CN111378732B - Mitochondrial genome sequencing primer, kit and method - Google Patents
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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, determined the complete sequence of the human mitochondrial genome in 1981, 16569bp, compared to the nuclear DNA, mtDNA has its own advantages as a "molecular clock" for phylogenetic generation of organisms: the mutation rate is high and is about 10 times of that of nuclear DNA, so that a large amount of nucleotide substitutions can be quickly accumulated even among species which diverge in the near period, and comparative analysis can be carried out; ② mtDNA is maternally inherited and no DNA recombination occurs, therefore, individuals with the same mtDNA sequence must be from a common female ancestor human mitochondrial DNA (mtDNA), containing a total of 37 genes, 22 of which encode transfer ribonucleic acids (tRNA), 2 of which encode ribosomal ribonucleic acids (12S and 16S rRNA), and 13 of which encode polypeptides. Mitochondria, a two-layer membrane-coated organelle present in most cells, are structures that produce energy in cells, and are the major site of aerobic respiration of cells, and are called "power house". Mitochondrial DNA mutations are associated with many human diseases, which occur due to abnormal mitochondrial function and most of which affect energy-demanding organs such as muscle, brain, heart, so it is important to detect mitochondrial gene mutations, and since analysis of mitochondrial genome requires analysis of 37 genes, prenatal mitochondrial genome analysis of mothers has great significance for birth health babies because mitochondrial genes belong to maternal inheritance. For patients with suspected mitochondrial disease, the ideal genetic diagnostic method is to find the relevant gene mutations that result in defects in mitochondrial structure and function. These gene mutations may occur in mtDNA or nuclear genes, and the mitochondrial inheritance pattern may be autosomal recessive inheritance, X-linked inheritance, maternal inheritance, or some may be new mutations. As mitochondrial diseases relate to a plurality of genes, only a few common mitochondrial gene loci can be selected for mutation and deletion screening in clinic at present, the positive rate is low, and most patients cannot obtain accurate etiology diagnosis.
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 mutation or structural variation can also be detected.
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 human mitochondrial gene variation and preparing diagnostic agent for diseases related to human mitochondrial defect.
Compared with the prior art, the invention has the beneficial effects that:
a) simple and fast operation, low initial template amount and covering of the whole mitochondria. 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 embodiments or the prior art descriptions 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 of 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 have a barcode sequence attached to them for use in differentiating 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. Various types of cell cultures are potentially useful as the cells may be from any tissue. However, fibroblasts, brain tissue, muscle cells and platelets are preferred sources of donor mitochondria. Platelets are most preferred because they are rich in mtDNA and lack 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 application of the primer pair combination product or the kit in preparing a diagnostic agent for diseases related to 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 related to a disease associated with a mitochondrial defect may be a gene 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 the products or methods provided by the present invention may also be useful for diseases with structural or topographical defects or deformities, for example, in the search for drugs which can determine 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, 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) product purification: taking 3 pairs of primer PCR products of a sample to carry out 0.9 XAmpXP magnetic bead purification, and determining the concentration of the purified product of each pair of primers.
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 reads loading was performed using the pacbio platform Smrtlink5-1 analytical protocol.
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-tail 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 incubate for 20 minutes at 65 ℃.
1.2 magnetic bead purification
Add 1 × AMPure beads to DNA, incubate for 15min at room temperature, adsorb for 5min on a magnetic rack at room temperature, and aspirate the supernatant.
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 5 '→ 3' from R terminal:
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 amplified products was measured by Agilent 2100Bioanalyzer and showed the highest peak at about 17000bp, which was consistent 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 the steps described in the second and third examples, and the results show that the sequencing only comprises 7% -10% of full-length sequences, more than 90% of the full-length sequences are not full-length sequences, and the sequencing reads distribution graph is shown in FIG. 4.
Due to the fact that the non-full-length sequence cannot be used, great waste of sequencing data is caused. 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 the steps as described in the first, second and third examples are 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.
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Claims (7)
1. The primer pair combination for detecting the mitochondrial genome comprises 3 groups of amplification primer pairs, which are respectively as follows:
the amino acid sequence of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6.
2. The primer pair combination according to claim 1, wherein some or all of the primers of the amplification primer pair are linked with a barcode sequence for use in differentiating different samples upon 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; 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 an end-repair enzyme, an end-repair buffer, an amplification or ligation adaptor, and a DNA ligase.
6. A method of sequencing a mitochondrial genome, comprising:
a) amplifying human mitochondrial genomic DNA to obtain an amplification product by using the primer pair combination of claim 1 or 2 or the kit of any one of claims 3 to 5; and
b) detecting the amplification product by using a third-generation sequencing platform;
c) splicing sequences of sequencing results;
when third-generation sequencing is carried out, connecting the two ends of the amplification product with barcode sequences for distinguishing different samples to construct a library model of third-generation sequencing and carrying out computer sequencing; the third generation sequencing is carried out by any platform of PacBio sequence, PromethION, MinION and GridION.
7. Use of the primer pair combination of claim 1 or 2 or the kit of any one of claims 3 to 5 for the preparation of a diagnostic agent for a disease associated with human mitochondrial defects; the human mitochondrial defect related diseases are mitochondrial myopathy encephalopathy with lactic acidosis and stroke-like attack, mitochondrial diabetes or Leber hereditary optic neuropathy.
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