms specific alleles then.Carry out suitable control experiment simultaneously to verify this result.
1.C. method of attachment
This embodiment relate to utilization with the single stranded DNA template annealing before being connected between two kinds of primers.The enforcement of present embodiment is based on such understanding: can generate strong duplex with being connected of two kinds of primers that ssDNA mates fully, but therefore withstand higher temperatures washing (Tm that is higher than each primer).And the mispairing template will be difficult to tolerate the temperature washing that is higher than primer Tm, so itself can dissociate out and finally washed off from duplex.
Make two kinds of primers located adjacent one another, wherein a kind of primer is allele-specific or heterologous sequence primer, and it has a pleomorphism site at 3 ' end, at 5 ' end a biotin labeling is arranged.Second kind of primer is to connect primer, the phosphate group that it has a mediation to connect at 5 ' end.Suppose that two kinds of primers were joined together before hybridizing with the ssDNA template, but the inventive method does not rely on this hypothesis.20 μ l reaction mixtures contain the special ssDNA of 10 μ l (100ng), the various primers of 1 μ l (1 μ M), 2 μ l 10X connection damping fluid and 10U Taq ligase enzyme.
90 ℃ of down heating 2 minutes, then 37 ℃ of following incubations 30 minutes, this moment was by adding the EDTA termination reaction in thermal cycler for mixture.Use QIAQUICK
The column purification mixture is removed all do not have bonded primer and vitamin Hs, and they are the non-specific factors in the allele-specific PCR reaction.
As mentioned above, the complex body of purifying combines with the magnetic bead of streptavidin bag quilt.Under the strict wash conditions of height, wash complex body.Control the severity of washing by the temperature (55-95 ℃) that improves lavation buffer solution, so that reach the threshold temperature that separates the allele-specific dna fragmentation.Utilize the primer of the identification allelic pleomorphism site of catching,, further verify the template of wash-out by allele-specific PCR.
2. the hybridizing method measured of haplotype
The different oligonucleotide of HLA A locus-specific polymorphism and not on the same group globule (Luminex) coupling that is used for hybridizing method.The template of selection and the hybridization of oligonucleotide coupling globule is to provide sequence homology completely.Carry out the coupling of globule and specific oligonucleotide according to manufacturer's explanation (Luminex Corp.).Luminex globule-probe conjugate and the PCR fragment hybridization for preparing above.Being used to separate the segmental probe sequence of allele-specific PCR is:
L5′A107A 1AGGTATTTCT
ACACCTCCGTG
L5′A107C 1AGGTATTTCT
CCACATCCGTG
Wash the pcr template that does not have hybridization off, and from the Luminex globule the PCR fragment of wash-out and 5 ' A107A or 5 ' A107C specific hybrid.Oligonucleotide with the different sizes that do not have transcribed spacer (being to contain other 20 bases at random in the middle of the oligonucleotide sequence) are arranged,, and hybridize, measure not homoallelic specificity with different templates with different globule group couplings.The different oligonucleotide with link coupled on the globule of numeral in the primer sign are associated, and show the pleomorphism site of specific alleles.For example, 107A or C represent pleomorphism site at 107 bit base places, and wherein each allelotrope has an A or C at 107.
The hybridization program is as follows: the ssDNA of 17 μ l then adds 33 μ l specific oligonucleotide link coupled globules (5000 globules/every kind of oligonucleotide) (with the template complementation) 95 ℃ of following sex change 5 minutes, and 55 ℃ of following incubations 30 minutes.When use had the oligonucleotide of transcribed spacer, hybridization temperature was increased to 65 ℃, to guarantee specificity.The abundant eddy current of globule mixture is mixed and supersound process, before adding ssDNA, rise to the hybridization temperature of requirement.After the hybridization, that mixture is centrifugal under 2000xg; With 1.5X TMAC (3M TMAC, 0.1%SDS, 50mM Tris-Cl, pH8.0,4mM EDTA pH 8.0) washed twice, use 1ml, the reject supernatant liquor at every turn.
The H that adds 20 μ l to complex body
2O caught template 5 minutes at 95 ℃ of following wash-outs and oligonucleotide coupling magnetic bead bonded.Template to 1 μ l wash-out is carried out asymmetric PCR, obtains the wash-out template of bigger abundance, is used for second and takes turns hybridization.
With with catch template complementary second cover globule and carry out second and take turns hybridization, as the test of validation template accuracy.Add 120ng streptavidin-phycoerythrin (SA-PE) and under hybridization temperature again after the incubation 5 minutes to each test tube, working sample on Luminex 100 flow cytometers.The fluorescent signal amount that obtains is the true performance that vitamin H and SA-PE react to each other.This method is a kind of quantitative measuring method, and the amount of positive signal is represented with the maximum number that obtains for given reaction.
Second to take turns other allelotrope-specificity Luminex globule-probe that hybridization uses as follows:
Be used to confirm the isolating Luminex globule-probe of allele-specific:
L5′A107A 1AGGTATTTCTACACCTCCGTG
L5′A107C 1AGGTATTTCTCCACATCCGTG
L5′A153A 1CTTCATCGCAGTGGGCTAC
L5′A153C 1CTTCATCGCCGTGGGCTAC
L5′A249T 1GCAGGAGGGTCCGGAGTAT
L5′A249G 1GCAGGAGGGGCCGGAGTAT
L5′A291C 1GAAGGCCCACTCACAGACT
L5′A291G 1GAAGGCCCA
GTCACAGACT
Allele-specific reaction pattern after the hybridization of table 1. expection
| The template DNA title | Luminex globule-probe reaction pattern | ||||||
| HLA-A allelotrope | L5’A107A | L5’A107C | L5’A249G | L5’A249T | L5’A291C | L5’A291G | |
| UCLA 210 (homozygote) | A*0206，- | + | - | - | + | + | - |
| UCLA 230 (heterozygote) | A*2402101 | + | + | + | - | + | - |
| A*3401 | + | - | + | - | - | + | |
| UCLA 243 (homozygote) | A*2402101， - | - | + | + | - | + | - |
Allele-specific reaction pattern after the observed hybridization of table 2.
| The template DNA title | Probe | L5’A107A | L5’A107C | L5’A249G | L5’A249T | L5’A291C | L5’A291G |
| UCLA 210 (homozygote) | L5’A107A | (+)166 | (-)50 | (-)124 | (+)279 | (+)234 | (-)21 |
| L5’A107C | (-)152 | (-)60 | (-)137 | (-)330 | (-)223 | (-)29 | |
| UCLA 230 (heterozygote) | L5’A107A | (+)63 | (+)111 | (+)90 | (-)56 | (+)94 | (-)27 |
| L5’A107C | (-)52 | (+)87 | (+)70 | (-)55 | (+)57 | (-)13 | |
| UCLA 243 (homozygote) | L5’A107A | (-)13 | (-)57 | (-)37 | (-)23 | (+)96 | (-)14 |
| L5’A107C | (-)15 | (+)83 | (+)60 | (-)36 | (+)124 | (-)13 | |
| Negative control | 7 | 9 | 14 | 19 | 6 | 9 |
Table 3. uses the observed hybridization of negative control allele-specific reaction pattern afterwards
| The template DNA title | There is not probe (contrast) | L5’A107A | L5’A107C | L5’A249G | L5’A249T | L5’A291C | L5’A291G |
| UCLA 210 (homozygote) | (+)65 | (-)29 | (-)65 | (+)124 | (+)97 | (-)19 | |
| UCLA 230 (heterozygote) | (+)63 | (+)111 | (+)90 | (-)56 | (+)30 | (-)68 | |
| UCLA 243 (homozygote) | (-)12 | (-)216 | (-)100 | (-)23 | (+)213 | (-)10 | |
| Negative control | 7 | 9 | 14 | 19 | 6 | 9 |
Result in the table has proved the hybridization of successful allele-specific above, because allele-specific number big than non-allelic genes specific reaction.
Separate as those skilled in the art are sensible, for any He all purposes, particularly just provide written description, all scopes disclosed herein comprise any and all possible secondary scope and combination thereof.Understand easily, all scopes of listing are that same range as is divided into half that equates at least, the abundant disclosure and description of 1/3rd, 1/4th, 1/5th, ten/first-class.As non-limitative example, each scope discussed here is divided into down 1/3rd easily, middle(-)third and last three/first-class.Those skilled in the art also understand, all for example " at the most ", " at least ", " greater than ", " less than " etc. description be meant the scope that can then be divided into secondary scope as mentioned above.
Although just described several embodiment preferred, those of ordinary skills understand and can modify and change this embodiment and do not break away from center of the present invention spirit and scope.Therefore, above-described embodiment preferred is aspect all and illustrates rather than limit the scope of the invention, scope of the present invention is indicated by claims, rather than limit by the specification sheets of front, and drop within the claim implication and equivalency range within all changes be included in the scope of the invention.
Following reference hereby incorporated by reference in present patent application:
Jorde, L.B.: American Journal of Human Genetics (Am.J.Hum.Genet.) 56, pp.11-14,1995;
Thomson, G.: American Journal of Human Genetics (Am.J.Hum.Genet.) 57, pp.474-486,1995;
Ruano, G., Kidd, K.K. and Stephens, J.C.: periodical (Proc.Natl.Acad.Sci.USA) 87 of institute of NAS, pp.6296-6300,1990;
Ruano, G. and Kidd, K.K.: nucleic acids research (Nucleic Acids Res.) 19, pp.6877-6882,1991;
Beloin, S.M., Tishkoff, S.A., Bentley, K.L., Kidd, K.K. and Ruano, G.: nucleic acids research (Nucleic Acids Res.) 24, pp.4841-4843,1996;
Gilles, P.N., Wu, D.J., Foster, C.B., Dillon, P.J. and Chanock, S.J.: distinguish (Single nucleotide polymorphic discrimination by an electronicdot blot assay on semiconductor microchips) by single nucleotide polymorphism to the electronics dot blotting analysis of semi-conductor chip, Nature Biotechnol (NatureBiotechnol.) 17, pp.365-370,1999;
Little, D.P., Braun, A., O ' Donnell, M.J. and Koster, H.: the microminiaturized array mass spectrum (Mass spectrometry fromminiaturized arrays for full comparative DNA analysis) that full comparison dna is analyzed, natural drug (Nature Med.) 3, pp.357-362,1997;
Marshal, R.D., Koonts, J. and Sklar, J.: with of the detection (Detection of mutations by cleavageof DNA heteroduplexes with bacteriophage resolvases) of phage resolvase crack DNA allos duplex to sudden change, natural genetics (Nature Genet.) 9, pp.177-183,1995;
Nauck, M.S., Gierens, H., Nauck, M.A., Marz, W. and Wieland, H.: the hybridization probe with the fluorophore mark on light circulator is measured (Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on theLightcycler) to HPA's 1 (HPA-1) rapid gene type, Brit.J.Haematol.105, pp.803-810,1999;
Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P. and Fodor, S.P.A.: the oligonucleotide arrays (Light-generated oligonucleotide arrays for rapid DNAsequence analysis) that is used for the generation light of rapid DNA sequencing analysis, institute of NAS periodical (Proc.Natl.Acad.Sci.USA), 1994;
Southern, the E.M.:DNA chip: extensive and oligonucleotide hybridization carries out sequential analysis (DNA chips:Analysis sequence by hybridization tooligonucleotides on a large scale), genetics is (Trends Genet.) 12 dynamically, pp.110-115,1996;
Syvanen, A.C., Aalto-Setala, K., Harju, L., Kontula, K. and Soderlund, H.: the primer guiding Nucleotide in the apolipoprotein E gene type is measured inserts test (A primer-guided nucleotide incorporation assay in thegenotyping of apolipoproteinE), genome (Genomics) 8, pp.684-692,1990;
Tyagi, S. and Kramer, F.R.: molecular indexes: fluorescigenic probe during hybridization (Molecular beacons:Probes that fluoresce upon hybridization), Nature Biotechnol (Nature Biotechnol), 14, pp.303-308,1996.
Sequence table
＜110〉Liu Xiangjun
＜120〉allelic measuring method
<130>034928/0112
<140>US 09/943,416
<141>2001-08-30
<150>US 60/228,994
<151>2000-08-30
<160>18
<170>PatentIn version 3.1
<210>1
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>1
acagcgacgc cgcgagcca 19
<210>2
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>2
cctcgctctg gttgtagta 19
<210>3
<211>17
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>3
agcgacgccg cgagcca 17
<210>4
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>4
ccaagagcgc aggtcctcg 19
<210>5
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>5
ccaagagcgc aggtcctct 19
<210>6
<211>21
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>6
aggtatttct acacctccgt g 21
<210>7
<211>21
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>7
aggtatttct ccacatccgt g 21
<210>8
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>8
cttcatcgca gtgggctac 19
<210>9
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>9
cttcatcgcc gtgggctac 19
<210>10
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>10
gcaggagggt ccggagtat 19
<210>11
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>11
gcaggagggg ccggagtat 19
<210>12
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>12
gaaggcccac tcacagact 19
<210>13
<211>19
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>13
gaaggcccag tcacagact 19
<210>14
<211>23
<212>DNA
＜213〉the unknown
<220>
＜223〉probe
<400>14
tgtacgtagc agtcagtagt agc 23
<210>15
<211>23
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>15
gctactactg actgctacgt aca 23
<210>16
<211>23
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>16
tgtacgtagc aatcagtagt agc 23
<210>17
<211>23
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>17
gctactactg attgctacgt aca 23
<210>18
<211>12
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>18
tgtacgtagc aa 12
Claims (12)
1. one kind is passed through the allelic method of hybridization detection, comprising:
With target oligonucleotide and the oligonucleotide hybridization that is coupled to not on the globule on the same group, form complex body, wherein saidly be coupled to not on the same group that the oligonucleotide of globule is the oligonucleotide that has and do not have transcribed spacer; With
Analyze described complex body, determine not homoallelic specificity.
2. the method for claim 1 further comprises and separates the allele-specific nucleic acid fragment.
3. the method for claim 2, wherein said separation allele-specific nucleic acid fragment comprise uses the oligonucleotide that is coupled to not the specific polymorphism on the globule on the same group.
4. each method of claim 1-3 further comprises the oligonucleotide of specific polymorphism and not globule coupling on the same group.
5. each method of claim 1-4 further comprises having and not have the oligonucleotide of transcribed spacer and not globule coupling on the same group.
6. each method of claim 1-5 further comprises obtaining to comprise a plurality of allelic target nucleic acid samples, and each allelotrope has the unique heterologous sequence site of a cover.
7. each method of claim 1-6 further comprises the described target nucleic acid sample of amplification.
8. each method of claim 1-7 comprises that further with described target nucleic acid sex change be single-chain nucleic acid.
9. each method of claim 1-8, further comprise by making the hybridization of described target oligonucleotide and second group of globule and verifying the sequence of described target oligonucleotide, wherein said second group of globule and described target oligonucleotide complementation by the hybridization of the described target oligonucleotide of cells were tested by flow cytometry.
10. each method of claim 1-9, wherein said target oligonucleotide is a HLA allelotrope.
11. each method of claim 1-10, wherein said and the oligonucleotide link coupled that has and do not have a transcribed spacer not on the same group globule and different oligonucleotide put together, and can identify by the fluorescence colorimetric that participates in the one or more globules in one group of globule.
12. one kind contains and the composition of globule link coupled oligonucleotide on the same group not, wherein said with not on the same group globule link coupled oligonucleotide be the oligonucleotide that has and do not have transcribed spacer.
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| US22899400P | 2000-08-30 | 2000-08-30 | |
| US60/228,994 | 2000-08-30 |
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| JP (1) | JP2004520812A (en) |
| CN (1) | CN1293204C (en) |
| AU (1) | AU2001289177A1 (en) |
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| JP2005525787A (en) * | 2001-10-24 | 2005-09-02 | シンギュレックス・インコーポレイテッド | Detection method of gene haplotype by interaction with probe |
| NZ578982A (en) | 2001-11-13 | 2011-03-31 | Univ Pennsylvania | A method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby |
| EP3339430A1 (en) | 2001-12-17 | 2018-06-27 | The Trustees of The University of Pennsylvania | Adeno-associated virus (aav) serotype 9 sequences, vectors containing same, and uses thereof |
| ES2717377T3 (en) | 2001-12-17 | 2019-06-20 | Univ Pennsylvania | Sequences of serotype 8 of adeno-associated virus (AAV), vectors containing them and uses thereof |
| JP3752466B2 (en) | 2002-04-24 | 2006-03-08 | 株式会社日立製作所 | Genetic testing method |
| EP1536021A1 (en) * | 2003-11-27 | 2005-06-01 | Consortium National de Recherche en Genomique (CNRG) | Method for HLA typing |
| GB0406863D0 (en) * | 2004-03-26 | 2004-04-28 | Qiagen As | Nucleic acid sequencing |
| US20050282195A1 (en) * | 2004-04-30 | 2005-12-22 | Applera Corporation | Compositions, methods, and kits for (MIS)ligating oligonucleotides |
| GB0600116D0 (en) * | 2006-01-05 | 2006-02-15 | Univ Cardiff | Allele-specific sequencing |
| US8198509B2 (en) * | 2007-08-07 | 2012-06-12 | Monsanto Technology Llc | Methods and compositions for selecting soybean plants resistant to southern root knot nematode |
| EP2411543B1 (en) * | 2009-03-27 | 2017-04-19 | Life Technologies Corporation | Methods, compositions, and kits for detecting allelic variants |
| JP2011072222A (en) * | 2009-09-29 | 2011-04-14 | Kitasato Otsuka Biomedical Assay Kenkyusho:Kk | Method for detecting target nucleic acid |
| US8623603B2 (en) | 2010-03-08 | 2014-01-07 | Dana-Farber Cancer Institute, Inc. | Full cold-PCR enrichment with reference blocking sequence |
| JP2014507950A (en) * | 2011-02-28 | 2014-04-03 | トランスゲノミック，インク． | Kits and methods for sequencing target DNA in mixed populations |
| DK2691541T3 (en) | 2011-03-31 | 2018-01-22 | Dana Farber Cancer Inst Inc | PROCEDURE FOR ENRICHMENT OF SINGLE DRAWED MUTANTS SEQUENCES FROM A MIXTURE OF WILD TYPE AND MUTANTS |
| US9133490B2 (en) | 2012-05-16 | 2015-09-15 | Transgenomic, Inc. | Step-up method for COLD-PCR enrichment |
| US10913977B2 (en) | 2013-07-24 | 2021-02-09 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to enable enrichment of minor DNA alleles by limiting denaturation time in PCR or simply enable enrichment of minor DNA alleles by limiting the denaturation time in PCR |
| BR112016020688B1 (en) | 2014-03-09 | 2024-01-30 | The Trustees Of The University Of Pennsylvania | RECOMBINANT VIRAL VECTORS, RECOMBINANT ADENOASSOCIATED VIRUS, PHARMACEUTICAL COMPOSITION, AS WELL AS USE THEREOF |
| CA3046953A1 (en) | 2016-12-12 | 2018-06-21 | Dana Farber Cancer Institute, Inc. | Compositions and methods for molecular barcoding of dna molecules prior to mutation enrichment and/or mutation detection |
| WO2019023243A1 (en) | 2017-07-24 | 2019-01-31 | Dana-Farber Cancer Institute, Inc. | Methods and compositions for selecting and amplifying dna targets in a single reaction mixture |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1998013527A2 (en) * | 1996-09-24 | 1998-04-02 | Rapigene, Inc. | Compositions and methods for enhancing hybridization specificity |
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| DK0457824T4 (en) * | 1989-02-13 | 2004-10-11 | Geneco Pty Ltd | Detection of a nucleic acid sequence or a change therein |
| US5702885A (en) * | 1990-06-27 | 1997-12-30 | The Blood Center Research Foundation, Inc. | Method for HLA typing |
| JPH06509946A (en) * | 1992-06-17 | 1994-11-10 | シティ・オブ・ホープ | How to detect and identify nucleic acids |
| GB9503808D0 (en) * | 1995-02-24 | 1995-04-12 | Univ Nottingham | Detection assay |
| EP2368897B1 (en) * | 1996-02-09 | 2016-10-19 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays |
-
2001
- 2001-08-30 WO PCT/US2001/041956 patent/WO2002018659A2/en active Application Filing
- 2001-08-30 CA CA002421078A patent/CA2421078A1/en not_active Abandoned
- 2001-08-30 JP JP2002522564A patent/JP2004520812A/en not_active Withdrawn
- 2001-08-30 CN CNB018183670A patent/CN1293204C/en not_active Expired - Fee Related
- 2001-08-30 AU AU2001289177A patent/AU2001289177A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998013527A2 (en) * | 1996-09-24 | 1998-04-02 | Rapigene, Inc. | Compositions and methods for enhancing hybridization specificity |
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| WO2002018659A3 (en) | 2003-07-31 |
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| AU2001289177A1 (en) | 2002-03-13 |
| WO2002018659A2 (en) | 2002-03-07 |
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