CN111378732A - Mitochondrial genome sequencing primer, kit and method - Google Patents
Mitochondrial genome sequencing primer, kit and method Download PDFInfo
- Publication number
- CN111378732A CN111378732A CN201811610869.2A CN201811610869A CN111378732A CN 111378732 A CN111378732 A CN 111378732A CN 201811610869 A CN201811610869 A CN 201811610869A CN 111378732 A CN111378732 A CN 111378732A
- Authority
- CN
- China
- Prior art keywords
- mitochondrial
- kit
- sequencing
- primer
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of mitochondrial disease diagnosis and sequencing, in particular to a mitochondrial genome sequencing primer, a kit and a method. The primer and the kit provided by the invention are combined with a third-generation sequencing method, so that the human mitochondrial genome can be effectively sequenced, and mutation in the human mitochondrial genome can be analyzed.
Description
Technical Field
The invention relates to the field of mitochondrial disease diagnosis and sequencing, in particular to a mitochondrial genome sequencing primer, a kit and a method.
Background
Anderson, et al, 1981, determined the complete sequence of the human mitochondrial genome, 16569bp, compared to nuclear DNA, mtDNA as the organism phylogenetic "molecular clock" (molecular clock) has its own advantages of high ① mutation rate, about 10 times that of nuclear DNA, thus accumulating a large number of nucleotide substitutions rapidly even between species that diverge in the near term, comparative analysis can be performed, ② mtDNA is maternally inherited and does not undergo DNA recombination, therefore, individuals with the same mtDNA sequence must be from a common female ancestor human mitochondrial DNA (mtDNA), contain 37 genes in total, 22 of these 37 genes encode transfer ribonucleic acid (tRNA), 2 encode ribosomal ribonucleic acids (12S and 16S rRNA), 13 encode polypeptides, mitochondria are organelles enclosed by two membranes, are structures that make energy in most cells, are major structures that make oxygen breathing in cells, are called "potush", 13 encode polypeptide, mitochondria, are important mutations in the genome, and are important for the majority of patients, because of the genetic mutation of nuclear DNA, the genetic mutation of the genetic gene is a few, because of the genetic mutation of the nuclear DNA, the genetic gene, the genetic mutation of the genetic defect of the nuclear DNA, the genetic gene, because of the genetic gene, the genetic mutation of the genetic defect of the genetic gene, the genetic disease, the genetic gene, the genetic defect of the genetic gene, the genetic disease, because of the genetic disease, because of the genetic disease, the genetic disease.
The analysis and determination of mitochondrial genome needs to analyze 37 genes, and since the mitochondrial gene belongs to maternal inheritance, prenatal mitochondrial genome analysis of mothers is of great significance for the birth of healthy babies.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention relates to a primer pair combination product, which comprises the following primer pairs:
SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6.
The primer pair can carry out segmented amplification on human mitochondrial genome (mtDNA), an amplification product can cover the full length of the mtDNA, and the PCR products of the three pairs of primers are overlapped, so that the primer pair can ensure that if mutation or structural variation occurs at the position of the primer, the primer pair can also detect the mutation or structural variation.
According to one aspect of the invention, the invention also relates to a kit comprising a primer pair combination as described above.
According to one aspect of the invention, the invention also relates to a mitochondrial genome sequencing method comprising:
a) preparing a composition as described above; and
b) connecting the two ends of the amplicon in the composition with barcode sequences for distinguishing different samples to construct a library model of the third-generation sequencing and computer sequencing;
c) optionally, the sequence of the sequencing result is spliced.
The invention also relates to the application of the product and the method in detecting the human mitochondrial gene variation and preparing the diagnostic agent of the diseases related to the human mitochondrial defect.
Compared with the prior art, the invention has the beneficial effects that:
a) simple and rapid operation, low initial template amount and covering of the whole mitochondrial length. The number of reactions is reduced, so that when the full-length mitochondria can be effectively detected, the requirement on the initial amount of a sample is lower, and the requirements on the purity and the integrity of a template are also reduced.
b) The detection sensitivity is high, low-frequency mutation can be detected, known mutation sites can be detected, and unknown mutations can also be found. The PCR reaction can amplify low-frequency mutation with low copy number so as to detect the mutation, and the PCR products of the three pairs of primers are overlapped so as to ensure that the mutation or structural variation at the position of the primers can be detected.
Since genomic DNA is used as a PCR template, which includes a large amount of nuclear genomic DNA, and the ratio of mitochondrial DNA is low, the requirement for specificity of primers is very high. Meanwhile, high-frequency mutation regions are present in the mitochondrial genome and are positioned in hypervariable sequence (HVS) I, II and III, the HVS I is positioned in 16024-16365 of the mtDNA sequence, the HVS II is positioned in 57-372 of the mtDNA sequence, the HVS III is positioned in 438-574 of the mtDNA sequence, the designed primer needs to avoid the high-frequency mutation regions, and if the primer is designed in the high-frequency mutation regions, the position of the primer is mutated and is not beneficial to detection. The experiment optimizes 8 pairs of primers, wherein the primers comprise 5 pairs of primers with non-specific amplification and 3 pairs of primers with low amplification efficiency and weak amplification bands. The 3 pairs of primers used in the invention overcome the problems of non-specific amplification and low amplification efficiency of the primers, and avoid high-frequency mutation regions. In order to ensure the consistency of the sequencing depth, the amplification products of 3 pairs of primers need to be consistent in length. In order to allow the amplification products to splice into the complete mitochondrial genome, there needs to be sufficient overlap between each of the 3 pairs of primers. If the overlap is too much and exceeds 600bp, under the condition of the same data volume, the sequence can be accurately judged as long as a certain sequencing depth exists, and the overlap region sequence belongs to repeated sequencing, which inevitably causes data waste. By combining the above considerations, it can be ensured that the position variation of the site can be detected and the specific primers can be found, so that overlap between the selected primers is 500bp in 400-.
c) No interference of other sequences such as genome and the like, and reduced false positive rate
d) Different samples are distinguished by different barcode primers, and the method is suitable for SNP and indel analysis of large-scale samples.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the result of electrophoresis of PCR products according to one embodiment of the present invention; 1.2 and 3 are products obtained by amplifying a sample by using a primer A, B, C respectively, and D15000 is a DNA marker;
FIG. 2 shows the result of electrophoresis of PCR products according to one embodiment of the present invention; 1.2 are products obtained by amplifying two samples by using a primer D, and D15000 is a DNA marker;
FIG. 3 is a diagram of primer D amplification product Agilent 2100Bioanalyzer analysis, in accordance with an embodiment of the present invention;
FIG. 4 is a graph showing the sequencing reads profile of the amplification product of primer D in one embodiment of the present invention; the abscissa represents the length of reads, the ordinate represents the number of reads, and reads near the position 17000 of the abscissa are the full-length reads of the target mitochondria;
FIG. 5 is a graph showing the sequencing reads profile of the amplification product from primer A, B, C, according to one embodiment of the present invention; the abscissa represents the reads length, the ordinate represents the number of reads, and the reads near the abscissa 6000 is the target length reads.
Detailed Description
The invention relates to a primer pair combination product, which comprises the following primer pairs:
SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6.
In some embodiments, some or all of the primers are ligated with barcode sequences to be used to distinguish between different samples during amplification.
According to one aspect of the invention, the invention also relates to a kit comprising a primer pair combination as described above.
In some embodiments, the kit further comprises one or more of a genomic DNA extraction system, PCR reaction buffer, nuclease-free water, DNA polymerase, molecular weight marker.
In some embodiments, the water is nuclease-free water, such as double distilled or deionized water.
In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragment.
In some embodiments, the kit further comprises one or more of a terminal repair enzyme, a terminal repair buffer, an amplification or ligation adaptor, a DNA ligase.
The primer pair combination product or the kit can be combined with third-generation sequencing to achieve a better technical effect.
According to an aspect of the present invention, the present invention also relates to a composition comprising an amplicon resulting from amplification of human mitochondrial genomic DNA or human whole genomic DNA with the primer pair combination as described above, or with the kit as described above.
The composition is usually in the form of a solution, and the solvent may be water or a common buffer agent.
In some embodiments, the human mitochondrial genomic DNA or human whole genomic DNA is from human fibroblasts, amniotic fluid, villi, brain tissue, muscle cells, oocytes, and platelets.
In the present specification, the sample means: a sample comprising the human mitochondrial genome. The sample is not particularly limited and may be isolated from any tissue or cell source. As cells can be from any tissue, various types of cell cultures are potential targets for use. However, fibroblasts, brain tissue, muscle cells and platelets are preferred sources of donor mitochondria. Platelets are most preferred because of their abundance in mtDNA and lack of nuclear DNA. This preference is not meant to be limiting as to the range of cell types that can be used as donor sources.
According to one aspect of the invention, the invention also relates to a mitochondrial genome sequencing method comprising:
a) preparing a composition as described above; and
b) detecting the amplification product by using a third-generation sequencing platform;
c) optionally, the sequence of the sequencing result is spliced.
In some embodiments, when performing third generation sequencing, both ends of the amplicons in the composition are ligated with barcode sequences that are used to distinguish different samples to construct a library model for third generation sequencing and are machine sequenced;
in some embodiments, the third generation sequencing is performed using a PacBio sequence, PromethION, MinION, gridios platform; the PacBio queue or PromethION platform is preferred.
According to one aspect of the invention, the invention also relates to the use of a primer pair combination as described above, a kit as described above, or a method as described above, for detecting human mitochondrial gene variation;
in some embodiments, the human mitochondrial gene variation comprises an insertion, deletion, replication, inversion, translocation, SNP;
the use may be of non-diagnostic interest, e.g. in the field of maternal inheritance or ethnic distribution, human evolution, etc. (typically the use of SNPs), or in the identification of cellular and animal models of mitochondrial defect-related diseases (if homology is high for humans).
The application may also be diagnostic:
the use of a primer pair combination as described above, or of a kit as described above, for the preparation of a diagnostic agent for a disease associated with human mitochondrial defects;
in some embodiments, the human mitochondrial defect-associated disease comprises chronic progressive extraocular muscle paralysis, Keams-Sayre syndrome, lactic acid-toxic mitochondrial encephalomyopathy, mitochondrial encephalomyopathy with hyperlactacidemia and stroke-like seizure syndrome, myoclonic epilepsy with sarcopenia red fiber syndrome, parkinson's disease, Leigh syndrome, diabetes, and mitochondrial deafness.
The present invention also relates to a method for detecting a disease associated with a human mitochondrial defect, comprising:
a) preparing a composition as described above; and
b) determining the presence of at least one mitochondrial mutation or gene associated with the disease.
The gene of a disease associated with a mitochondrial defect may be known to those skilled in the art, and examples thereof include a cytochrome c oxidase gene, a COX gene, and the like.
In addition, although the present invention is primarily directed to the diagnosis of metabolic defect diseases, it is not limited thereto. It is envisaged that for diseases with structural or topographical defects or abnormalities, the products or methods provided by the present invention may also be useful, for example, in the search for drugs that can identify specific aspects of such diseases. In addition, some particular individuals contain or are suspected of containing particularly effective or highly efficient mitochondria, and the products or methods of the invention can be used to study such mitochondria. All of these and similar applications are within the scope of the present invention, and the term "mitochondrial defect" as used herein should not be understood to exclude such embodiments.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example DNA extraction and PCR amplification
DNA extraction experimental method
Genomic DNA is extracted from Blood using a Kit, for example, the TIANAmp Blood DNA Kit Blood genomic DNA extraction Kit (DP348), according to the Kit instructions:
1. adding Buffer CL into the blood sample, mixing uniformly and centrifuging;
2. discarding the supernatant, adding Buffer GS, mixing uniformly and centrifuging;
3. adding Buffer GB and protease K, mixing uniformly, and incubating;
4. standing at room temperature, adding Buffer BD, and mixing uniformly;
5. passing through adsorption column CG2, standing at room temperature, centrifuging, and removing filtrate;
6. adding Buffer BD into an adsorption column CG2, centrifuging, and discarding the filtrate;
7. adding Buffer PW into an adsorption column CG2, centrifuging, and removing the filtrate;
8. repeating the step 7;
9. centrifuging, and air drying adsorption column CG 2;
10. buffer TB was added to the adsorption column CG2, and the mixture was allowed to stand at room temperature and centrifuged. Adding the filtrate into adsorption column CG2 again, standing at room temperature, centrifuging, and collecting the filtrate. The filtrate contained human whole genome DNA.
Second, PCR amplification
3 pairs of primers were designed in-line with primer 3.
Primer names and sequences are given in the following table:
the amplification was carried out for 11 samples synthesizing the corresponding barcode primers, see table below for the barcode sequences, highlighted in italics, underlined form:
PCR amplification experiments:
reaction procedure:
the detection result of the PCR product by 1% agarose gel electrophoresis is shown in figure 1, wherein 1, 2 and 3 are products obtained by respectively amplifying a sample by using the primer A, B, C, the size of the products is in line with the expectation, and the specificity is good.
Third, PCR product purification and sample mixing
And (3) purifying the product, namely taking 3 pairs of primer PCR products of one sample to purify 0.9 × AmpXP magnetic beads, and determining the concentration of the product after each pair of primers is purified.
Mixing samples: and determining the mixing ratio of 3 pairs of primer products according to the concentration, mixing other samples according to the ratio, and purifying the whole sample once.
EXAMPLE two three generations of sequencing Using PacBio sequence
Firstly, building a library by using a library building kit
1. Repairing the mixed library
Preparing a repairing solution:
mixing, centrifuging, and placing into a PCR thermal cycler for repair reaction under the following specific conditions:
2. joint connection
The ligation solution system was as follows:
mixing, centrifuging, and performing ligation reaction in a PCR thermal cycler under the following specific conditions:
3. purification of
Purifying with AMPure XP magnetic bead, eluting with double distilled water, and storing in refrigerator at-20 deg.c.
Second, sequencing
Third, bioinformatics analysis of sequencing data
Call ccs reads: ccs readserving is carried out by applying a pacbio platform Smrtlink5-1 analysis flow.
Splitting the barcode: the corresponding connection of the barcode sequence and the primer sequence is used as new barcode reads, and the application of the lima software is used for carrying out the barcode resolution.
3. And (3) sequence alignment: the lima results were sorted by sample and each sample sequence was aligned to the hg38 reference genome using the software graphpap.
Call SNP site: and (3) carrying out SNP locus calling on each sample by using software samtools mpieup and bcfttools, filtering the SNP locus, comparing the filtered locus with second generation data, keeping high consistency between PB data and the second generation data, and having high confidence level in terms of base accuracy and SNP locus calling rate.
EXAMPLE three Using the PromethION platform of Oxford Nanopore Technology for three generations of sequencing
First, use and build storehouse kit and build storehouse
1. Library preparation
1.1 end repair and A-tailed ligation
DNA is taken and placed on ice, NEB end repair and A tail connection reagent are added, and the mixture is mixed evenly. 20 deg.C
Incubate for 40 minutes and at 65 ℃ for 20 minutes.
1.2 magnetic bead purification
1 × AMPure beads were added to the DNA, incubated for 15min at room temperature, magnetically mounted for 5min at room temperature, and the supernatant was discarded.
Adding 80% ethanol, adsorbing with magnetic frame, discarding supernatant, and repeating once. And (5) drying at room temperature.
Ultra Pure Water was added and the elution was performed by pipetting at 37 ℃.
Standing for 5min on a magnetic frame, and sucking supernatant to obtain purified DNA.
1.3 ligation sequencing linker
Adding NEB T4DNA fast connection buffer, NEB T4DNA fast ligase and adaptor, mixing, and incubating at 20 deg.C for 20 min.
1.4 magnetic bead purification, step 1.2
2. Sample loading and sequencing
3. Analysis of letter of birth
3.1 basefilling and barcode resolution
3.2 sequence alignment: the baseharvesting barcode results were sorted by sample and each sample sequence was aligned to the hg38 reference genome using minimap 2.
3.3Call SNP site: carrying out SNP locus calling on each sample by using samtools mpieup and bcfttools, filtering the SNP locus, comparing the locus obtained by filtering with second-generation and pb data, and having high reliability in terms of base accuracy and SNP locus calling rate.
Example four
Using the method and procedure as described in example one, the only difference is that only one pair of primers (primer HMt-D) is used to amplify mitochondria, the primer sequences being:
primer sequence containing barcode sequence 5 '→ 3' on F end:
CGCTCTGTCACGTCTGTTAACCTCTACTTCTACCTACGCC；
primer sequence containing barcode sequence at R end 5 '→ 3':
GCGTGTGAGAGTCTGCGTGTGATTGAGGTGGAGTAGATTA
the agarose gel electrophoresis detection result is shown in fig. 2, wherein 1 and 2 are products obtained by amplifying two samples respectively by using the primers, the size of the products accords with the expectation, the specificity is good, and the primers can amplify the full length of mitochondria.
The distribution of fragments of the amplification product was measured using an Agilent 2100Bioanalyzer and showed a peak at about 17000bp, which coincided with the full length of mitochondria and almost no amplification of small fragments, see FIG. 3 for details. Detecting the absorbance of the amplified product by using nanodrop as 260/280-1.82; 260/230 is 2.02, also in the acceptable range.
The amplification products are subjected to third-generation sequencing by using the methods and steps described in the second and third examples, and the results show that the full-length sequence obtained by sequencing only accounts for 7% -10%, and more than 90% of the full-length sequence is not full-length sequence, and the sequencing reads distribution graph is shown in FIG. 4.
Due to the fact that the non-full-length sequence cannot be used, the sequencing data are greatly wasted. In order to ensure the accuracy of the sequencing result, the sequencing times are only increased to obtain more full-length sequences, which greatly increases the detection cost.
When the mitochondria are amplified and sequenced by using three pairs of primers by adopting the method and the steps as described in the first, second and third examples, the sequencing target sequence accounts for more than 90 percent, and the sequencing reads distribution chart is shown in figure 5. Therefore, although more primers are used, the utilization rate of reads obtained by sequencing is greatly improved, and the cost is reduced on the premise of ensuring the accuracy.
EXAMPLE V application in preparation of diagnostic agent for diseases related to human mitochondrial defects
Application in diagnosis of mitochondrial myopathy encephalopathy with lactic acidosis and stroke-like attacks (MELAS) and mitochondrial diabetes (MIDD)
The method and procedure described in examples one, two and three were used to test human peripheral blood samples, and the test results are shown in the following table:
mitochondrial genome heterogeneity mutation m3243A > G was detected in peripheral blood of the subjects, with the proportion of mutations being about 51%, which are defined pathogenic mutation sites associated with mitochondrial myopathy encephalopathy with lactic acidosis and stroke-like episodes (MELAS) and mitochondrial diabetes (MIDD).
Inputting the bam file obtained by sequencing into IGV software, comparing the bam file with a mitochondrial reference genome, and finding that the base A is mutated into G at the position m.3243, thereby further determining the result.
Second, application in diagnosis of Leber Hereditary Optic Neuropathy (LHON)
The method and procedure described in examples one, two and three were used to test human peripheral blood samples, and the test results are shown in the following table:
a mitochondrial genome homogeneity mutation m.11778G > A, which is a well-defined pathogenic mutation site associated with Leber's Hereditary Optic Neuropathy (LHON), was detected in the subject's peripheral blood.
Inputting the bam file obtained by sequencing into IGV software, comparing the bam file with a mitochondrial reference genome, and finding that the base G is mutated into A at the position m.11778, thereby further determining the result.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Beijing hope group Biotechnology Co., Ltd
<120> mitochondrial genome sequencing primer, kit and method
<160>6
<170>PatentIn version 3.3
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<400>1
agccgcagac ctcctcattc taa 23
<210>2
<211>24
<212>DNA
<213> Artificial sequence
<400>2
ttagatatgg ggagtagtgt gatt 24
<210>3
<211>27
<212>DNA
<213> Artificial sequence
<400>3
ctactaccgc attcctacta ctcaact 27
<210>4
<211>25
<212>DNA
<213> Artificial sequence
<400>4
gttgtgagtg taaattagtg cgatg 25
<210>5
<211>24
<212>DNA
<213> Artificial sequence
<400>5
ttactaccac tgacatgact ttcc 24
<210>6
<211>23
<212>DNA
<213> Artificial sequence
<400>6
ggggtgactg ttaaaagtgc ata 23
Claims (10)
1. A primer pair combination comprising the following primer pairs:
SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6.
2. The primer pair combination of claim 1 wherein some or all of the primers have a barcode sequence attached thereto for use in differentiating between different samples during amplification.
3. A kit comprising the primer-pair combination of claim 1 or 2.
4. The kit of claim 3, wherein the kit further comprises one or more of a genomic DNA extraction system, PCR reaction buffer, nuclease-free water, DNA polymerase, molecular weight marker;
preferably, the DNA polymerase is selected from any one of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase and Klenow fragment.
5. The kit of claim 3, further comprising one or more of a terminal repair enzyme, a terminal repair buffer, an amplification or ligation adaptor, and a DNA ligase.
6. A composition comprising an amplicon obtained by amplifying human mitochondrial genomic DNA or human whole genomic DNA using the primer set combination of claim 1 or 2 or the kit of any one of claims 3 to 5.
7. The composition according to claim 6, wherein the human mitochondrial genomic DNA or human whole genomic DNA is derived from human fibroblasts, amniotic fluid, villi, brain tissue, muscle cells, oocytes, platelets, and the like.
8. A method of sequencing a mitochondrial genome, comprising:
a) preparing the composition of claim 6 or 7; and
b) detecting the amplification product by using a third-generation sequencing platform;
c) optionally, splicing the sequence of the sequencing result;
preferably, when third-generation sequencing is carried out, the two ends of the amplicon in the composition are connected with barcode sequences for distinguishing different samples so as to construct a library model of third-generation sequencing and machine sequencing;
preferably, the third generation sequencing is performed using the platform PacBio sequence, PromethION, MinION, GridION.
9. Use of a primer set combination according to claim 1 or 2, or a kit according to any one of claims 3 to 5, or a method according to claim 8 for detecting human mitochondrial gene variation;
preferably, the human mitochondrial gene variation comprises an insertion, deletion, duplication, inversion, translocation, SNP.
10. Use of a primer set combination product according to claim 1 or 2, or a kit according to any one of claims 3 to 5, for the preparation of a diagnostic agent for a disease associated with human mitochondrial defects;
preferably, the human mitochondrial defect associated diseases include chronic progressive extraocular paralysis, Keams-Sayre syndrome, lactic acid-toxic mitochondrial encephalomyopathy, mitochondrial encephalomyopathy with hyperlactacidemia and stroke-like seizure syndrome, myoclonic epilepsy with broken muscle red fiber syndrome, Parkinson's disease, Leigh syndrome, diabetes and mitochondrial deafness.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811610869.2A CN111378732B (en) | 2018-12-27 | 2018-12-27 | Mitochondrial genome sequencing primer, kit and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811610869.2A CN111378732B (en) | 2018-12-27 | 2018-12-27 | Mitochondrial genome sequencing primer, kit and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111378732A true CN111378732A (en) | 2020-07-07 |
| CN111378732B CN111378732B (en) | 2022-08-23 |
Family
ID=71216372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811610869.2A Active CN111378732B (en) | 2018-12-27 | 2018-12-27 | Mitochondrial genome sequencing primer, kit and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111378732B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676280A (en) * | 2020-07-29 | 2020-09-18 | 中国人民解放军军事科学院军事医学研究院 | Primer combination for mitochondrial hypervariable region composite amplification based on high-throughput sequencing technology, sequencing method and application of primer combination |
| CN116875718A (en) * | 2023-09-07 | 2023-10-13 | 南京诺因生物科技有限公司 | Construction method and kit of mycobacterium tuberculosis drug-resistant mutation site sequencing library |
| CN117070511A (en) * | 2023-08-16 | 2023-11-17 | 广州卿泽生物科技有限公司 | Primer group for amplifying mitochondrial whole genome and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010058514A (en) * | 1999-12-30 | 2001-07-06 | 이홍규 | PCR primers for use in analyzing mitochondrial DNA in human and a quantitative analysis kit using them |
| US20040191769A1 (en) * | 2001-07-24 | 2004-09-30 | Transgenomic, Inc. | Methods, compositions, and kits for mutation detection in mitochondrial DNA |
| CN103173441A (en) * | 2013-02-05 | 2013-06-26 | 深圳华大基因研究院 | Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid) |
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN106755456A (en) * | 2017-01-09 | 2017-05-31 | 北京圣谷智汇医学检验所有限公司 | For the primer combination of mitochondria full-length genome detection and kit |
| CN108220393A (en) * | 2018-01-26 | 2018-06-29 | 广州海思医疗科技有限公司 | A kind of method of high-throughput quick detection mitochondrial gene mutation or missing |
| CN108949942A (en) * | 2018-07-17 | 2018-12-07 | 浙江大学 | A kind of mitochondria genome sequencing method based on high-flux sequence |
-
2018
- 2018-12-27 CN CN201811610869.2A patent/CN111378732B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010058514A (en) * | 1999-12-30 | 2001-07-06 | 이홍규 | PCR primers for use in analyzing mitochondrial DNA in human and a quantitative analysis kit using them |
| US20040191769A1 (en) * | 2001-07-24 | 2004-09-30 | Transgenomic, Inc. | Methods, compositions, and kits for mutation detection in mitochondrial DNA |
| CN103173441A (en) * | 2013-02-05 | 2013-06-26 | 深圳华大基因研究院 | Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid) |
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN106755456A (en) * | 2017-01-09 | 2017-05-31 | 北京圣谷智汇医学检验所有限公司 | For the primer combination of mitochondria full-length genome detection and kit |
| CN108220393A (en) * | 2018-01-26 | 2018-06-29 | 广州海思医疗科技有限公司 | A kind of method of high-throughput quick detection mitochondrial gene mutation or missing |
| CN108949942A (en) * | 2018-07-17 | 2018-12-07 | 浙江大学 | A kind of mitochondria genome sequencing method based on high-flux sequence |
Non-Patent Citations (1)
| Title |
|---|
| 李天杰等: "动物线粒体基因组测序方法的研究进展", 《天津医药》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676280A (en) * | 2020-07-29 | 2020-09-18 | 中国人民解放军军事科学院军事医学研究院 | Primer combination for mitochondrial hypervariable region composite amplification based on high-throughput sequencing technology, sequencing method and application of primer combination |
| CN117070511A (en) * | 2023-08-16 | 2023-11-17 | 广州卿泽生物科技有限公司 | Primer group for amplifying mitochondrial whole genome and application thereof |
| CN117070511B (en) * | 2023-08-16 | 2024-02-02 | 广州卿泽生物科技有限公司 | Primer group for amplifying human mitochondrial whole genome and application thereof |
| CN116875718A (en) * | 2023-09-07 | 2023-10-13 | 南京诺因生物科技有限公司 | Construction method and kit of mycobacterium tuberculosis drug-resistant mutation site sequencing library |
| CN116875718B (en) * | 2023-09-07 | 2023-12-01 | 南京诺因生物科技有限公司 | Construction method and kit of mycobacterium tuberculosis drug-resistant mutation site sequencing library |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111378732B (en) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220228197A1 (en) | Method for determining copy number variations | |
| Zhou et al. | Deep sequencing of the MHC region in the Chinese population contributes to studies of complex disease | |
| EP3169803B1 (en) | Non-invasive prenatal diagnosis of fetal genetic condition using cellular dna and cell free dna | |
| KR101974492B1 (en) | Method for determining the presence or absence of different aneuploidies in a sample | |
| JP6867045B2 (en) | Single molecule sequencing of plasma DNA | |
| CN111378732B (en) | Mitochondrial genome sequencing primer, kit and method | |
| GB2484764A (en) | Foetal chromosomal aneuploidy diagnosis by sequence tag detection and normalisation | |
| TW201639967A (en) | Method, kit, device and system of detecting fetal genetic information | |
| AU2011365507A1 (en) | Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies | |
| CN113699231B (en) | Alpha-thalassemia-related gene detection kit | |
| CN112442530B (en) | Method for detecting CAH related true and false gene | |
| He et al. | Applications of Oxford nanopore sequencing in Schizosaccharomyces pombe | |
| CN111808937A (en) | Fut 1508 dupT allele of Bombay blood group and detection method and application thereof | |
| CN111020710A (en) | ctDNA high-throughput detection of hematopoietic and lymphoid tissue tumors | |
| CN110863041A (en) | Mutant gene related to thalassemia and detection reagent and application thereof | |
| WO2019061199A1 (en) | A primer combination for performing simultaneous amplification of target region and whole genome, gene amplification method and application thereof | |
| CN111378738A (en) | Detection primer, kit and method for HTT and JPH3 genes | |
| CN110819710A (en) | High-throughput sequencing detection of myeloid tumors | |
| CN102199658B (en) | Gene detection chip of common mutations of diphosphate glucuronyl transferase 1A and use method thereof | |
| CN116121383A (en) | Composition for clinical diagnosis and treatment of hematological malignant tumor and application thereof | |
| CN117757912A (en) | Primer composition, kit and sequencing method for PKD1 and PKD2 genes | |
| Shooter | Application of Next Generation Sequencing In the Characterisation of Variants Causing Haemoglobinopathies | |
| CN111118113A (en) | High throughput sequencing assay for hemophagocytic syndrome | |
| CN117757911A (en) | Probe composition, kit and sequencing method for nephrotic gene | |
| CN111676280A (en) | Primer combination for mitochondrial hypervariable region composite amplification based on high-throughput sequencing technology, sequencing method and application of primer combination |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |