CN108315399A - Chondriogen detection kit and application method - Google Patents
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Abstract
A kind of chondriogen detection kit of present invention offer and its application method, the chondriogen detection kit include two groups of primer pairs, the reagent for PCR amplification, and the reagent for sanger sequencings.Chondriogen detection kit provided by the invention has the characteristics that low testing cost, easy to operate, high sensitivity, specificity are high, can be widely used in the forensic sciences such as individual identification, Relationship iden- tification.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of chondriogen detection reagent based on sanger sequencings
Box and application method.
Background technology
Human mtdna is almost present in various tissues, cell, it is the human inheritance being uniquely distributed in outside core
Substance has the function of self-replacation, transcription and coding.Mitochondrial DNA follows matrilinear inheritance rule, i.e., before excluding to be mutated
It puts, same maternal individual mitochondrial DNA sequence is identical.Therefore now mitochondrial DNA detection is increasingly used in method
Doctor field, and show increasingly important role：（1）Maternal Relationship iden- tification：Whether same maternal family is derived from；
（2）Individual identification：It plays a significant role in difficult, degradation sample establishing identity.
Human mtdna（mt DNA）In double-stranded circular, total length 16569nt is divided into code area and control zone；
Control zone is made of 1022bp (16024-16569bp and 1-576bp).16024-16365,73-340 and 438- therein
Tri- regions 574bp are polymorphic district occurred frequently, are referred to as 1st areas Gao Bian (hypervariable region I, HV I), high change
2nd area (hypervariable region II, HV II) and 3rd areas Gao Bian (hypervariable region III, HV III).
The high polymorphism in these three regions, there are the difference in height opposite sex, is known medical jurisprudence individual in Different Individual and different maternal families
Not and relationship identification has great importance.
In order to which the These characteristics of mitochondria are applied to medical law fields, existing a variety of chondriogen sequencing approaches, such as Shen
Please number be 201510863948.4 patent of invention, provide it is a kind of based on sanger be sequenced human mitochondria gene group side
Method, although the method avoids the cumbersome experimental implementation processes of extraction higher degree mtDNA, but need to utilize about 20 groups
Primer pair carries out segmentation amplification under same PCR reaction conditions, it is difficult to realize that each segment can by high-quality amplify
Come, that is, be difficult to realize the unicity of amplified production, needs all to carry out every group of amplified production agarose in this patent of the owner and coagulate
Gel electrophoresis recycles, in this way, then considerably increasing the workload of mitochondrial DNA amplification and recycling.
Application No. is 201410482243.3 patents of invention, provide one kind and are realized to mitochondria based on two generation sequencing technologies
Overall length be sequenced, reach the purpose of individual identification in medical jurisprudence.But the method is based on two generation sequencing technologies, not only detects into
This height, and sensitivity need to be improved（The template quantity needed in this patent is at least 5ng；It is straight when blood cake is as detection sample
Diameter is not less than 1cm）.
Therefore, it is highly desirable exploitation it is a kind of it is easy to operate, testing cost is low, the chondriogen detection reagent of high sensitivity
Box can be applied more broadly in the medical law fields such as relationship identification, individual identification.
Invention content
In view of the above-mentioned problems, the object of the present invention is to provide a kind of chondriogen detection kit, including two groups of primers
To, for the reagent of PCR amplification, and for sanger sequencing reagent.Two groups of primer pairs are：
First group of primer pair：Nucleotide sequence such as SEQ ID NO：Such as SEQ ID of sense primer and nucleotide sequence shown in 1
NO：Downstream primer shown in 2；Second group of primer pair：Nucleotide sequence such as SEQ ID NO：Sense primer and nucleotide shown in 3
Sequence such as SEQ ID NO：Downstream primer shown in 4.
Reagent for PCR amplification specifically includes：Archaeal dna polymerase, dNTP and PCR reaction buffers.
As an optimization, above-mentioned chondriogen detection kit further includes the reagent extracted for DNA, to hair, is referred to
The samples such as first, tooth, blood cake, mouth desquamated cells are into pedestrian's Genome DNA extraction.
It is a further object of the present invention to provide the application methods of above-mentioned chondriogen detection kit, specifically include following
Step：
S1, PCR amplification, acquisition include two groups of genetic fragments of mitochondria control region information：Using people's total DNA as template, with first
Group primer pair is primer, and first group of genetic fragment is obtained after PCR amplification；Using people's total DNA as template, with second group of primer pair
For primer, second group of genetic fragment is obtained after PCR amplification；
S2, sanger bidirectional sequencings are carried out respectively to two groups of genetic fragments described in S1, obtains two groups of sequencing data results；
S3, comparison result：Two groups of sequencing data results in S2 are compared with Anderson standard sequences respectively, it is special to obtain gene
Sign point.
Since everyone has individual difference, gene obtained by the above method in the gene expression characteristics point of mitochondria control region
Characteristic point can be used as the individual identification of medical law fields.Since the high polymorphism of mitochondria control region is in Different Individual and difference
In maternal family there are difference in height the opposite sex, if the gene expression characteristics point of two or more different people is compared, can be used for into
Maternal Relationship iden- tification between row institute comparison crowd.
Further, in step S2, the response parameter of PCR amplifications is：95 DEG C of pre-degeneration, 5min；Denaturation 95
DEG C, 45s；55 DEG C of annealing, 45s；Extend 72 DEG C, 1min；Extend 72 DEG C eventually, 5min；Recurring number 35-45.To obtain height
The amplified production of quality, recurring number are preferably 35 cycles.
Third object of the present invention is to provide a kind of chondriogen detection kits in legal medical expert's individual identification product
Application.
Fourth object of the present invention is to provide a kind of chondriogen detection kit in the maternal affiliation mirror of legal medical expert
Application in fixed output quota product.
Compared with prior art, the invention has the advantages that：
1, chondriogen detection kit of the invention uses generation sanger bidirectional sequencings, testing cost low.
2, chondriogen detection kit provided by the invention carries out innovative designs to two groups of primer pairs, can not only be
Two groups of special PCR products are amplified under the conditions of same PCR so that two groups of amplified productions include all letters of mitochondria control region
Breath, it is sufficient to carry out the application of the forensic sciences such as individual identity identification, Relationship iden- tification；And by common two of mitochondria
Heterogeneity points are individually placed to be expanded in different PCR, avoid in sanger bidirectional sequencings, two heterogeneous points because
The base Information Problems being unable to get in same amplified production between two heterogeneous points.
3, high sensitivity.Chondriogen detection kit provided by the invention, when detection template used behave it is total
DNA avoids the cumbersome experimental implementation processes of extraction higher degree mtDNA；And detecting DNA profiling amount used can be down to
The blood cake of 0.063ng, a diameter of 2-3mm can be used as the detection sample of the present invention, be advantageous to apply in extraction genome
Amount is few（Such as hair, nail）Forensic science.
4, specificity is high.Chondriogen detection kit provided by the invention can specifically expand for the total DNA of people
Increase and target fragment, to the DNA of other species such as rabbit, pig, cannot amplify desired specificities product, conducive to often having
Popularization and application in terms of the forensic science of the unknown sample in source.
5, easy to operate.Chondriogen detection kit provided by the invention, it is only necessary to two groups of primers, in same PCR
Under the conditions of can amplify to high-quality purpose product, amplified production specificity is high, without the side of agarose gel electrophoresis
Formula carries out being further purified for amplified production, time saving and energy saving, simple operation.
Description of the drawings
Fig. 1 is：Under the conditions of different templates amount, using first group of primer pair as primer, the electrophoresis result after PCR amplification
Figure；
Fig. 2 is：Under the conditions of different templates amount, using second group of primer pair as primer, the electrophoresis result figure after PCR amplification；
Fig. 3 is：Using the DNA in different genera source as template, using first group of primer pair as primer, the electrophoresis after PCR amplification
Result figure；
Fig. 4 is：Using the DNA in different genera source as template, using second group of primer pair as primer, the electrophoresis after PCR amplification
Result figure.
Specific implementation mode
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose, rather than the limitation scope of the invention.
Embodiment 1
A kind of chondriogen detection kit, including two groups of primer pairs, the reagent for PCR amplification, and it is used for sanger
The reagent of sequencing.Two groups of primer pairs are：
Reagent for PCR amplification specifically includes：Archaeal dna polymerase, dNTP and PCR reaction buffers.
As an optimization, above-mentioned chondriogen detection kit further includes the reagent extracted for DNA, to hair, is referred to
The samples such as first, tooth, blood cake, mouth desquamated cells are into pedestrian's Genome DNA extraction.
Embodiment 2
A kind of application method of chondriogen detection kit described in embodiment 1, specifically includes following steps：
S1, PCR amplification, acquisition include two groups of genetic fragments of mitochondria control region information：
（1）Primer sequence：
（2）PCR components：One mixed system of each pair of primer
（3）PCR programs：
95 DEG C of pre-degeneration, 5min；95 DEG C of denaturation, 45s；55 DEG C of annealing, 45s；Extend 72 DEG C, 1min；Extend 72 eventually
DEG C, 5min；Recurring number 35-45.
S2, sanger bidirectional sequencings are carried out respectively to two groups of genetic fragments in S1, obtains two groups of sequencing data results；
S3, comparison result：Two groups of sequencing data results in S2 are compared with Anderson standard sequences respectively, it is special to obtain gene
Sign point.
Due to mitochondria control region high polymorphism in Different Individual and different maternal families there are difference in height the opposite sex,
If the gene expression characteristics point of two or more different people is compared, it can be used for maternal Relationship iden- tification.Due to everyone
In the gene expression characteristics point of mitochondria control region there is individual difference, gene expression characteristics point obtained by the above method can be used to legal medical expert
The individual identification in field.
3 sensitivity of embodiment is verified
Using the chondriogen detection kit of embodiment 1, using the application method of embodiment 2, in the S1 steps of embodiment 2
In, use people's total DNA of following various concentration to be expanded for template（The template quantity of each concentration uses 5 groups of samples）：
Using first group of primer pair as primer（Sequence such as SEQ ID NO：1 and SEQ ID NO：Shown in 2）, after PCR amplification, obtain
The amplified fragments arrived carry out gel electrophoresis, and electrophoresis result is as shown in Figure 1.
Using second group of primer pair as primer（Sequence such as SEQ ID NO：3 and SEQ ID NO：Shown in 4）, by PCR amplification
Afterwards, the amplified fragments obtained carry out gel electrophoresis, and electrophoresis result is as shown in Figure 2.
Found out by Fig. 1 and Fig. 2, in the PCR system of 25 μ L, when people's total DNA template quantity is 0.0625ng, can be amplified
Two groups of special genetic fragments, wherein when people's total DNA template quantity is 0.03ng, first group of primer pair can also amplify its is right
The gene-specific fragments answered.Illustrate the high sensitivity of chondriogen detection kit provided by the invention, a diameter of 2-3mm's
Blood cake can be used as the detection sample of the present invention, be advantageous to apply few in extraction genome amount（Such as hair, nail）'s
Forensic science.
4 specificity verification of embodiment
Using the chondriogen detection kit of embodiment 1, using the application method of embodiment 2, in the S1 steps of embodiment 2
In, the total DNA that rabbit, pig, fish, chicken, ox and people is respectively adopted is that template expands（The template in each source uses 3 groups of samples
This）.
Using first group of primer pair as primer（Sequence such as SEQ ID NO：1 and SEQ ID NO：Shown in 2）, by PCR amplification
Afterwards, the amplified fragments obtained carry out gel electrophoresis, and electrophoresis result is as shown in Figure 3.
Using second group of primer pair as primer（Sequence such as SEQ ID NO：3 and SEQ ID NO：Shown in 4）, by PCR amplification
Afterwards, the amplified fragments obtained carry out gel electrophoresis, and electrophoresis result is as shown in Figure 4.
Mitochondria detection kit provided by the invention and its application method are utilized it can be seen from Fig. 3 and Fig. 4, in addition to
Outside people's total DNA, other kind total DNAs cannot amplify two groups of special genetic fragments, illustrate line grain provided by the invention
The species specificity of body gene detecting kit is high.
Embodiment 5
Now using the peripheral blood of man's first as sample, detected using the chondriogen described in embodiment 1 based on sanger sequencings
Application method described in kit and embodiment 2, is detected the chondriogen of man's first, exists to the kit of the present invention
Application in legal medical expert's individual identification product is described further.
The present embodiment is the chondriogen detection kit using embodiment 1, using the application method of embodiment 2,
It in the S1 steps of embodiment 2, is expanded as template using the total DNA of man's first, it includes mitochondria control region letter to be obtained after amplification
Two groups of genetic fragments of breath.
Sanger bidirectional sequencings are carried out respectively to two groups of above-mentioned genetic fragments, obtain two groups of sequencing data results.
Comparison result：Two groups of sequencing data results above are compared with Anderson standard sequences respectively, obtain part
Gene expression characteristics point is as follows：
Since everyone has individual difference, the gene expression characteristics that the present embodiment obtains in the gene expression characteristics point of mitochondria control region
Put the individual identification that can be used as man's first in medical law fields.
Embodiment 6
Now for identifying whether man's first and woman's second are same maternal family, to kit of the invention in the maternal parent of legal medical expert
Application in edge relationship identification product is described further.
According to the method for embodiment 5, by S1 steps, " using the total DNA of man's first as template " changes into " with the total of woman's second
DNA is template " it is expanded, other experiment reagents, condition etc. are constant, obtain the gene expression characteristics point of woman's second.
The gene expression characteristics point for man's first that embodiment 5 is obtained is clicked through with the gene expression characteristics of woman's second obtained by the above method
Row comparison, result are as follows：
As can be seen from the above table：Man's first and woman's second are identical in mitochondrial DNA hypervariable region sequence height, illustrate that the two is same
Maternal family.
Embodiment 7
Now for identifying whether man's first and woman third are same maternal family, to kit of the invention in the maternal parent of legal medical expert
Application in edge relationship identification product is described further.
According to the method for embodiment 5, by S1 steps, " using the total DNA of man's first as template " changes into " with the total of woman third
DNA is template " it is expanded, other experiment reagents, condition etc. are constant, obtain the gene expression characteristics point of woman third.
The gene expression characteristics point for man's first that embodiment 5 is obtained is clicked through with the gene expression characteristics of woman third obtained by the above method
Row comparison, partial results are as follows：
As can be seen from the above table：Both man's first and woman third are different at mitochondrial DNA hypervariable region sequence at least 7, illustrate
Not same maternal family.
It should be noted that the preferable embodiment of the present invention is given in the specification and its attached drawing of the present invention, but
It is that the present invention can be realized by many different forms, however it is not limited to embodiment described in this specification, these realities
Mode is applied not as the additional limitation to the content of present invention, the purpose of providing these embodiments is that making in disclosure of the invention
The understanding of appearance is more thorough and comprehensive.However after the above for having read the present invention, those skilled in the art can be to this hair
Bright to make various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.
Sequence table
<110>Chengdu Genegle Biological Technology Co., Ltd.
Sichuan gene lattice judicial expertise institute
<120>Chondriogen detection kit and application method
<130> 2018.01.18
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atcctaatac caactatctc 20
<210> 2
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctatcaccct attaaccact 20
<210> 3
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
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<213>Artificial sequence (Artificial Sequence)
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Claims (7)
1. a kind of chondriogen detection kit, which is characterized in that the kit includes two groups of primer pairs, expands for PCR
The reagent of increasing, and the reagent for sanger sequencings；Two groups of primer pairs are：
First group of primer pair：Nucleotide sequence such as SEQ ID NO：Such as SEQ ID of sense primer and nucleotide sequence shown in 1
NO：Downstream primer shown in 2；Second group of primer pair：Nucleotide sequence such as SEQ ID NO：Sense primer and nucleotide shown in 3
Sequence such as SEQ ID NO：Downstream primer shown in 4.
2. chondriogen detection kit according to claim 1, which is characterized in that described for PCR amplification
Reagent includes：Archaeal dna polymerase, dNTP and PCR reaction buffers.
3. chondriogen detection kit according to claim 1, which is characterized in that the kit further includes using
In the reagent of DNA extractions.
4. a kind of application method of claim 1-2 any one of them chondriogen detection kit, which is characterized in that packet
Include following steps：
S1, PCR amplification, acquisition include two groups of genetic fragments of mitochondria control region information：Using people's total DNA as template, described
One group of primer pair is primer, and first group of genetic fragment is obtained after PCR amplification；Using people's total DNA as template, described second group is drawn
Object after PCR amplification to for primer, obtaining second group of genetic fragment；
S2, sanger bidirectional sequencings are carried out respectively to two groups of genetic fragments described in S1, obtains two groups of sequencing data results；
S3, comparison result：Two groups of sequencing data results in S2 are compared with Anderson standard sequences respectively, it is special to obtain gene
Sign point.
5. the application method of chondriogen detection kit according to claim 3, which is characterized in that in step S2,
The response parameter of PCR amplification is：95 DEG C of pre-degeneration, 5min；95 DEG C of denaturation, 45s；55 DEG C of annealing, 45s；Prolong
72 DEG C are stretched, 1min；Extend 72 DEG C eventually, 5min；Recurring number 35-45.
6. application of the chondriogen detection kit described in claim 1 in legal medical expert's individual identification product.
7. application of the chondriogen detection kit described in claim 1 in the maternal Relationship iden- tification product of legal medical expert.
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