CN108220414A - ApoE genetic tests primer sets, detection kit and detection method - Google Patents
ApoE genetic tests primer sets, detection kit and detection method Download PDFInfo
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Abstract
The present invention relates to genetic engineering and biology field, a kind of ApoE genetic tests primer sets, including at least one of rs429358 primer sets and rs7412 primer sets, the rs429358 primer sets are used to expand the nucleic acid fragment in the site containing rs429358；The rs7412 primer sets are used to expand the nucleic acid fragment in the site containing rs7412.The present invention also provides a kind of ApoE gene detecting kits and detection methods.ApoE genetic tests primer sets using the present invention expand the nucleic acid fragment containing site to be measured, reduce the possibility for expanding multiple target sites, improve the specificity expanded to the nucleic acid fragment containing site to be measured and sensitivity, reduce false positive rate；Meanwhile using second generation high throughput gene sequencing technology so that the detection cycle for treating location point is short, and flux is high.
Description
Technical field
The present invention relates to genetic engineering and biology field, more specifically to a kind of ApoE gene primers group,
Detection kit and detection method.
Background technology
ApoE genes and a variety of cardiovascular and cerebrovascular diseases are closely related, the apo E of coding（Apolipoprotein
E, ApoE）Participate in the entire occurrence and development process of the cardiovascular and cerebrovascular diseases such as hyperlipemia, atherosclerosis, coronary heart disease, ApoE
The main reason for gene pleiomorphism is individual difference in these diseases early stage and evolution.
ApoE genetic models are proposed according to ApoE phenotypes, it is believed that the synthesis of ApoE is by being located at three on a gene loci
A allele is controlled, i.e. E2, E3 and E4, each allele corresponds to a main isomer and generates three kinds of homozygotes
(E2/2, E3/3, E4/4) and three kinds of heterozygotes (E2/3, E2/4, E3/4) totally six kinds of common phenotypics.ApoE3/3 types are also known as wild
Type.ApoE is a polymorphism albumen, and there are three common isomers, i.e. E2, E3 and E4.The 112 of the amino acid sequence of ApoE
Position（Cys112Arg, rs429358, hereinafter referred to as 358）With 158（Arg158Cys, rs7412, hereinafter referred to as 412）Two kinds of ammonia
Base acid residue, that is, arginine（Arg）And cysteine（Cys）Exchange determine the type of isomers.ApoE4 is in the two positions
Put all is Arg；E2 is Cys；It is Arg person is ApoE3 isomers that 112, which are Cys and 158,.In general population, gene frequency
Rate E3 is distributed highest, ApoE3/3 Phenotype Distributions about 70%.
In addition both at home and abroad it is multinomial the study found that ApoE genes be the Alzheimer disease being currently known it is most close heredity mark
Will object, the neurological susceptibility of ApoE4 carrier's Alzheimer diseases increase.After ApoE mutation, change with the affinity of LDL-R
Become.Individual its for carrying an E4 allele develops into individual about 3-4 times of the possibility of AD higher than no E4 allele.E4
Allele ratio in general population is about 15%, and the ratio in AD patient is up to 40%.ApoE4 is the important danger of AD
Dangerous factor.By the genotype for detecting ApoE, it may be appreciated that whether be AD Susceptible population, then from teiology delay AD generation,
Development；And inspect periodically, so as to early diagnosis, to the greatest extent early treatment, damage caused by reduction disease.
Be detected by the ApoE genes to patient, obtain the genotype of specific site, using genetic test result as
The method of the information of intermediate result is not belonging to the diagnostic method of disease；The selection of drug is determined with the testing result of genotype,
It can realize personalized medicine, avoid to immunosuppressor adverse reaction.
At present to the detection of gene mutation and gene pleiomorphism, it is common have direct sequencing, gene chip hybridization method,
Taqman fluorescence probe method, allele specific amplification method（Amplification Refractory Mutation
System Real Time PCR, ARMS-RT PCR）, allele specific amplification method（Amplification
Refractory Mutation System Real Time PCR, ARMS-RT PCR）Deng.Above method sensitivity is low, false sun
Property rate is high, it is longer to take, flux is small.
Therefore, it is necessary to a kind of kits and detection method of new detection ApoE gene mutation so as to ApoE bases
Because of the high sensitivity of mutation, false positive rate is low, and detection cycle is short.
Invention content
The purpose of the present invention is to provide a kind of ApoE gene detecting kits and detection methods, it is intended to solve the prior art
The technical issues of middle ApoE genetic tests sensitivity is low, false positive rate is high, the period is long, and flux is small.
The present invention provides a kind of ApoE genetic tests primer sets, a kind of ApoE genetic tests primer sets, including
At least one of rs429358 primer sets and rs7412 primer sets；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets are for expanding the nucleic acid fragment in the site containing rs7412, including rs7412 sense primers and rs7412
Downstream primer, the rs7412 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID
NO:20 or SEQ ID NO:21 sequence, the rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or
SEQ ID NO:22 sequence.
Preferably, the rs429358 primer sets include ApoE primer sets and rs429358 inner primer groups；The rs7412
Primer sets include ApoE primer sets and rs7412 inner primer groups；The ApoE primer sets are described for expanding ApoE genetic fragments
Rs429358 inner primers group is used to expand the ApoE genetic fragments in the site containing rs429358；The rs7412 inner primers group is used to expand
Increase the ApoE genetic fragments in the site containing rs7412.
The present invention also provides a kind of ApoE gene detecting kits, the ApoE gene detecting kits include above-mentioned
A kind of ApoE genetic tests primer sets.
Preferably, the ApoE gene detecting kits further include connector, and sequence label is contained on the connector, described to connect
Amplified production of the head for directly with site containing rs429358 and/or rs7412 sites is connect.
Preferably, the ApoE gene detecting kits further include rs429358 sequencing primers and/or rs7412 sequencings are drawn
Object, the rs429358 sequencing primers have SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:
32、SEQ ID NO:33、SEQ ID NO:34 or SEQ ID NO:35 sequence；The rs7412 sequencing primers have SEQ ID
NO:36、SEQ ID NO:37 or SEQ ID NO:38 sequence.
Preferably, the ApoE gene detecting kits further include the oligonucleotide probe of unstressed configuration label.
The present invention also provides a kind of ApoE gene testers, include the following steps：
A, using rs429358 primer sets, the nucleic acid fragment in the site containing rs429358 is expanded, obtains rs429358 amplification productions
Object；Or rs7412 primer sets are utilized, the nucleic acid fragment in the site containing rs7412 is expanded, obtains rs7412 amplified productions；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets include rs7412 sense primers and rs7412 downstream primers, and the rs7412 sense primers have
SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, it is described
Rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or SEQ ID NO:22 sequence；
B, the jointing on the rs429358 amplified productions and/or rs7412 amplified productions obtains rs429358 libraries point
Son and/or rs7412 library molecules；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determine rs429358 and/or
The genotype in rs7412 sites.
Preferably, the step A includes the following steps：
A1, ApoE genetic fragments are expanded using ApoE primer sets, obtains the first amplified production, the ApoE primer sets are included on ApoE
Primer and ApoE downstream primers are swum, the ApoE sense primers have SEQ ID NO:1 or SEQ ID NO:2 sequence, it is described
ApoE downstream primers have SEQ ID NO:12 or SEQ ID NO:13 sequence；
A2, using the first amplified production as template, using rs429358 inner primers group amplification the site containing rs429358 ApoE genes
Segment, obtains the 2nd rs429358 amplified productions, the rs429358 inner primers group include in rs429358 sense primer and
Downstream primer in rs429358, sense primer has SEQ ID NO in the rs429358:3、SEQ ID NO:4、SEQ ID
NO:5 or SEQ ID NO:6 sequence, downstream primer has SEQ ID NO in the rs429358:14 or SEQ ID NO:15
Sequence；Or sense primer has SEQ ID NO in the rs429358:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID
NO:10 or SEQ ID NO:Downstream primer has SEQ ID NO in 11, the rs429358:16、SEQ ID NO:17 or SEQ
ID NO:18 sequence；Using the ApoE genetic fragments in rs7412 inner primers group amplification site containing rs7412, the 2nd rs7412 is obtained
Amplified production, the rs7412 inner primers group include downstream primer in sense primer in rs7412 and rs7412, the rs7412
Interior sense primer has SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, downstream in the rs7412
Primer has SEQ ID NO:22 sequence.
Preferably, the step B includes the following steps：
B1, the jointing on the 2nd rs429358 amplified productions and/or the 2nd rs7412 amplified productions, obtain
Rs429358 library molecules and/or rs7412 library molecules；
B2, by the biotin labeling on connector, the rs429358 library molecules and/or rs7412 library molecules are fixed on
On magnetic bead containing Avidin；It is fixed on magnetic bead is addressable on solid phase carrier.
Preferably, rs429358 sequencing primers and/or rs7412 sequencing primers are used in step C to the text containing site to be measured
Library molecule is sequenced, and the rs429358 sequencing primers have SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:
31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34 or SEQ ID NO:35 sequence；The rs7412 sequencings
Primer has SEQ ID NO:36、SEQ ID NO:37 or SEQ ID NO:38 sequence.
The nucleic acid fragment containing site to be measured is expanded using ApoE genetic tests primer sets provided by the invention, reduces amplification
The possibility of multiple target sites improves the specificity expanded to the nucleic acid fragment containing site to be measured and sensitivity, reduces vacation
Positive rate；Meanwhile using second generation high throughput gene sequencing technology so that the detection cycle for treating location point is short, and flux is high.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.
The present invention proposes first embodiment, a kind of ApoE genetic tests primer sets, including rs429358 primer sets and
At least one of rs7412 primer sets；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets are for expanding the nucleic acid fragment in the site containing rs7412, including rs7412 sense primers and rs7412
Downstream primer, the rs7412 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID
NO:20 or SEQ ID NO:21 sequence, the rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or
SEQ ID NO:22 sequence.
This programme provide ApoE genetic test primer sets, can specific amplification site containing rs429358 and/or
The nucleic acid fragment of rs7412 reduces the possibility for expanding non-target fragment so as to rs429358 sites and/or
The high sensitivity of rs7412 site primers, false positive rate are low.
Preferably, the rs429358 primer sets include ApoE primer sets and rs429358 inner primer groups；The rs7412
Primer sets include ApoE primer sets and rs7412 inner primer groups；The ApoE primer sets are described for expanding ApoE genetic fragments
Rs429358 inner primers group is used to expand the ApoE genetic fragments in the site containing rs429358；The rs7412 inner primers group is used to expand
Increase the ApoE genetic fragments in the site containing rs7412.
The ApoE primer sets include ApoE sense primers and ApoE downstream primers；The ApoE sense primers have SEQ
ID NO:1 or SEQ ID NO:2 sequence, the ApoE downstream primers have SEQ ID NO:12 or SEQ ID NO:13 sequence
Row.
The rs429358 inner primers group includes downstream primer in sense primer in rs429358 and rs429358；It is described
Sense primer has SEQ ID NO in rs429358:3、SEQ ID NO:4、SEQ ID NO:5 or SEQ ID NO:6 sequence,
Downstream primer has SEQ ID NO in the rs429358:14 or SEQ ID NO:15 sequence；Or the rs429358 on
Swimming primer has SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10 or SEQ ID NO:11 sequence
It arranges, downstream primer has SEQ ID NO in the rs429358:16、SEQ ID NO:17 or SEQ ID NO:18 sequence.
The rs7412 inner primers group includes downstream primer in sense primer in rs7412 and rs7412；In the rs7412
Sense primer has SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, downstream is drawn in the rs7412
Object has SEQ ID NO:22 sequence.
The two groups of ApoE genetic test primer sets provided using this programme are treated location point and carry out two-wheeled nested amplification, due to
The sequence all complementary with two groups of primer sets is seldom simultaneously, so as to reduce the possibility of non-specific amplification, increases to be measured
The susceptibility of site primer.In the present solution, SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ
ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:On 21
Comprising ACCU sequences, the nucleic acid fragment containing site to be measured is expanded using the ApoE gene primers group of this programme, is obtained
Comprising ACCU sequences on amplified production, amplified production is cut using the enzyme of specificity cutting uracil, viscosity can be formed
End, cohesive end joint efficiency higher during follow-up jointing.
The present invention proposes second embodiment, and a kind of ApoE gene detecting kits are examined containing any of the above-described kind of ApoE gene
Survey primer sets.
The kit of the present invention is capable of the nucleic acid fragment of specific amplification site containing rs429358 and/or rs7412, reduces
The possibility of the non-target fragment of amplification, so as to the high sensitivity in rs429358 sites and/or rs7412 site primers,
False positive rate is low.
Location point is treated using two groups of ApoE genetic test primer sets and carries out two-wheeled nested amplification, due to drawing simultaneously with two groups
The all complementary sequence of object group is seldom, so as to reduce the possibility of non-specific amplification, increases to treat and surveys the quick of site primer
Sensitivity.
Preferably, the kit further includes the oligonucleotide probe of unstressed configuration label.
It should be noted that the oligonucleotide probe of the unstressed configuration label and the fluorescence connecting used in sequencing technologies
Probe is compared, and sequence is identical, differs only in unstressed configuration label.The kit of this programme, adds in nothing in sequencing procedure
The oligonucleotide probe of fluorescent marker can effectively reduce the error signal occurred in connection sequencing, improve and treat location point
The accuracy of detection.
Preferably, the kit further includes the enzyme of specificity cutting uracil, the enzyme of the specificity cutting uracil
For USER enzymes, RNase H or UDG enzymes.
Due to the SEQ ID NO of the second wheel amplification:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ
ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21 sequences
On comprising ACCU sequences, therefore also comprising ACCU sequences on obtained amplified production, using the enzyme of specificity cutting uracil
Amplified production is cut, forms cohesive end, cohesive end joint efficiency higher during follow-up jointing.
Preferably, the kit further includes connector, contains sequence label on the connector, the connector for directly with
The amplified production in the site containing rs429358 and/or rs7412 sites connects.
It should be noted that kit provided by the invention, is carried out separately the amplification step in different sites to be measured,
Multiple amplified productions containing different sites to be measured may be mixed together and be carried out at the same time sequencing.When kit using the present invention
When being carried out at the same time detection to the rs429358 sites of same sample and rs7412 sites in same system, and containing rs429358
The connector of the amplified production connection of point and the connector that is connect with the amplified production in the site containing rs7412, sequence label thereon can be with
It is identical, it can also be different.When the sequence label difference on connector, can be treated by the detection of butt joint sequence label to distinguish
The genotype of location point when the sequence label on connector is identical, can be distinguished by the difference of used sequencing primer
The genotype in site to be measured.
When kit using the present invention is to simultaneously examining the same site to be measured of different samples in same system
During survey, since the sequencing primer used in sequencing procedure is identical, it is therefore desirable to use the connector containing different sequence labels
Different samples is connected respectively, by the way that sequence label is sequenced, can effectively be distinguished without carrying out repeatedly sequencing to sample
The sequencing result of different samples.
Diversified forms may be used in the connector of the present invention, including but not limited to flat end fitting, protruding terminus connector, bifurcated
Connector or the connector containing loop-stem structure.
Preferably, the connector is protruding terminus connector, and the protruding terminus is cut with the amplified production through specificity
The cohesive end complete complementary pairing formed after the cleavage of uracil, this programme cause the connection between amplified production and connector
It is more efficient.
Preferably, contain biotin labeling on the connector, for it to be made to be fixed on the magnetic bead containing Avidin.
It is furthermore preferred that the connector includes at least one of connector A, connector B, connector C, the connector A is by SEQ
ID NO:23 and SEQ ID NO:The double-stranded nucleic acid molecule of 24 compositions；The connector B is by SEQ ID NO:25 and SEQ ID
NO:The double-stranded nucleic acid molecule of 26 compositions；The connector C is by SEQ ID NO:27 and SEQ ID NO:The double-strandednucleic acid of 28 compositions
Molecule；NNNN in the connector is sequence label, and the N is A, G, C or T.
Preferably, the kit further includes rs429358 sequencing primers and/or rs7412 sequencing primers, described
Rs429358 sequencing primers have SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ
ID NO:33、SEQ ID NO:34 or SEQ ID NO:35 sequence；The rs7412 sequencing primers have SEQ ID NO:36、
SEQ ID NO:37 or SEQ ID NO:38 sequence.
It is furthermore preferred that on the sequencing primer for linking probe one end away from site to be measured be 1 to 9bp, more preferably 1
To 5bp.This programme causes in sequencing procedure, it is only necessary to carry out the genotype that primary connection sequencing can determine site to be measured.
Preferably, the kit further includes ligase.Under the action of ligase, between the amplified production and connector
The connection that can stablize.
Preferably, the kit further includes connection buffer solution.
The present invention proposes 3rd embodiment, and a kind of ApoE gene testers include the following steps：
A, using rs429358 primer sets, the nucleic acid fragment in the site containing rs429358 is expanded, obtains rs429358 amplification productions
Object；Or rs7412 primer sets are utilized, the nucleic acid fragment in the site containing rs7412 is expanded, obtains rs7412 amplified productions；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets include rs7412 sense primers and rs7412 downstream primers, and the rs7412 sense primers have
SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, it is described
Rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or SEQ ID NO:22 sequence；
B, the jointing on the rs429358 amplified productions and/or rs7412 amplified productions obtains rs429358 libraries point
Son and/or rs7412 library molecules；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determine rs429358 and/or
The genotype in rs7412 sites.
Preferably, the step A includes the following steps：
A1, ApoE genetic fragments are expanded using ApoE primer sets, obtains the first amplified production, the ApoE primer sets are included on ApoE
Primer and ApoE downstream primers are swum, the ApoE sense primers have SEQ ID NO:1 or SEQ ID NO:2 sequence, it is described
ApoE downstream primers have SEQ ID NO:12 or SEQ ID NO:13 sequence；
A2, using the first amplified production as template, using rs429358 inner primers group amplification the site containing rs429358 ApoE genes
Segment, obtains the 2nd rs429358 amplified productions, the rs429358 inner primers group include in rs429358 sense primer and
Downstream primer in rs429358, sense primer has SEQ ID NO in the rs429358:3、SEQ ID NO:4、SEQ ID
NO:5 or SEQ ID NO:6 sequence, downstream primer has SEQ ID NO in the rs429358:14 or SEQ ID NO:15
Sequence；Or sense primer has SEQ ID NO in the rs429358:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID
NO:10 or SEQ ID NO:Downstream primer has SEQ ID NO in 11, the rs429358:16、SEQ ID NO:17 or SEQ
ID NO:18 sequence；Using the ApoE genetic fragments in rs7412 inner primers group amplification site containing rs7412, the 2nd rs7412 is obtained
Amplified production, the rs7412 inner primers group include downstream primer in sense primer in rs7412 and rs7412, the rs7412
Interior sense primer has SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, downstream in the rs7412
Primer has SEQ ID NO:22 sequence.
This programme carries out two-wheeled specificity expansion by designing two pairs of amplimer groups, to the nucleic acid fragment containing site to be measured
Increase, can effectively ensure the dirt of the non-specific amplification not caused by primer pairing specificity is not strong in the second amplified production
Dye, reduces the possibility for expanding multiple target sites, increases specificity and the sensitivity of amplification.
Preferably, it is further included in the step B to the 2nd rs429358 amplified productions and/or the 2nd rs7412 amplified productions
The step of middle enzyme for adding in specificity cutting uracil is cut, digestion of the amplified production through specificity cutting uracil
Cut generation cohesive end.Due to SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:
16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20 and SEQ ID NO:It is wrapped on 21
Sequence containing ACCU, after obtaining the 2nd rs429358 amplified productions and/or the 2nd rs7412 amplified productions by amplification, specificity is cut
Cut the di-phosphate ester between the 2nd rs429358 amplified productions of cleavage and/or the 2nd rs7412 amplified productions G and U of uracil
Key, forms the cohesive end that phosphate group is contained in 5 ' ends, which is conducive to the jointing during subsequent reactions.
Preferably, the enzyme of the specificity cutting uracil is USER enzymes, RNase H or UDG enzymes.
Preferably, the step B includes the following steps：
B1, the jointing on the 2nd rs429358 amplified productions and/or the 2nd rs7412 amplified productions, obtain
Rs429358 library molecules and/or rs7412 library molecules；
B2, by the biotin labeling on connector, the rs429358 library molecules and/or rs7412 library molecules are fixed on
On magnetic bead containing Avidin；It is fixed on magnetic bead is addressable on solid phase carrier.
Preferably, contain sequence label on the connector, the connector for directly with the site containing rs429358 and/
Or the amplified production connection in rs7412 sites.
It should be noted that the detection method that this programme provides, the amplification step to different sites to be measured is to separate progress
, multiple amplified productions containing different sites to be measured may be mixed together and be carried out at the same time sequencing.When right in same system
When the rs429358 sites and rs7412 sites of same sample are carried out at the same time detection, connect with the amplified production in the site containing rs429358
The connector connect and the connector being connect with the amplified production in the site containing rs7412, sequence label thereon can be identical, can not also
Together.When the sequence label difference on connector, the gene in site to be measured can be distinguished by the detection of butt joint sequence label
Type when the sequence label on connector is identical, can distinguish the base in site to be measured by the difference of used sequencing primer
Because of type.
When being detected simultaneously to the same site to be measured of different samples in same system, due in sequencing procedure
The sequencing primer used is identical, it is therefore desirable to connect different samples respectively using the connector containing different sequence labels, lead to
It crosses and sequence label is sequenced, the sequencing result of different samples can be effectively distinguished without carrying out repeatedly sequencing to sample.
Diversified forms may be used in the connector of the present invention, including but not limited to flat end fitting, protruding terminus connector, bifurcated
Connector or the connector containing loop-stem structure.
Preferably, the connector is protruding terminus connector, and the protruding terminus is cut with the amplified production through specificity
The cohesive end complete complementary pairing formed after the cleavage of uracil, this programme cause the connection between amplified production and connector
It is more efficient.
It is furthermore preferred that the connector includes at least one of connector A, connector B, connector C, the connector A is by SEQ
ID NO:23 and SEQ ID NO:The double-stranded nucleic acid molecule of 24 compositions；The connector B is by SEQ ID NO:25 and SEQ ID
NO:The double-stranded nucleic acid molecule of 26 compositions；The connector C is by SEQ ID NO:27 and SEQ ID NO:The double-strandednucleic acid of 28 compositions
Molecule；NNNN in the connector is sequence label, and the N is A, G, C or T.
Preferably, the step C includes the following steps：
C1, the oligonucleotide probe marked using unstressed configuration, carry out rs429358 library molecules and/or rs7412 library molecules
Primary connection sequencing；
C2, sequencing is attached to rs429358 library molecules and/or rs7412 library molecules using fluorescence probe, determined
The genotype in rs429358 and/or rs7412 sites.
It should be noted that the oligonucleotide probe of the unstressed configuration label and the fluorescence connecting used in sequencing technologies
Probe is compared, and sequence is identical, differs only in unstressed configuration label.The detection method of this programme, adds in sequencing procedure
The oligonucleotide probe of unstressed configuration label can effectively reduce the error signal occurred in connection sequencing, improve and treat location
The accuracy of point detection.
Connection sequencing each time includes the following steps：Sequencing primer is anchored, and is rinsed（Remove the extra sequencing not being anchored
Primer）, linking probe, flushing（Remove excess probes, ligase etc.）, adopt figure（Obtain position corresponding to fluorescent marker on probe
Sequence information）, denaturation elution connection product（To connect the anchoring of sequencing primer in sequencing reaction next time）.In step C1
Connection sequencing reaction because use unstressed configuration mark oligonucleotide probe, therefore can without adopting figure step, with
Experimental procedure is reduced, improves conventional efficient.
The present invention provides fourth embodiment, a kind of ApoE gene testers, using the poba gene group DNA of two people as
Template detects the rs429358 sites in each template ApoE genetic fragments and rs7412 sites respectively.It establishes according to the following steps anti-
Answer system：
A1, for above-mentioned two sample, it is each to prepare two amplification ApoE genetic fragments totally 4 reaction systems：The blood-based of 50ng
Because of a group DNA；5U/μLde Ex Taq 0.25μL；2×long Taq Mix（Shenzhen HYK Gene Technology Co., Ltd. produces）
10μL；10 μM of 2 μ L of ApoE sense primers；10 μM of 2 μ L of ApoE downstream primers；Each 4 μ L of dNTP of 2.5mM；Add deionized water
To 50 μ L；Mixing simultaneously centrifuges.Then centrifuge tube is placed in PCR instrument, response procedures is set：Continue 3 minutes under the conditions of 94 DEG C；94
It is for 15 seconds under the conditions of DEG C, it is for 20 seconds under the conditions of 57 DEG C, continue 45 seconds under the conditions of 72 DEG C, altogether 20 cycles；72 DEG C of conditions
Under continue 3 minutes；After the completion of amplified reaction, the first amplified production is obtained.Wherein, ApoE sense primers group is SEQ ID NO:1,
ApoE downstream primers are SEQ ID NO:12；
A2, using the first amplified production of above-mentioned two sample as template, each one amplification site containing rs429358 of configuration with
The reaction system of the ApoE genetic fragments in rs7412 sites, the system in one rs429358 sites of amplified sample is amplification system 1, is expanded
The system for increasing one rs7412 sites of sample is amplification system 2, and the system in two rs429358 sites of amplified sample is amplification system 3,
The system in two rs7412 sites of amplified sample is amplification system 4, and reaction system component is as follows：The first amplified productions of 100ng；10μM
Inner primer group totally 4 μ L；Each 4 μ L of dNTP of 2.5mM；5U/μLde Ex Taq 0.25μL；2×long Taq Mix（Shenzhen China
Yin Kang Gene Tech. Company Limited produces）10μL；Add deionized water to 50 μ L；Mixing simultaneously centrifuges.Centrifuge tube is placed in PCR instrument
In, response procedures are set：Continue 3 minutes under the conditions of 94 DEG C；It is for 15 seconds under the conditions of 94 DEG C, it is for 20 seconds under the conditions of 57 DEG C, 72
Continue 45 seconds under the conditions of DEG C, altogether 30 cycles；Continue 3 minutes under the conditions of 72 DEG C；After the completion of PCR reactions, the second amplification production is obtained
Object.Wherein, it is SEQ ID NO for the inner primer group of amplification system 1 and system 3:3 and SEQ ID NO:14；For expanding body
Be 2 and system 4 inner primer group be SEQ ID NO:19 and SEQ ID NO:22；
It after the completion of second expands, is detected through Page gel electrophoresis, contains target molecule in the second amplified production of two samples,
And electrophoretogram is shown without miscellaneous band, band is single；Illustrate that required amplified production has successfully been obtained in second of amplification.
B1, for the second amplified production obtained in amplification system 1-4, prepare cutting-coupled reaction system 1-4 respectively：
The 10 μ L of USER enzymes of second amplified production, 20 μ L, T4 ligase buffer solution, 8 μ L, T4 DNA ligase, 2 μ L, 1U/ μ L, 10 μM
2 μ L of connector；Add deionized water to 40 μ L；25 DEG C are reacted 20 minutes, are obtained connection product, that is, are treated sequencing library molecule；
B2, library molecule is combined respectively with the magnetic bead that Avidin is modified so that library molecule is fixed on magnetic bead surfaces.
The load sample piece that above-mentioned product point sample to isothiocyano is modified（Slide）, in 37 DEG C of fixed 1h, that is, complete to be fixed on magnetic bead
Treat that the addressable of sequencing library molecule is fixed；
Wherein buffer solution for Tris containing 400mM, 100mM MgCl2,100mM DTT, 5mM ATP, the solution of pH value 7.8；
Connector is by SEQ ID NO:29 and SEQ ID NO:30 compositions.The label of the connector used in cutting-coupled reaction system 1
Sequence is AGTC；The sequence label of the connector used in cutting-coupled reaction system 2 is ACAC；Cutting-coupled reaction system 3
The sequence label of the middle connector used is CAGT；The sequence label of the connector used in cutting-coupled reaction system 4 is GCCG.
C, the high-throughput gene sequencer of Shenzhen HYK Gene Technology Co., Ltd. is used in this embodiment
Pstar II A sequenators, and be sequenced using connection PCR sequencing PCR.In sequencing procedure, for rs429358 sites, used
Sequencing primer be SEQ ID NO:29；For rs7412 sites, used sequencing primer is SEQ ID NO:36.Sequencing knot
Fruit shows the genotype in the rs429358 sites of sample one as wild-type homozygote TT, the gene in the rs7412 sites of sample one
Type is saltant type homozygote TT, and the genotype in the rs429358 sites of sample two is saltant type heterozygote TC；The gene of sample two
Type is wild-type homozygote CC.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>ApoE genetic tests primer sets, detection kit and detection method
<130>
<160> 38
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
aggcctacaa atcggaactg g 21
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
cactgtgcga caccctcc 18
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
accucggaca tggaggtcgt g 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
accucggaga tggacgacgt g 21
<210> 5
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<212> DNA
<213>Artificial sequence
<400> 5
accucggaca tggaggacgt g 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
accucggata tggagtacgt g 21
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence
<400> 7
cggacatgga ggacgtg 17
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence
<400> 8
cggatatgga gtacgtg 17
<210> 9
<211> 17
<212> DNA
<213>Artificial sequence
<400> 9
cggacatgga ggtcgtg 17
<210> 10
<211> 15
<212> DNA
<213>Artificial sequence
<400> 10
cacggctgtc caagg 15
<210> 11
<211> 17
<212> DNA
<213>Artificial sequence
<400> 11
cggagatgga cgacgtg 17
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
ctcgaaccag ctcttgaggc 20
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<400> 13
gctgcccatc tcctccatc 19
<210> 14
<211> 16
<212> DNA
<213>Artificial sequence
<400> 14
ggtactgcac caggcg 16
<210> 15
<211> 16
<212> DNA
<213>Artificial sequence
<400> 15
gtactgcacc aggcgg 16
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence
<400> 16
accucaccag gcggcagc 18
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence
<400> 17
accucaccag gcgaccgc 18
<210> 18
<211> 18
<212> DNA
<213>Artificial sequence
<400> 18
accucaccag gcagccgc 18
<210> 19
<211> 21
<212> DNA
<213>Artificial sequence
<400> 19
accuccgatg acccgcagaa g 21
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence
<400> 20
accuccgatg acctgaagaa g 21
<210> 21
<211> 21
<212> DNA
<213>Artificial sequence
<400> 21
accuccgatg acctccagaa g 21
<210> 22
<211> 16
<212> DNA
<213>Artificial sequence
<400> 22
tggtacactg ccaggc 16
<210> 23
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (23)..(26)
<223> n is a, c, g, or t
<400> 23
cctccctgca gtctctatgg gcnnnnctgc tagtcgctga agtagtacct 50
<210> 24
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (20)..(23)
<223> n is a, c, g, or t
<400> 24
ctacttcagc gactagcagn nnngcccata gagactgcag g 41
<210> 25
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (23)..(26)
<223> n is a, c, g, or t
<400> 25
cctccctgca gtctctatgg gcnnnnctgc tagtcgctgt tgtgatacct 50
<210> 26
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (20)..(23)
<223> n is a, c, g, or t
<400> 26
tcacaacagc gactagcagn nnngcccata gagactgcag g 41
<210> 27
<211> 50
<212> DNA
<213>Artificial sequence
<400> 27
cctccctgca gtctctatgg gcagtcctac ttgtcgctgt tgtgatacct 50
<210> 28
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (20)..(23)
<223> n is a, c, g, or t
<400> 28
tcacaacagc gacaagtagn nnngcccata gagactgcag g 41
<210> 29
<211> 17
<212> DNA
<213>Artificial sequence
<400> 29
cacgacctcc atgtccg 17
<210> 30
<211> 17
<212> DNA
<213>Artificial sequence
<400> 30
cacgtcgtcc atctccg 17
<210> 31
<211> 17
<212> DNA
<213>Artificial sequence
<400> 31
cacgtcctcc atgtccg 17
<210> 32
<211> 17
<212> DNA
<213>Artificial sequence
<400> 32
cacgtactcc atatccg 17
<210> 33
<211> 14
<212> DNA
<213>Artificial sequence
<400> 33
gctgccgcct ggtg 14
<210> 34
<211> 14
<212> DNA
<213>Artificial sequence
<400> 34
gcggtcgcct ggtg 14
<210> 35
<211> 14
<212> DNA
<213>Artificial sequence
<400> 35
gcggctgcct ggtg 14
<210> 36
<211> 17
<212> DNA
<213>Artificial sequence
<400> 36
cttctgcggg tcatcgg 17
<210> 37
<211> 17
<212> DNA
<213>Artificial sequence
<400> 37
cttcttcagg tcatcgg 17
<210> 38
<211> 17
<212> DNA
<213>Artificial sequence
<400> 38
cttctggagg tcatcgg 17
Claims (10)
1. a kind of ApoE genetic tests primer sets, which is characterized in that including in rs429358 primer sets and rs7412 primer sets
It is at least one；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets are for expanding the nucleic acid fragment in the site containing rs7412, including rs7412 sense primers and rs7412
Downstream primer, the rs7412 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID
NO:20 or SEQ ID NO:21 sequence, the rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or
SEQ ID NO:22 sequence.
2. ApoE genetic tests primer sets according to claim 1, which is characterized in that the rs429358 primer sets include
ApoE primer sets and rs429358 inner primer groups；The rs7412 primer sets include ApoE primer sets and rs7412 inner primer groups；
The ApoE primer sets are for expanding ApoE genetic fragments, and the rs429358 inner primers group is for amplification site containing rs429358
ApoE genetic fragments；The rs7412 inner primers group is used to expand the ApoE genetic fragments in the site containing rs7412.
3. a kind of ApoE gene detecting kits, which is characterized in that the ApoE gene detecting kits include claim 1 or
ApoE genetic test primer sets described in 2.
4. ApoE gene detecting kits according to claim 3, which is characterized in that the ApoE gene detecting kits
Further include connector, contain sequence label on the connector, the connector for directly with site containing rs429358 and/or rs7412
The amplified production connection in site.
5. ApoE gene detecting kits according to claim 3, which is characterized in that the ApoE gene detecting kits
Rs429358 sequencing primers and/or rs7412 sequencing primers are further included, the rs429358 sequencing primers have SEQ ID NO:
29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34 or SEQ ID
NO:35 sequence；The rs7412 sequencing primers have SEQ ID NO:36、SEQ ID NO:37 or SEQ ID NO:38 sequence
Row.
6. ApoE gene detecting kits according to claim 3, which is characterized in that the ApoE gene detecting kits
Further include the oligonucleotide probe of unstressed configuration label.
7. a kind of ApoE gene testers, which is characterized in that include the following steps：
A, using rs429358 primer sets, the nucleic acid fragment in the site containing rs429358 is expanded, obtains rs429358 amplification productions
Object；Or rs7412 primer sets are utilized, the nucleic acid fragment in the site containing rs7412 is expanded, obtains rs7412 amplified productions；
The rs429358 primer sets for expanding the nucleic acid fragment in the site containing rs429358, including rs429358 sense primers and
Rs429358 downstream primers, the rs429358 sense primers have SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、
SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ
ID NO:10 or SEQ ID NO:11 sequence, the rs429358 downstream primers have SEQ ID NO:12、SEQ ID NO:
13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 or SEQ ID NO:18 sequence；
The rs7412 primer sets include rs7412 sense primers and rs7412 downstream primers, and the rs7412 sense primers have
SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, it is described
Rs7412 downstream primers have SEQ ID NO:12、SEQ ID NO:13 or SEQ ID NO:22 sequence；
B, the jointing on the rs429358 amplified productions and/or rs7412 amplified productions obtains rs429358 libraries point
Son and/or rs7412 library molecules；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determine rs429358 and/or
The genotype in rs7412 sites.
8. ApoE gene testers according to claim 7, which is characterized in that the step A includes the following steps：
A1, ApoE genetic fragments are expanded using ApoE primer sets, obtains the first amplified production, the ApoE primer sets are included on ApoE
Primer and ApoE downstream primers are swum, the ApoE sense primers have SEQ ID NO:1 or SEQ ID NO:2 sequence, it is described
ApoE downstream primers have SEQ ID NO:12 or SEQ ID NO:13 sequence；
A2, using the first amplified production as template, using rs429358 inner primers group amplification the site containing rs429358 ApoE genes
Segment, obtains the 2nd rs429358 amplified productions, the rs429358 inner primers group include in rs429358 sense primer and
Downstream primer in rs429358, sense primer has SEQ ID NO in the rs429358:3、SEQ ID NO:4、SEQ ID
NO:5 or SEQ ID NO:6 sequence, downstream primer has SEQ ID NO in the rs429358:14 or SEQ ID NO:15
Sequence；Or sense primer has SEQ ID NO in the rs429358:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID
NO:10 or SEQ ID NO:Downstream primer has SEQ ID NO in 11, the rs429358:16、SEQ ID NO:17 or SEQ
ID NO:18 sequence；Using the ApoE genetic fragments in rs7412 inner primers group amplification site containing rs7412, the 2nd rs7412 is obtained
Amplified production, the rs7412 inner primers group include downstream primer in sense primer in rs7412 and rs7412, the rs7412
Interior sense primer has SEQ ID NO:19、SEQ ID NO:20 or SEQ ID NO:21 sequence, downstream in the rs7412
Primer has SEQ ID NO:22 sequence.
9. ApoE gene testers according to claim 8, which is characterized in that the step B includes the following steps：
B1, the jointing on the 2nd rs429358 amplified productions and/or the 2nd rs7412 amplified productions, obtain
Rs429358 library molecules and/or rs7412 library molecules；
B2, by the biotin labeling on connector, the rs429358 library molecules and/or rs7412 library molecules are fixed on
On magnetic bead containing Avidin；It is fixed on magnetic bead is addressable on solid phase carrier.
10. ApoE gene testers according to claim 7, which is characterized in that be sequenced in step C using rs429358
The library molecule containing site to be measured is sequenced in primer and/or rs7412 sequencing primers, the rs429358 sequencing primers tool
There are SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:
34 or SEQ ID NO:35 sequence；The rs7412 sequencing primers have SEQ ID NO:36、SEQ ID NO:37 or SEQ
ID NO:38 sequence.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029159A (en) * | 2019-02-26 | 2019-07-19 | 成都华青精准医疗科技有限公司 | A kind of nucleotide sequence group and its application for the detection of ApoE Genotyping |
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| CN105886607A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | MTHFR gene detection method and kit |
| CN105886608A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | ApoE gene primer group, detection kit and detection method |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886607A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | MTHFR gene detection method and kit |
| CN105886608A (en) * | 2015-12-22 | 2016-08-24 | 武汉康昕瑞基因健康科技有限公司 | ApoE gene primer group, detection kit and detection method |
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| CN110029159A (en) * | 2019-02-26 | 2019-07-19 | 成都华青精准医疗科技有限公司 | A kind of nucleotide sequence group and its application for the detection of ApoE Genotyping |
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