CN106701748A - Primer, method and kit used for amplifying hyper variable region of human mitochondrial genome - Google Patents
Primer, method and kit used for amplifying hyper variable region of human mitochondrial genome Download PDFInfo
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- CN106701748A CN106701748A CN201510789800.0A CN201510789800A CN106701748A CN 106701748 A CN106701748 A CN 106701748A CN 201510789800 A CN201510789800 A CN 201510789800A CN 106701748 A CN106701748 A CN 106701748A
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- primer sets
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Abstract
The invention provides a primer group capable of performing PCR (polymerase chain reaction) amplification with high quality on human mitochondrial genomes of various origins, a kit comprising the primer group and used for PCR amplifying and a PCR amplifying method using the primer group. The primer group provided by the invention is composed of an upstream primer and a downstream primer, wherein a nucleotide sequence of the upstream primer is as shown in SEQ ID NO:1; and a nucleotide sequence of the downstream primer is as shown in SEQ ID NO:2.
Description
Technical field
The present invention relates to a kind of primer sets for PCR amplifications.Specifically, the present invention relates to primer sets, the kit comprising the primer sets and the PCR amplification method using the primer sets of the hypervariable region of PCR amplification human mitochondria gene groups.
Background technology
Mitochondria is the important organelle in eukaryotic, is the place of energy production and conversion, also participates in the synthesis of aliphatic acid and the synthesis of a small amount of protein.Normal mitochondrial function depends on the collective effect of karyogene and chondriogen, and the mutation in any genome may all cause mitochondria dysfunction.Therefore, need to determine the ratio of different mitochondria in sample (such as normally with mutation) in some cases.
Human mitochondria gene group is the double-stranded cyclic DNA molecule (also referred to as mitochondrial DNA) that length is 16,569bp, is made up of code area and noncoding region.Wherein, code area includes 37 genes.Noncoding region, it is called control zone or D ring regions (Displacement-loop region, D-Loop), it is made up of 1,122bp, there are 2 length in the region is respectively the hypervariable region of 400bp, that is HVI areas (hypervariable region I, 16,024-16, and HVII areas (hypervariable region II, 1-576) 569).Current research finds that the sequence of coding region and hypervariable region is analyzed by sequencing means can improve the reliability of mitochondrial DNA data.
It is to amplify the hypervariable region from the sample comprising it to come to the premise that human mitochondria gene group hypervariable region is sequenced.If the quality of the hypervariable region for amplifying is not high enough, this can bring adverse effect to follow-up sequencing library construction and the sequencing of two generations.
The hypervariable region amplified to high-quality, amplification primers are crucial.But, for the hypervariable region, in theory can be countless as the sequence of its amplimer, selected from these primers one group can high-quality expand the primer sets not a duck soup of the hypervariable region.
The content of the invention
In view of above-mentioned the deficiencies in the prior art, the primer sets of high-quality PCR amplifications, the PCR reagent for amplification box comprising the primer sets and the PCR amplification method using the primer sets are carried out it is an object of the invention to provide a kind of hypervariable region to human mitochondria gene group.
The present inventor has made intensive studies to solve above-mentioned technical problem, as a result finds：Using by nucleotide sequence such as SEQ ID NO：Sense primer and nucleotide sequence such as SEQ ID NO shown in 1：The primer sets of the anti-sense primer composition shown in 2 can enter to high-quality performing PCR amplification, so as to complete the present invention as primer to human mitochondria gene group.
That is, the present invention includes：
1. a kind of primer sets, it is made up of sense primer and anti-sense primer, the nucleotide sequence such as SEQ ID NO of the sense primer：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer：Shown in 2.
2. primer sets according to item 1, its hypervariable region for being used to expand human mitochondria gene group, the hypervariable region is HVI areas and HVII areas.
3. a kind of kit for expanding the hypervariable region of human mitochondria gene group, it includes the primer sets described in item 1 or 2.
4. it is a kind of expand human mitochondria gene group hypervariable region method, its using the primer sets described in item 1 or 2 enter as primer performing PCR expand.
5. the method according to item 4, its user's mitochondrial genomes is used as template.
6. the method according to item 4 or 5, wherein, the denaturation temperature of the PCR amplifications is 93 DEG C -98 DEG C.
7. the method according to any one of item 4~6, wherein, the annealing temperature of the PCR amplifications is 52 DEG C -62 DEG C.
8. the method according to any one of item 4~7, wherein, the elongating temperature of the PCR amplifications is 72 DEG C.
Invention effect
By using primer sets of the invention, performing PCR amplification can be entered to the hypervariable region high-quality of human mitochondria gene group.
Brief description of the drawings
Fig. 1 is that display enters the result that performing PCR is expanded to 11 kinds of human mitochondrial DNA samples using the primer sets of embodiment 1, and leftmost side swimming lane is molecular weight standard DL2000.
Fig. 2 is that 11 kinds of human mitochondrial DNA samples are entered with the result that performing PCR is expanded using the primer sets of comparative example 1, and leftmost side swimming lane is molecular weight standard DL2000.
The specific embodiment of invention
In an aspect, the present invention provides a kind of primer sets (primer sets of the invention), and it is made up of sense primer and anti-sense primer, the nucleotide sequence such as SEQ ID NO of the sense primer：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer：Shown in 2.
SEQ ID NO：1
5’-ATTGGACAAGTAGCATCCG-3’
SEQ ID NO：2
5’-TGTGAGCCCGTCTAAACAT-3’
The primer sets can be used in the PCR of the hypervariable region for human mitochondria gene group amplifications as primer.In this manual, the hypervariable region of the human mitochondrion is homo mitochondrion gene Zu HVI areas (hypervariable region I, 16,024-16,569) and HVII areas (hypervariable region II, 1-576).
In another aspect, the present invention provides a kind of kit (kit of the invention) for expanding the hypervariable region of human mitochondria gene group, and it includes primer sets of the invention.For kit of the invention, primer sets of the invention are necessary, wherein, sense primer and anti-sense primer can be attached separately in different vessels, it is also possible in same container.In addition to comprising primer sets of the invention, kit of the invention can also include other reagents for example for PCR amplifications, including but not limited to selected from dNTPS、MgC12, Taq enzyme, PCR reaction buffers, distilled water (ddH2) etc. O at least one in, those skilled in the art can suitably select as needed.
Primer sets of the invention are applicable to the PCR amplifications of the hypervariable region of the human mitochondria gene group in various sources, and can obtain the amplified production of high-quality.Here, " high-quality " refers to：When entering row agarose gel electrophoresis to amplified production and checking, band single (such as purpose band accounts for more than 99%, preferably more than 99.5%, more preferably substantially 100%) and without disperse.Therefore, in another aspect, the present invention provides a kind of method (amplification method of the invention) of the hypervariable region for expanding human mitochondria gene group, and it enters performing PCR as primer and expands using primer sets of the invention.
PCR (polymerase chain reaction) amplifications are well known to those skilled in the art, and it is typically realized by certain PCR response procedures (temperature cycles).The PCR response procedures such as are typically denatured, anneal, extending at the step.
For amplification method of the invention, denaturing step can be carried out 0.1~10 minute (preferably 0.5~2 minute) at 93-98 DEG C (preferably 94-96 DEG C)；Annealing steps can be carried out 0.1~10 minute (preferably 0.5~2 minute) at 52-62 DEG C (preferably 54-56 DEG C)；Extending step can be carried out 0.1~10 minute (preferably 1~5 minute) at 72 DEG C；The circulation being made up of above-mentioned denaturation, annealing, extension step can be carried out 10-50 times (preferably 25-35 times).Preferably, template before experience said temperature circulation can carry out the heat treatment of (preferably 5~20 minutes) in 1~60 minute at 93-98 DEG C (preferably 94-96 DEG C), and the amplified production after experience said temperature circulation can be incubated 0-12 hours in 4 DEG C.
Amplification method of the invention can use various samples as template to carry out.In this manual, sample refers to：The sample of the hypervariable region comprising human mitochondria gene group.The sample is not particularly limited, as long as it includes the hypervariable region of human mitochondria gene group, sample can be such as human mitochondria gene group (mitochondrial DNA), can also be such as animal vegetable tissue comprising mitochondrial DNA, animal and plant cells or cell mass, microorganism etc., specifically such as blood, liver, musculature, egg mother cell etc..Preferably, the sample behaviour mitochondrial genomes.
Generally mitochondria can be extracted and purified from the sample comprising it, make the state for being more suitable for being expanded.It is (for example may be referred to " Wang Wen, Shi Liming; a kind of improved animal mitochondria DNA extracting method; zoological research, 1993 (2) ") well known by persons skilled in the art that mitochondrial method is extracted from animal vegetable tissue, animal and plant cells or microorganism.Additionally, the method that mitochondrial DNA is extracted from mitochondria is well known by persons skilled in the art (such as to may be referred to that " Zang Yingan, fourth are risen, and mitochondrial method is extracted from animal tissue.Chinese Veterinary Journal, 2006 (42) 2:61-65”；Or refer to green skies company cell mitochondrial separating kit (article No.：C3601 product description)).It is of course also possible to do not extract mitochondrial DNA and directly using the sample in itself or the mitochondria from sample enter performing PCR amplification.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is for explaining the present invention, not limitation of the invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Embodiment
1
Use primer sets of the invention
PCR
Amplification mitochondria
DNA
Hypervariable region
The PCR amplifications of the hypervariable region of mitochondrial DNA are carried out as follows.
The full length sequence of the hypervariable region according to mitochondrial DNA, devises a pair of specific primers, and nucleotide sequence is as follows：
HV-F：5-attggacaagtagcatccg-3(SEQ ID NO：1)
HV-R：5-tgtgagcccgtctaaacat-3(SEQ ID NO：2)
Enter performing PCR using above-mentioned primer sets to expand, obtain pcr amplification product.
PCR reaction systems such as table 1:
Table 1
| Composition | Usage amount (μ L) |
| 10×buffer(Mg2+free) | 2.5 |
| dNTP(2.5mM) | 2 |
| MgCl2 | 1.5 |
| Taq enzyme | 0.2 |
| HV-F | 1 |
| HV-R | 1 |
| ddH2O | 15.8 |
| Mitochondrial DNA (50ng/ μ L) | 1 |
| Total | 25 |
PCR response procedures：95 DEG C 10 minutes；(95 DEG C 1 minute, 55 DEG C 45 seconds, 72 DEG C 2 minutes) 30cycles；72 DEG C 10 minutes.
PCR amplifications are as shown in Figure 1.From electrophoresis result it can be seen that, for the 11 kinds of human mitochondrial DNA samples for preparing, amplification has obtained purpose band, and band it is single, without disperse.
Comparative example
1
Use other primer sets
PCR
Amplification mitochondria
DNA
Hypervariable region
As embodiment, PCR amplifications are carried out to 11 kinds of human mitochondrial DNA samples, unlike, amplimer is changed to
HV-F：ACTAATACACCAGTCTTGTAAA
HV-R：ATGCTTGCATGTGTAATCTTAC
PCR amplifications are as shown in Figure 2.Be can see from electrophoresis result, for 11 kinds of human mitochondrial DNA samples, some fail amplification and obtain purpose band, although some amplifications have obtained purpose band, band is not single, have disperse.
Also, it should be noted that on the premise of it can implement and substantially not run counter to purport of the invention, in this manual as a certain technical scheme composition part described by any technical characteristic or the combination of technical characteristic be equally readily adaptable for use in other technical schemes；Also, on the premise of it can implement and substantially not run counter to purport of the invention, as different technologies scheme composition part described by technical characteristic between can also in any way be combined and constitute other technical schemes.The present invention be also contained in it is above-mentioned in the case of by combination obtained from technical scheme, and these technical schemes equivalent to record in this manual.
Described above has shown and described the preferred embodiments of the present invention, as previously described, it should be understood that the present invention is not limited to form disclosed herein, it is not to be taken as the exclusion to other embodiment, and can be used for various other combinations, modification and environment, and can be modified by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of the present invention, then all should be in the protection domain of appended claims of the present invention.
Industrial applicibility
According to the present invention, there is provided a kind of human mitochondria gene group for various sources can enter to high-quality primer sets, the PCR reagent for amplification box comprising the primer sets and the PCR amplification method using the primer sets of performing PCR amplification.
Claims (8)
1. a kind of primer sets, it is made up of sense primer and anti-sense primer, the nucleosides of the sense primer
Acid sequence such as SEQ ID NO：Shown in 1, the nucleotide sequence such as SEQ ID NO of the anti-sense primer：
Shown in 2.
2. primer sets according to claim 1, its height for being used to expand human mitochondria gene group
Variable region, the hypervariable region is HVI areas and HVII areas.
3. a kind of kit for expanding the hypervariable region of human mitochondria gene group, it includes right
It is required that the primer sets described in 1 or 2.
4. it is a kind of expand human mitochondria gene group hypervariable region method, its usage right requirement 1
Or the primer sets described in 2 are entered performing PCR and are expanded as primer.
5. method according to claim 4, its user's mitochondrial genomes is used as template.
6. method according to claim 4, wherein, the denaturation temperature of the PCR amplifications is
93 DEG C -98 DEG C, preferably 94 DEG C -96 DEG C.
7. method according to claim 4, wherein, the annealing temperature of the PCR amplifications is
52 DEG C -62 DEG C, preferably 54 DEG C -56 DEG C.
8. method according to claim 4, wherein, the elongating temperature of the PCR amplifications is 72 DEG C.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002068689A1 (en) * | 2001-02-24 | 2002-09-06 | Childrens Hospital Los Angeles | Method of detecting mitochondrial dysfunction |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002068689A1 (en) * | 2001-02-24 | 2002-09-06 | Childrens Hospital Los Angeles | Method of detecting mitochondrial dysfunction |
Non-Patent Citations (4)
| Title |
|---|
| FREGEL,R. 等: "GenBank 登录号：KJ411423.1", 《NCBI》 * |
| 左素娥 等: "人类线粒体DNA非编码区的序列分析与法医学应用", 《法医学理论与实践》 * |
| 张纯斌 等: "用PCR测序法对人类线粒体DNA多态区的研究", 《中国法医学杂志》 * |
| 程宝文: "PCR-RFLP检测线粒体 DNA D-环多态性", 《中国法医学杂志》 * |
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