CN113981054A - Human mitochondria whole genome short segment imbricate type amplification detection kit - Google Patents
Human mitochondria whole genome short segment imbricate type amplification detection kit Download PDFInfo
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Abstract
The invention discloses a primer composition. The invention also discloses a simple, economic, accurate and automatic human mitochondrial whole genome short segment imbricated amplification detection kit based on a high-throughput sequencing technology, and forensics adopt the kit to carry out mitochondrial whole genome detection, so that the identification force of difficult old samples and highly degraded samples can be effectively improved.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a human mitochondrial whole genome short segment imbricated amplification detection kit.
Background
In the middle of the 20 th century, researchers first isolated and identified the extra-nuclear mitochondrial genome (mtGenome). The human mitochondrial genome sequence, first published in 1981 and modified in 2001, is called the revised Cambridge reference sequence (rCRS), a circular genetic material 16569bp in length, functionally divided into a Control Region (CR) 1122bp in length and a coding region (CodR) 15447bp in length, wherein 3 hypervariable regions (HVS) are included in the control region and have good polymorphisms. Mitochondrial DNA plays an important role in the fields of human evolution of origin and forensic identity recognition. On one hand, since the characteristics of mitochondrial DNA are maternally inherited and recombination does not occur, the haplotype can be stably inherited to offspring, so that the genetic characteristics can be utilized to carry out human origin evolution research, such as deducing origin time and place, migration process and the like; on the other hand, compared with nuclear DNA, the mitochondrial DNA has the characteristics of high copy number, stable annular structure, double-layer membrane protection and the like, so that the mitochondrial DNA can still be stored to the maximum extent under severe conditions (such as high temperature, humidity and the like), and the mitochondrial DNA has unique advantages when being applied to forensic identity recognition, and particularly aims at the identification of difficult and old detection materials (such as ancient remains, ossified bodies and the like) and highly-degraded detection materials (such as bodies or parts of human tissues in large-scale disaster sites of fire, explosion, air crash, marine accident, terrorist attack and the like).
Initially, forensic geneticists established a detection system for mitochondrial genomes based on the first generation sequencing (Sanger sequencing) technology. First, by performing PCR on mitochondrial DNA for long-fragment amplification, the fragments partially overlap to cover the entire genome. Next, short fragments are sequenced within each long fragment, with the fragments partially overlapping, covering the corresponding long fragments. Finally, the mixture is separated by electrophoresis on a sequencer, and the fluorescence signal is detected for sequencing. However, the application of Sanger sequencing to detect mitochondrial genomes is not well promoted because of the following (1) high requirements for test materials: a large amount of test materials with good quality are needed to ensure that long-fragment products meeting the sequencing requirement are amplified and are not suitable for analysis of difficult old test materials and highly degraded test materials; (2) the cost is higher, and the flux is lower: the optimal read length of Sanger sequencing is about 600bp, each sequencing reaction can only detect a single short fragment, at least 60 reactions are required to be simultaneously carried out to cover a mitochondrial genome, and only 1-2 samples can be detected at one time by taking an ABI 3730xl sequencer with the maximum flux of 96 reactions as an example; (3) the data analysis is complex: in order to obtain mitochondrial genome information, each short fragment sequence needs to be spliced manually, and when a plurality of samples are analyzed simultaneously, splicing errors caused by human reasons often occur; (4) the sequencing effect of the special region is poor, so that the genetic information of the genome is lost: such as multimers (homopolymers), insertions/deletions (indels), heterogeneity (heterozygosity), etc., are difficult to interpret, and usually neglect to classify this partial region. In general, the overall process of Sanger sequencing mitochondrial genome detection system is time-consuming, labor-consuming and expensive, thus limiting its application in the field of forensic identification.
In 2005, the academic paper published in nature journal by 454 life science corporation marked the beginning of the Next Generation Sequencing (NGS) technology — the innovation of high throughput sequencing (MPS), which was a subversive change to the first generation sequencing technology. With the development and maturation of MPS technology, mitochondrial genome sequencing is increasingly being applied in the field of forensic genetics. Emerging technology is from scientific research to practical application, and meets the requirements of the prior field, and the detection method is particularly remarkable. Firstly, the method for constructing the detection system, particularly the method for capturing the target fragment by PCR amplification (such as a long-fragment amplification method, a medium-fragment amplification method or a short-fragment amplification method), is the key for embodying the application range and detection capability of the system to different test materials. Currently, the majority of the methods used are long-fragment amplification methods, which are very suitable for high-quality and high-copy samples (such as peripheral blood and saliva), but are not suitable for analysis of difficult old samples (such as old blood stains) and highly degraded samples (such as hair). Secondly, the sequencing platform is selected, a large number of special regions (such as aggregates, heterogeneity and the like) are distributed in the mitochondrial genome, and a platform with a stable sequencing principle and sufficient sequencing depth is selected, so that more complete and accurate genetic information can be obtained as far as possible. Although foreign sequencing companies have successively introduced kits for detecting mitochondrial genomes by amplifying short fragments, the existing sequencing platforms need to be upgraded again for payment and the detection is expensive. Thirdly, in the data analysis method, the data generated by the MPS platform belongs to a big data class, and the intervention of the biological information related analysis tool is often required. The data which can be simply, repeatedly and massively analyzed is processed by a computer, meanwhile, manpower resources are released to carry out key analysis on difficult and complicated data, human errors are reduced, and the complementation of human-computer advantages is formed. However, the MPS platform is more concerned with obtaining the original data, and is less concerned with the secondary analysis of the data in practical applications.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to overcome the drawbacks of the prior art and to provide a short-segment imbricated amplification primer composition for capturing the whole genome of human mitochondria.
The invention also aims to provide a detection kit for simply, economically, accurately and automatically carrying out large-scale parallel determination of human mitochondrial whole genome based on a high-throughput sequencing technology, and forensics can effectively improve the identification force of difficult old samples and highly degraded samples by carrying out mitochondrial whole genome detection by adopting the kit.
To achieve these objects and other advantages in accordance with the present invention, there is provided a primer composition comprising:
the primer composition I comprises a primer 1-a primer 65, the nucleotide sequence of the primer 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the primer 2 is shown as SEQ ID NO. 2, the nucleotide sequence of the primer 3 is shown as SEQ ID NO. 3, the nucleotide sequence of the primer 4 is shown as SEQ ID NO. 4, the nucleotide sequence of the primer 5 is shown as SEQ ID NO. 5, the nucleotide sequence of the primer 6 is shown as SEQ ID NO. 6, the nucleotide sequence of the primer 7 is shown as SEQ ID NO. 7, the nucleotide sequence of the primer 8 is shown as SEQ ID NO. 8, the nucleotide sequence of the primer 9 is shown as SEQ ID NO. 9, the nucleotide sequence of the primer 10 is shown as SEQ ID NO. 10, the nucleotide sequence of the primer 11 is shown as SEQ ID NO. 11, and the nucleotide sequence of the primer 12 is shown as SEQ ID NO. 12, The nucleotide sequence of the primer 13 is shown as SEQ ID NO. 13, the nucleotide sequence of the primer 14 is shown as SEQ ID NO. 14, the nucleotide sequence of the primer 15 is shown as SEQ ID NO. 15, the nucleotide sequence of the primer 16 is shown as SEQ ID NO. 16, the nucleotide sequence of the primer 17 is shown as SEQ ID NO. 17, the nucleotide sequence of the primer 18 is shown as SEQ ID NO. 18, the nucleotide sequence of the primer 19 is shown as SEQ ID NO. 19, the nucleotide sequence of the primer 20 is shown as SEQ ID NO. 20, the nucleotide sequence of the primer 21 is shown as SEQ ID NO. 21, the nucleotide sequence of the primer 22 is shown as SEQ ID NO. 22, the nucleotide sequence of the primer 23 is shown as SEQ ID NO. 23, the nucleotide sequence of the primer 24 is shown as SEQ ID NO. 24, and the nucleotide sequence of the primer 25 is shown as SEQ ID NO. 25, The nucleotide sequence of the primer 26 is shown as SEQ ID NO. 26, the nucleotide sequence of the primer 27 is shown as SEQ ID NO. 27, the nucleotide sequence of the primer 28 is shown as SEQ ID NO. 28, the nucleotide sequence of the primer 29 is shown as SEQ ID NO. 29, the nucleotide sequence of the primer 30 is shown as SEQ ID NO. 30, the nucleotide sequence of the primer 31 is shown as SEQ ID NO. 31, the nucleotide sequence of the primer 32 is shown as SEQ ID NO. 32, the nucleotide sequence of the primer 33 is shown as SEQ ID NO. 33, the nucleotide sequence of the primer 34 is shown as SEQ ID NO. 34, the nucleotide sequence of the primer 35 is shown as SEQ ID NO. 35, the nucleotide sequence of the primer 36 is shown as SEQ ID NO. 36, the nucleotide sequence of the primer 37 is shown as SEQ ID NO. 37, and the nucleotide sequence of the primer 38 is shown as SEQ ID NO. 38, The nucleotide sequence of the primer 39 is shown as SEQ ID NO. 39, the nucleotide sequence of the primer 40 is shown as SEQ ID NO. 40, the nucleotide sequence of the primer 41 is shown as SEQ ID NO. 41, the nucleotide sequence of the primer 42 is shown as SEQ ID NO. 42, the nucleotide sequence of the primer 43 is shown as SEQ ID NO. 43, the nucleotide sequence of the primer 44 is shown as SEQ ID NO. 44, the nucleotide sequence of the primer 45 is shown as SEQ ID NO. 45, the nucleotide sequence of the primer 46 is shown as SEQ ID NO. 46, the nucleotide sequence of the primer 47 is shown as SEQ ID NO. 47, the nucleotide sequence of the primer 48 is shown as SEQ ID NO. 48, the nucleotide sequence of the primer 49 is shown as SEQ ID NO. 49, the nucleotide sequence of the primer 50 is shown as SEQ ID NO. 50, and the nucleotide sequence of the primer 51 is shown as SEQ ID NO. 51, The nucleotide sequence of the primer 52 is shown as SEQ ID NO:52 and the nucleotide sequence of the primer 53 is shown as SEQ ID NO:53, and the nucleotide sequence of the primer 54 is shown as SEQ ID NO:54 and the nucleotide sequence of the primer 55 is shown as SEQ ID NO:55 and the nucleotide sequence of the primer 56 is shown as SEQ ID NO:56 and the nucleotide sequence of the primer 57 is shown as SEQ ID NO:57 and the nucleotide sequence of the primer 58 is shown as SEQ ID NO:58 and the nucleotide sequence of the primer 59 is shown as SEQ ID NO:59, and the nucleotide sequence of the primer 60 is shown as SEQ ID NO:60 and the nucleotide sequence of the primer 61 is shown as SEQ ID NO:61 and the nucleotide sequence of primer 62 is shown as SEQ ID NO:62 and the nucleotide sequence of the primer 63 is shown as SEQ ID NO:63 and the nucleotide sequence of the primer 64 is shown as SEQ ID NO:64 and the nucleotide sequence of primer 65 is shown in SEQ ID NO: 65;
the primer composition II comprises a primer 66-a primer 130, the nucleotide sequence of the primer 66 is shown as SEQ ID NO 66, the nucleotide sequence of the primer 67 is shown as SEQ ID NO 67, the nucleotide sequence of the primer 68 is shown as SEQ ID NO 68, the nucleotide sequence of the primer 69 is shown as SEQ ID NO 69, the nucleotide sequence of the primer 70 is shown as SEQ ID NO 70, the nucleotide sequence of the primer 71 is shown as SEQ ID NO 71, the nucleotide sequence of the primer 72 is shown as SEQ ID NO 72, the nucleotide sequence of the primer 73 is shown as SEQ ID NO 73, the nucleotide sequence of the primer 74 is shown as SEQ ID NO 74, the nucleotide sequence of the primer 75 is shown as SEQ ID NO 75, the nucleotide sequence of the primer 76 is shown as SEQ ID NO 76, and the nucleotide sequence of the primer 77 is shown as SEQ ID NO 77, The nucleotide sequence of the primer 78 is shown as SEQ ID NO. 78, the nucleotide sequence of the primer 79 is shown as SEQ ID NO. 79, the nucleotide sequence of the primer 80 is shown as SEQ ID NO. 80, the nucleotide sequence of the primer 81 is shown as SEQ ID NO. 81, the nucleotide sequence of the primer 82 is shown as SEQ ID NO. 82, the nucleotide sequence of the primer 83 is shown as SEQ ID NO. 83, the nucleotide sequence of the primer 84 is shown as SEQ ID NO. 84, the nucleotide sequence of the primer 85 is shown as SEQ ID NO. 85, the nucleotide sequence of the primer 86 is shown as SEQ ID NO. 86, the nucleotide sequence of the primer 87 is shown as SEQ ID NO. 87, the nucleotide sequence of the primer 88 is shown as SEQ ID NO. 88, the nucleotide sequence of the primer 89 is shown as SEQ ID NO. 89, and the nucleotide sequence of the primer 90 is shown as SEQ ID NO. 90, The nucleotide sequence of the primer 91 is shown as SEQ ID NO. 91, the nucleotide sequence of the primer 92 is shown as SEQ ID NO. 92, the nucleotide sequence of the primer 93 is shown as SEQ ID NO. 93, the nucleotide sequence of the primer 94 is shown as SEQ ID NO. 94, the nucleotide sequence of the primer 95 is shown as SEQ ID NO. 95, the nucleotide sequence of the primer 96 is shown as SEQ ID NO. 96, the nucleotide sequence of the primer 97 is shown as SEQ ID NO. 97, the nucleotide sequence of the primer 98 is shown as SEQ ID NO. 98, the nucleotide sequence of the primer 99 is shown as SEQ ID NO. 99, the nucleotide sequence of the primer 100 is shown as SEQ ID NO. 100, the nucleotide sequence of the primer 101 is shown as SEQ ID NO. 101, the nucleotide sequence of the primer 102 is shown as SEQ ID NO. 102, and the nucleotide sequence of the primer 103 is shown as SEQ ID NO. 103, the nucleotide sequence of the primer 100 is shown as SEQ ID NO. 100, the nucleotide sequence of the primer 101 is shown as SEQ ID NO. 101, the nucleotide sequence of the primer 102 is shown as SEQ ID NO. 102, The nucleotide sequence of the primer 104 is shown as SEQ ID NO. 104, the nucleotide sequence of the primer 105 is shown as SEQ ID NO. 105, the nucleotide sequence of the primer 106 is shown as SEQ ID NO. 106, the nucleotide sequence of the primer 107 is shown as SEQ ID NO. 107, the nucleotide sequence of the primer 108 is shown as SEQ ID NO. 108, the nucleotide sequence of the primer 109 is shown as SEQ ID NO. 109, the nucleotide sequence of the primer 110 is shown as SEQ ID NO. 110, the nucleotide sequence of the primer 111 is shown as SEQ ID NO. 111, the nucleotide sequence of the primer 112 is shown as SEQ ID NO. 112, the nucleotide sequence of the primer 113 is shown as SEQ ID NO. 113, the nucleotide sequence of the primer 114 is shown as SEQ ID NO. 114, the nucleotide sequence of the primer 115 is shown as SEQ ID NO. 115, and the nucleotide sequence of the primer 116 is shown as SEQ ID NO. 116, The nucleotide sequence of the primer 117 is shown as SEQ ID NO. 117, the nucleotide sequence of the primer 118 is shown as SEQ ID NO. 118, the nucleotide sequence of the primer 119 is shown as SEQ ID NO. 119, the nucleotide sequence of the primer 120 is shown as SEQ ID NO. 120, the nucleotide sequence of the primer 121 is shown as SEQ ID NO. 121, the nucleotide sequence of the primer 122 is shown as SEQ ID NO. 122, the nucleotide sequence of the primer 123 is shown as SEQ ID NO. 123, the nucleotide sequence of the primer 124 is shown as SEQ ID NO. 124, the nucleotide sequence of the primer 125 is shown as SEQ ID NO. 125, the nucleotide sequence of the primer 126 is shown as SEQ ID NO. 126, the nucleotide sequence of the primer 127 is shown as SEQ ID NO. 127, the nucleotide sequence of the primer 128 is shown as SEQ ID NO. 128, and the nucleotide sequence of the primer 129 is shown as SEQ ID NO. 129, The nucleotide sequence of the primer 130 is shown as SEQ ID NO: 130.
A human mitochondrial whole genome short segment imbricate amplification detection kit is characterized by comprising the primer composition.
Preferably, the reaction system is:
reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s, and 40 cycles; delay at 72 ℃ for 10min, end, 4 ℃.
Preferably, the concentration of the sample genomic DNA is 10-200 pg/μ l.
The invention at least comprises the following beneficial effects:
1. the short-segment imbricated amplification primer composition provided by the invention can be used for capturing the human mitochondrial whole genome.
2. The invention provides a detection kit for simply, economically, accurately and automatically carrying out large-scale parallel determination of human mitochondrial whole genome based on a high-throughput sequencing technology, and forensics adopt the kit to carry out mitochondrial whole genome detection, so that the identification force of difficult old test materials and highly degraded test materials can be effectively improved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a short segment imbricated human mitochondrial whole genome primer composition for amplification;
FIG. 2 is a graph of 1 example of mitochondrial whole genome high throughput sequencing results of 3 examples of old blood trace samples analyzed by a Manifest file (A: old blood trace sample 1; B: old blood trace sample 1; C: old blood trace sample 1);
FIG. 3 is a graph of 1 example of the results of high throughput sequencing of 1ng of 9947A mitochondrial genome-wide using BED files;
FIG. 4 is a graph comparing the identity of 1 example of 50pg standard DNA 9947A mitochondrial whole genome high throughput sequencing and 1ng of standard DNA 9947A mitochondrial Sanger sequencing (A: high throughput sequencing; B: Sanger sequencing).
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
A human mitochondria whole genome short segment imbricate type amplification detection kit based on high-throughput sequencing technology comprises the following steps:
1. primer composition: the invention provides a primer group 1 and a primer group 2 compound for amplifying mitochondrial genomes, on one hand, corresponding degenerate primers (such as 30aF-30aR, 30bF and 30bR) are designed according to the polymorphism characteristics of Chinese population, and the success rate of primer combination is ensured; on the other hand, it is necessary to amplify fragments with overlapping portions to ensure coverage of the entire mitochondrial genome (FIG. 1). The sequence of the primer composition 1 is SEQ ID NO: 1-SEQ ID NO:65 are shown in table 1. The sequence of the primer composition 2 is SEQ ID NO: 66-SEQ ID NO:130 are shown in table 2.
Table 1: primer set 1 composition
Degenerate primer
Table 2: primer set 2 composition
Degenerate primer
2. Short segment imbricate type amplification detection kit: the invention also provides a detection kit based on high-throughput sequencing, which contains the primer combination 1 and the primer combination 2 and is used for amplifying target fragments (Mix1 and Mix2) of the primer combination 1 and the primer combination 2.
(1) Reaction system: the total volume was 50. mu.l, the composition was as follows:
preferably, the genomic DNA of the sample is collected from human biological samples such as blood (plaque), saliva (plaque), semen (plaque), hair, tissue and the like; alternatively, the sample genomic DNA may be standard DNA, such as female standard DNA 9947A, male standard DNA 2800M, and the like.
(2) Reaction conditions are as follows:
3. library construction and high throughput sequencing
(1) The amplification products Mix1 and Mix2 from step 2 were purified and quantified, diluted to 0.2 ng/. mu.l, and mixed in equal proportions (1:1) to Mix as Mix.
Preferably, the purification in the step 3(1) adopts
The magnetic beads are used for completing the quantification in the step 3(1), and a Qubit or qPCR mode can be adopted for the quantification.
(2) And (3) establishing a mitochondrial genome library comprising a plurality of samples by connecting the adapters and the tags by taking the Mix in the step 3(1) as an object.
(3) And (3) sequencing the library prepared in the step (3) and (2) on a high-throughput sequencing platform. Preparing a monoclonal template on the chip by bridge PCR or water-in-oil PCR; and (3) adding a substrate nucleotide with a mark by utilizing a strategy of synthesizing and sequencing, combining the substrate nucleotide with a template in a sequencing library under the action of a sequencing enzyme, collecting a fluorescent signal or an ion signal generated in the process and determining the base information of a detected site, and repeating the sequencing process after the last round of sequencing is completed to realize high-throughput sequencing. Preferably, the high throughput sequencing platform in step 3(3) comprises MiSeq FGx or Ion Torrent.
4. Data analysis
And (3) generating an original file in real time by means of a server and matched software, and performing secondary analysis on the sequencing file obtained in the step (3) by adopting a self-written Manifest file and a self-written BED file. Preferably, the Manifest file in the step 4 is used for performing result analysis on sequencing information in a FASTQ format (FIG. 2); the BED file was the result analysis for BAM format sequencing information (fig. 3).
The Manifest file format is as follows:
the BED file format is as follows:
example 1
In this example, the kit is used to detect mitochondrial whole genome of 3 old blood trace samples.
Old blood trace sample of unrelated individuals: supplied by volunteers and stored in the laboratory for more than 10 years.
Extracting sample DNA: obtaining genome DNA by a magnetic bead method or other extraction methods.
Sample DNA quantification: real-time fluorescent quantitative PCR method or other quantitative genome DNA method is adopted.
Haplotype grouping: EMMA or HaploGrep2 software was used.
And (3) sample detection results: the kit provided by the invention is used for detecting the whole genome mutation information and haplotype of 3 cases of old blood mark samples (the quantitative results are 246pg, 280pg and 120pg respectively) as shown in the following table:
the result of the embodiment shows that 3 cases of old blood trace samples all detect the mitochondrial whole genome sequence information, which cannot be known by the electrophoretic mitochondrial SNP detection technology. For example, the old blood mark sample 1 is detected by an electrophoresis kit to obtain only 16 mutations, and the haplotype is D4 e; the results of the kit and the high-throughput sequencing are shown in FIG. 2, 38 mutations can be obtained, the mutation is improved by 137% compared with the former, and the haplotype is D4e3, which is more accurate compared with the former. Similarly, the number of mutations detected by the electrophoresis kit in the old blood trace samples 2 and 3 is small, and haplotyping errors occur. The high-throughput sequencing technology obviously provides a more scientific and comprehensive detection means for mitochondrial whole genome analysis. The obtaining of the sequencing result provides more genetic evidence for common maternal identification cases and complex difficult cases.
Example 2
In this example, the kit is used to detect trace amount of detection material (taking 50pg of DNA 9947A as an example), and the consistency of the detection material and the result of the classical Sanger sequencing is compared.
Sample DNA quantification: real-time fluorescent quantitative PCR method or other quantitative genome DNA method is adopted.
The sample detection results are shown in fig. 4: 93G,195C,214G,263G,309.1C,309.2C,315.1C,750G,1438G,4135C,4769G,7645C,7861Y,8448C,8860G,9315C,13572C,13759a,15326G,16311C, 16519C; the haplotype group is H11b 1.
The results of this example show that 50pg of the standard DNA 9947A can detect the mitochondrial whole genome sequence information, and the mutation information is completely consistent with the Sanger sequencing result of 1ng of the standard DNA 9947A. Although the amplification detection of the standard was performed using the same primers in the high-throughput sequencing and the Sanger sequencing, the DNA starting template amount is only 1/20 of the Sanger sequencing method, and the high-throughput sequencing requires only 2 PCR reactions, while the Sanger sequencing requires 62 PCR reactions. The embodiment provides a simpler, economic, accurate and automatic detection means for mitochondrial whole genome sequencing, and is more suitable for detection of trace detection materials.
Example 3
This example is the detection of mitochondrial genome mutation heterogeneity using the kit provided by the present invention.
As shown in the results of fig. 4, the heterogeneity of 7861 th base mutation in the mitochondrial genome can be directly interpreted as 7861Y by the high throughput sequencing method of the present invention, whereas the base is misread as absent mutation (T7861) by Sanger sequencing method, which requires manual interpretation for correction.
Example 4
This example is an evaluation of the sensitivity of detection of a mitochondrial genome of a standard DNA 9947 using the kit provided by the present invention.
Sample DNA quantification: real-time fluorescent quantitative PCR method or other quantitative genome DNA method is adopted.
And (3) sample detection results: the kit provided by the invention is used for detecting the whole genome mutation information and haplotype of mitochondria of the standard substance DNA 9947A with different initial template amounts as shown in the following table:
the result of the embodiment shows that the DNA 9947A standard can detect the sequence information of the whole mitochondrial genome under the condition that the initial template amount is 50-1000 pg; at an initial template amount of 10pg, a deletion occurred between base 309 and base 315. However, this does not affect the correct partitioning of the single group. The embodiment provides an optimal detection range for mitochondrial whole genome sequencing, has the sensitivity as low as 50pg, and is suitable for detection of a small-volume micro-detection material.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
<110> China criminal police college
<120> human mitochondria whole genome short segment imbricate amplification detection kit
<160>130
<210>1
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>1
caaccaaacc ccaaagacac 20
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>2
ccggcttcta ttgacttggg 20
<210>3
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>3
aaaactgctc gccagaacac 20
<210>4
<211>18
<212>DNA
<213> Artificial sequence
<220>
<400>4
gtgtgtacgc gcttcagg 18
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>5
tggacgaacc agagtgtagc 20
<210>6
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>6
tgctacatag acgggtgtgc 20
<210>7
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>7
gcccacagaa ccctctaaat c 21
<210>8
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>8
cctgtgttgg gttgacagtg 20
<210>9
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>9
tgtatgaatg gctccacgag 20
<210>10
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>10
tgtcctgatc caacatcgag 20
<210>11
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>11
tacttcacaa agcgccttcc 20
<210>12
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>12
tagatgtggc gggttttagg 20
<210>13
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>13
aagtggctcc tttaacctct cc 22
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>14
gagttggtcg tagcggaatc 20
<210>15
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>15
ctatgagaat cgaacccatc c 21
<210>16
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>16
gatggcagct tctgtggaac 20
<210>17
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>17
gcctgcttct tctcacatga c 20
<210>18
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>18
ccctttcact tctgagtccc ag 22
<210>19
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>19
tagggagagg agggtggatg 20
<210>20
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>20
atcatcccca ccatcatagc 20
<210>21
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>21
aaagtggctg atttgcgttc 20
<210>22
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>22
tgcaattcaa tatgaaaatc acc 23
<210>23
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>23
gccaaagcct ccgattatg 19
<210>24
<211>18
<212>DNA
<213> Artificial sequence
<220>
<400>24
cctggagcct ccgtagac 18
<210>25
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>25
ttattccgaa gcctggtagg 20
<210>26
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>26
tggcttccta gggtttatcg 20
<210>27
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>27
tggcgtaggt ttggtctagg 20
<210>28
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>28
taccccgatg catacaccac 20
<210>29
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>29
gggaagtagc gtcttgtaga cc 22
<210>30
<211>27
<212>DNA
<213> Artificial sequence
<220>
<400>30
taactaatac taacatctca gacgctc 27
<210>31
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>31
gtttagacgt ccgggaattg 20
<210>32
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>32
gcccgtattt accctatagc ac 22
<210>33
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>33
gcccgtattt accctatagc ac 22
<210>34
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>34
gggcgcagtg attataggc 19
<210>35
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>35
tgttgtcgtg caggtagagg 20
<210>36
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>36
gcgatgtaac acgagaaagc 20
<210>37
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>37
tggtgaaggg agactcgaag 20
<210>38
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>38
cgcctgatac tggcattttg 20
<210>39
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>39
ttgtagggct catggtaggg 20
<210>40
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>40
accaaatgcc cctcatttac 20
<210>41
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>41
aattaggctg tgggtggttg 20
<210>42
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>42
cgccacttat ccagtgaacc 20
<210>43
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>43
aagtggagtc cgtaaagagg 20
<210>44
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>44
ctgcctacga caaacagacc 20
<210>45
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>45
gagggagtat agggctgtga c 21
<210>46
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>46
tcaaccccga catcattacc 20
<210>47
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>47
taaaggtgga tgcgacaatg 20
<210>48
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>48
cagacccaaa cattaatcag ttc 23
<210>49
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>49
ctatggctga ggggagtcag 20
<210>50
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>50
cactctgttc gcagcagtct g 21
<210>51
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>51
cagggaggta gcgatgagag 20
<210>52
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>52
ccccttccaa acaacaatcc 20
<210>53
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>53
ggttaggatg agtgggaaga ag 22
<210>54
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>54
taggatcctc ccgaatcaac 20
<210>55
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>55
tatgctttgt ttctgttgag tg 22
<210>56
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>56
atcctccaaa tcaccacagg 20
<210>57
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>57
atgtatggga tggcggatag 20
<210>58
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>58
cccattccga taaaatcacc 20
<210>59
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>59
actggttgtc ctccgattca gg 22
<210>60
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>60
gcacccaaag ctaagattct aat 23
<210>61
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>61
gtcaagggac ccctatctga 20
<210>62
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>62
ttaactccac cattagcacc 20
<210>63
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>63
ttgtgcggga tattgatttc ac 22
<210>64
<211>18
<212>DNA
<213> Artificial sequence
<220>
<400>64
gctccgggcc cataacac 18
<210>65
<211>24
<212>DNA
<213> Artificial sequence
<220>
<400>65
ttgttatgat gtctgtgtgg aaag 24
<210>66
<211>17
<212>DNA
<213> Artificial sequence
<220>
<400>66
tctgcacagc cgctttc 17
<210>67
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>67
cattttcagt gtattgcttt gag 23
<210>68
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>68
cccagggttg gtcaatttc 19
<210>69
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>69
agaggtggtg aggttgatcg 20
<210>70
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>70
gtggcaagaa atgggctac 19
<210>71
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>71
gccaggtttc aatttctatc g 21
<210>72
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>72
ccaagcataa tatagcaagg ac 22
<210>73
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>73
aattggtggc tgcttttagg 20
<210>74
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>74
cattctcctc cgcataagc 19
<210>75
<211>25
<212>DNA
<213> Artificial sequence
<220>
<400>75
aacatataac tgaactcctc acacc 25
<210>76
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>76
agggtcttct cgtcttgctg 20
<210>77
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>77
accaacggaa caagttaccc 20
<210>78
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>78
cgggctctgc catcttaac 19
<210>79
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>79
actacaaccc ttcgctgacg 20
<210>80
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>80
tgaagcctga gactagttcg g 21
<210>81
<211>26
<212>DNA
<213> Artificial sequence
<220>
<400>81
tctaggctat atacaactac gcaaag 26
<210>82
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>82
tggcaggagt aatcagaggt g 21
<210>83
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>83
acgcactctc ccctgaactc 20
<210>84
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>84
aggatggggt gtgataggtg 20
<210>85
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>85
ctctaccatc tttgcaggca c 21
<210>86
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>86
ttttgtcatg tgagaagaag cag 23
<210>87
<211>25
<212>DNA
<213> Artificial sequence
<220>
<400>87
tacgcaaaat cttagcatac tcctc 25
<210>88
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>88
tggggagtag tgtgattgag g 21
<210>89
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>89
gagccttcaa agccctcag 19
<210>90
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>90
ggctgagtga agcattggac 20
<210>91
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>91
gttatcgtca cagcccatgc 20
<210>92
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>92
ggcgtttggt attgggttat g 21
<210>93
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>93
tagctgctgg catcactata c 21
<210>94
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>94
tattgcttcc gtggagtgtg 20
<210>95
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>95
acgttgtagc ccacttccac 20
<210>96
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>96
tccaggttta tggagggttc 20
<210>97
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>97
aaagctggtt tcaagccaac 20
<210>98
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>98
agggatcgtt gacctcgtc 19
<210>99
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>99
ccttgacgtt gacaatcgag 20
<210>100
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>100
tttagttggg gcatttcact g 21
<210>101
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>101
ctgttcgctt cattcattgc 20
<210>102
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>102
tggtaagaag tgggctaggg 20
<210>103
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>103
aaatcgctgt cgccttaatc 20
<210>104
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>104
gattatcccg tatcgaaggc 20
<210>105
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>105
atccgtatta ctcgcatcag g 21
<210>106
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>106
accctcatca atagatggag aca 23
<210>107
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>107
atccacccct tacgagtgc 19
<210>108
<211>24
<212>DNA
<213> Artificial sequence
<220>
<400>108
ggatatgagg tgtgagcgat atac 24
<210>109
<211>22
<212>DNA
<213> Artificial sequence
<220>
<400>109
tgctaaaact aatcgtccca ac 22
<210>110
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>110
caaggtgggg ataagtgtgg 20
<210>111
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>111
ctcacaacac cctaggctca c 21
<210>112
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>112
gagggctatg tggctgattg 20
<210>113
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>113
cctcgctaac ctcgccttac 20
<210>114
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>114
ggggcatgag ttagcagttc 20
<210>115
<211>24
<212>DNA
<213> Artificial sequence
<220>
<400>115
gcacactact ataaccaccc taac 24
<210>116
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>116
cgatgaacag ttggaatagg ttg 23
<210>117
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>117
ccccactact aggcctcctc 20
<210>118
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>118
agaaggcgtg ggtacagatg 20
<210>119
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>119
attggcagcc tagcattagc 20
<210>120
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>120
ttgaggtcta gggctgttag aag 23
<210>121
<211>23
<212>DNA
<213> Artificial sequence
<220>
<400>121
acaatttcac agcaccaaat ctc 23
<210>122
<211>18
<212>DNA
<213> Artificial sequence
<220>
<400>122
tcaggggttg aggtcttg 18
<210>123
<211>19
<212>DNA
<213> Artificial sequence
<220>
<400>123
gaccacaccg ctaacaatc 19
<210>124
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>124
gattgatgaa aaggcggttg 20
<210>125
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>125
ctcccgtgag gccaaatatc 20
<210>126
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>126
gtttaagggg ttggctaggg 20
<210>127
<211>21
<212>DNA
<213> Artificial sequence
<220>
<400>127
tcctagcaat aatccccatc c 21
<210>128
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>128
ctgcttcccc atgaaagaac 20
<210>129
<211>25
<212>DNA
<213> Artificial sequence
<220>
<400>129
cttacaagca agtacagcaa tcaac 25
<210>130
<211>20
<212>DNA
<213> Artificial sequence
<220>
<400>130
aggaaccaga tgtcggatac 20
Claims (4)
1. A primer composition, comprising:
the primer composition I comprises a primer 1-a primer 65, the nucleotide sequence of the primer 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the primer 2 is shown as SEQ ID NO. 2, the nucleotide sequence of the primer 3 is shown as SEQ ID NO. 3, the nucleotide sequence of the primer 4 is shown as SEQ ID NO. 4, the nucleotide sequence of the primer 5 is shown as SEQ ID NO. 5, the nucleotide sequence of the primer 6 is shown as SEQ ID NO. 6, the nucleotide sequence of the primer 7 is shown as SEQ ID NO. 7, the nucleotide sequence of the primer 8 is shown as SEQ ID NO. 8, the nucleotide sequence of the primer 9 is shown as SEQ ID NO. 9, the nucleotide sequence of the primer 10 is shown as SEQ ID NO. 10, the nucleotide sequence of the primer 11 is shown as SEQ ID NO. 11, and the nucleotide sequence of the primer 12 is shown as SEQ ID NO. 12, The nucleotide sequence of the primer 13 is shown as SEQ ID NO. 13, the nucleotide sequence of the primer 14 is shown as SEQ ID NO. 14, the nucleotide sequence of the primer 15 is shown as SEQ ID NO. 15, the nucleotide sequence of the primer 16 is shown as SEQ ID NO. 16, the nucleotide sequence of the primer 17 is shown as SEQ ID NO. 17, the nucleotide sequence of the primer 18 is shown as SEQ ID NO. 18, the nucleotide sequence of the primer 19 is shown as SEQ ID NO. 19, the nucleotide sequence of the primer 20 is shown as SEQ ID NO. 20, the nucleotide sequence of the primer 21 is shown as SEQ ID NO. 21, the nucleotide sequence of the primer 22 is shown as SEQ ID NO. 22, the nucleotide sequence of the primer 23 is shown as SEQ ID NO. 23, the nucleotide sequence of the primer 24 is shown as SEQ ID NO. 24, and the nucleotide sequence of the primer 25 is shown as SEQ ID NO. 25, The nucleotide sequence of the primer 26 is shown as SEQ ID NO. 26, the nucleotide sequence of the primer 27 is shown as SEQ ID NO. 27, the nucleotide sequence of the primer 28 is shown as SEQ ID NO. 28, the nucleotide sequence of the primer 29 is shown as SEQ ID NO. 29, the nucleotide sequence of the primer 30 is shown as SEQ ID NO. 30, the nucleotide sequence of the primer 31 is shown as SEQ ID NO. 31, the nucleotide sequence of the primer 32 is shown as SEQ ID NO. 32, the nucleotide sequence of the primer 33 is shown as SEQ ID NO. 33, the nucleotide sequence of the primer 34 is shown as SEQ ID NO. 34, the nucleotide sequence of the primer 35 is shown as SEQ ID NO. 35, the nucleotide sequence of the primer 36 is shown as SEQ ID NO. 36, the nucleotide sequence of the primer 37 is shown as SEQ ID NO. 37, and the nucleotide sequence of the primer 38 is shown as SEQ ID NO. 38, The nucleotide sequence of the primer 39 is shown as SEQ ID NO. 39, the nucleotide sequence of the primer 40 is shown as SEQ ID NO. 40, the nucleotide sequence of the primer 41 is shown as SEQ ID NO. 41, the nucleotide sequence of the primer 42 is shown as SEQ ID NO. 42, the nucleotide sequence of the primer 43 is shown as SEQ ID NO. 43, the nucleotide sequence of the primer 44 is shown as SEQ ID NO. 44, the nucleotide sequence of the primer 45 is shown as SEQ ID NO. 45, the nucleotide sequence of the primer 46 is shown as SEQ ID NO. 46, the nucleotide sequence of the primer 47 is shown as SEQ ID NO. 47, the nucleotide sequence of the primer 48 is shown as SEQ ID NO. 48, the nucleotide sequence of the primer 49 is shown as SEQ ID NO. 49, the nucleotide sequence of the primer 50 is shown as SEQ ID NO. 50, and the nucleotide sequence of the primer 51 is shown as SEQ ID NO. 51, The nucleotide sequence of the primer 52 is shown as SEQ ID NO:52 and the nucleotide sequence of the primer 53 is shown as SEQ ID NO:53, and the nucleotide sequence of the primer 54 is shown as SEQ ID NO:54 and the nucleotide sequence of the primer 55 is shown as SEQ ID NO:55 and the nucleotide sequence of the primer 56 is shown as SEQ ID NO:56 and the nucleotide sequence of the primer 57 is shown as SEQ ID NO:57 and the nucleotide sequence of the primer 58 is shown as SEQ ID NO:58 and the nucleotide sequence of the primer 59 is shown as SEQ ID NO:59, and the nucleotide sequence of the primer 60 is shown as SEQ ID NO:60 and the nucleotide sequence of the primer 61 is shown as SEQ ID NO:61 and the nucleotide sequence of primer 62 is shown as SEQ ID NO:62 and the nucleotide sequence of the primer 63 is shown as SEQ ID NO:63 and the nucleotide sequence of the primer 64 is shown as SEQ ID NO:64 and the nucleotide sequence of primer 65 is shown in SEQ ID NO: 65;
the primer composition II comprises a primer 66-a primer 130, the nucleotide sequence of the primer 66 is shown as SEQ ID NO 66, the nucleotide sequence of the primer 67 is shown as SEQ ID NO 67, the nucleotide sequence of the primer 68 is shown as SEQ ID NO 68, the nucleotide sequence of the primer 69 is shown as SEQ ID NO 69, the nucleotide sequence of the primer 70 is shown as SEQ ID NO 70, the nucleotide sequence of the primer 71 is shown as SEQ ID NO 71, the nucleotide sequence of the primer 72 is shown as SEQ ID NO 72, the nucleotide sequence of the primer 73 is shown as SEQ ID NO 73, the nucleotide sequence of the primer 74 is shown as SEQ ID NO 74, the nucleotide sequence of the primer 75 is shown as SEQ ID NO 75, the nucleotide sequence of the primer 76 is shown as SEQ ID NO 76, and the nucleotide sequence of the primer 77 is shown as SEQ ID NO 77, The nucleotide sequence of the primer 78 is shown as SEQ ID NO. 78, the nucleotide sequence of the primer 79 is shown as SEQ ID NO. 79, the nucleotide sequence of the primer 80 is shown as SEQ ID NO. 80, the nucleotide sequence of the primer 81 is shown as SEQ ID NO. 81, the nucleotide sequence of the primer 82 is shown as SEQ ID NO. 82, the nucleotide sequence of the primer 83 is shown as SEQ ID NO. 83, the nucleotide sequence of the primer 84 is shown as SEQ ID NO. 84, the nucleotide sequence of the primer 85 is shown as SEQ ID NO. 85, the nucleotide sequence of the primer 86 is shown as SEQ ID NO. 86, the nucleotide sequence of the primer 87 is shown as SEQ ID NO. 87, the nucleotide sequence of the primer 88 is shown as SEQ ID NO. 88, the nucleotide sequence of the primer 89 is shown as SEQ ID NO. 89, and the nucleotide sequence of the primer 90 is shown as SEQ ID NO. 90, The nucleotide sequence of the primer 91 is shown as SEQ ID NO. 91, the nucleotide sequence of the primer 92 is shown as SEQ ID NO. 92, the nucleotide sequence of the primer 93 is shown as SEQ ID NO. 93, the nucleotide sequence of the primer 94 is shown as SEQ ID NO. 94, the nucleotide sequence of the primer 95 is shown as SEQ ID NO. 95, the nucleotide sequence of the primer 96 is shown as SEQ ID NO. 96, the nucleotide sequence of the primer 97 is shown as SEQ ID NO. 97, the nucleotide sequence of the primer 98 is shown as SEQ ID NO. 98, the nucleotide sequence of the primer 99 is shown as SEQ ID NO. 99, the nucleotide sequence of the primer 100 is shown as SEQ ID NO. 100, the nucleotide sequence of the primer 101 is shown as SEQ ID NO. 101, the nucleotide sequence of the primer 102 is shown as SEQ ID NO. 102, and the nucleotide sequence of the primer 103 is shown as SEQ ID NO. 103, the nucleotide sequence of the primer 100 is shown as SEQ ID NO. 100, the nucleotide sequence of the primer 101 is shown as SEQ ID NO. 101, the nucleotide sequence of the primer 102 is shown as SEQ ID NO. 102, The nucleotide sequence of the primer 104 is shown as SEQ ID NO. 104, the nucleotide sequence of the primer 105 is shown as SEQ ID NO. 105, the nucleotide sequence of the primer 106 is shown as SEQ ID NO. 106, the nucleotide sequence of the primer 107 is shown as SEQ ID NO. 107, the nucleotide sequence of the primer 108 is shown as SEQ ID NO. 108, the nucleotide sequence of the primer 109 is shown as SEQ ID NO. 109, the nucleotide sequence of the primer 110 is shown as SEQ ID NO. 110, the nucleotide sequence of the primer 111 is shown as SEQ ID NO. 111, the nucleotide sequence of the primer 112 is shown as SEQ ID NO. 112, the nucleotide sequence of the primer 113 is shown as SEQ ID NO. 113, the nucleotide sequence of the primer 114 is shown as SEQ ID NO. 114, the nucleotide sequence of the primer 115 is shown as SEQ ID NO. 115, and the nucleotide sequence of the primer 116 is shown as SEQ ID NO. 116, The nucleotide sequence of the primer 117 is shown as SEQ ID NO. 117, the nucleotide sequence of the primer 118 is shown as SEQ ID NO. 118, the nucleotide sequence of the primer 119 is shown as SEQ ID NO. 119, the nucleotide sequence of the primer 120 is shown as SEQ ID NO. 120, the nucleotide sequence of the primer 121 is shown as SEQ ID NO. 121, the nucleotide sequence of the primer 122 is shown as SEQ ID NO. 122, the nucleotide sequence of the primer 123 is shown as SEQ ID NO. 123, the nucleotide sequence of the primer 124 is shown as SEQ ID NO. 124, the nucleotide sequence of the primer 125 is shown as SEQ ID NO. 125, the nucleotide sequence of the primer 126 is shown as SEQ ID NO. 126, the nucleotide sequence of the primer 127 is shown as SEQ ID NO. 127, the nucleotide sequence of the primer 128 is shown as SEQ ID NO. 128, and the nucleotide sequence of the primer 129 is shown as SEQ ID NO. 129, The nucleotide sequence of the primer 130 is shown as SEQ ID NO: 130.
2. A human mitochondrial whole genome short segment shingled amplification detection kit comprising the primer composition of claim 1.
3. The human mitochondrial whole genome short segment shingled amplification detection kit of claim 2, wherein the reaction system is:
reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s, and 40 cycles; delay at 72 ℃ for 10min, end, 4 ℃.
4. The human mitochondrial whole genome short segment shingled amplification detection kit of claim 3, wherein the concentration of the sample genomic DNA is 10-200 pg/μ l.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399553A (en) * | 2016-11-09 | 2017-02-15 | 上海添音生物科技有限公司 | Human mitochondria whole genome high-throughput sequencing method based on multiple PCR |
| CN106701903A (en) * | 2015-11-17 | 2017-05-24 | 安诺优达基因科技(北京)有限公司 | Reagent kit for detecting mitochondrial heteroplasmy and detection method |
| 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 |
| CN108315399A (en) * | 2018-01-19 | 2018-07-24 | 成都新基因格生物科技有限公司 | Chondriogen detection kit and application method |
-
2021
- 2021-11-03 CN CN202111295036.3A patent/CN113981054B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701903A (en) * | 2015-11-17 | 2017-05-24 | 安诺优达基因科技(北京)有限公司 | Reagent kit for detecting mitochondrial heteroplasmy and detection method |
| CN106399553A (en) * | 2016-11-09 | 2017-02-15 | 上海添音生物科技有限公司 | Human mitochondria whole genome high-throughput sequencing method based on multiple PCR |
| 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 |
| CN108315399A (en) * | 2018-01-19 | 2018-07-24 | 成都新基因格生物科技有限公司 | Chondriogen detection kit and application method |
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