AU2020104121A4 - rapid batch determination method for the copy number of genome multicopy gene by PCR technology - Google Patents
rapid batch determination method for the copy number of genome multicopy gene by PCR technology Download PDFInfo
- Publication number
- AU2020104121A4 AU2020104121A4 AU2020104121A AU2020104121A AU2020104121A4 AU 2020104121 A4 AU2020104121 A4 AU 2020104121A4 AU 2020104121 A AU2020104121 A AU 2020104121A AU 2020104121 A AU2020104121 A AU 2020104121A AU 2020104121 A4 AU2020104121 A4 AU 2020104121A4
- Authority
- AU
- Australia
- Prior art keywords
- gene
- pcr
- multicopy
- same
- copy number
- 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.)
- Ceased
Links
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rapid batch determination method for the copy number of
genome multicopy gene by PCR technology, which comprises the following steps: respectively
performing PCR amplification on certain multicopy gene and certain known single copy gene
on the same tissue DNA sample, then purifying the products; After measuring the mass
concentration of PCR purified products of each gene, the mass concentration is converted into
molar concentration according to its nucleic acid sequence; Then, preparing the standard curves
of these genes by the same RT-PCR reaction with the PCR products after gradient dilution and
the tissue DNA samples as the templates, so as to obtain the concentration of each gene in the
DNA samples. Under the same extraction efficiency, the copy number of multicopy gene is the
number of multicopy gene divided by the number of single copy gene in the same volume.
Therefore, the copy numbers of seven different multicopy gene Y in the same biological
individual can be determined by one 96-well RT-PCR reaction. The copy number of 11
different multicopy gene Y can also be determined by one 384-well RT-PCR reaction. This is
how to realized the rapid batch measurement efficiency. The following contents take 96-well
RT-PCR reaction as the example.
Description
A rapid batch determination method for the copy number of genome multicopy
gene by PCR technology
[01] The invention relates to a rapid batch determination method for the copy number of genome multicopy gene by PCR technology and RT-PCR technology, which belongs to the field of molecular biology according to the international patent classification table (IPC).
[02] Copy-number variant (CNV), which is also known as copy-number polymorphism (CNP), is a variation of DNA fragments with a size ranging from 1kb to 3Mb, which is widely distributed in the human genome. The total number of nucleotides covered by CNV greatly exceeds the total number of single nucleotide polymorphisms (SNPs), which greatly enriches the diversity of genetic variation in the genome. CNV can be of great biological significance for species-specific genome composition, species evolution and phylogeny, and gene expression and regulation in certain specific regions of genome. It has been found that the frequency of CNV is much higher than that of chromosome structural variation, and the total number of nucleotides covered in the whole genome greatly exceeds the total number of SNPs. Therefore, researchers believe that CNV may be closely related to phenotypic variation, and it play an important role in the evolution and development of species. At the same time, researchers also believe that this kind of variation is the most important variation in the genome, which is the main cause of phenotypic differences among individuals and of various genetic diseases and disease susceptibility. For example, the pathogenesis of autism, schizophrenia and Crohn's disease is that the copy number of normal multicopy gene suddenly changes, which leads to the occurrence of diseases. Therefore, the rapid and accurate determination of the copy number of multicopy gene in organisms can provide biological methods for the diagnosis and treatment of some human diseases and scientific means for the study of physiological mechanisms of related phenomena in life sciences.
[03] The current methods for determining gene copy number are as follows:
[04] 1. Genome sequencing. All gene sequences of the whole genome are determined by various sequencing methods, and the obtained sequences are compared with the target gene sequences, so as to obtain the copy number of the target gene.
[05] 2. Gene chip technology. Tens of thousands or even millions of gene probes with specific sequences are regularly arranged and fixed on supports such as silicon wafers and slides of 2cm2 by microarray technology to form a two-dimensional DNA probe array. When the nucleic acid sequence with fluorescence label in the solution is complementary matched with the nucleic acid probe at the corresponding position on the gene chip, a group of probe sequences with completely complementary sequences can be obtained by determining the probe position with the strongest fluorescence intensity. Based on this, the copy number of the target gene can be recombined.
[06] 3. DNA renaturation kinetics. It refers to the dynamic analysis of the process of DNA thermal denaturation and recovery (renaturation). According to the speed of DNA renaturation, eukaryotic DNA is divided into three different gene tissues: single sequence, medium repeat sequence and high repeat sequence, so as to obtain the copy number of the target gene.
[07] 4. Biological hybridization. Transgenic animal and plant individuals and non-transgenic animal and plant individuals are hybridized with each other, and the copy number of target genes in transgenic animal and plant individuals is calculated according to the number of transgenic animal and plant individuals of offspring and Mendel's law.
[08] 5. Southern blotting. The DNA of transgenic individuals is hydrolyzed by restriction endonuclease, then separated by agarose gel electrophoresis, and then transferred to nitrocellulose or nylon membrane. The target gene is labeled with radioisotope or other methods, and then hybridized with DNA printed on the membrane. According to the value of radioactive band, the copy number of target gene can be deduced.
[09] 6. Fluorescence quantitative method. Real-time PCR, also known as TapMan PCR, utilizes a pair of PCR primers and probes of genes and DNA polymerase to amplify target genes exponentially by repeated hot and cold cycles. Because PCR products can be quantified by fluorescence signals, and the fluorescence threshold cycle number (Ct) is negatively correlated with the initial concentration logarithm of the amplified gene, the copy number of the target gene can be determined by the target gene and the known internal reference gene.
[010] The main defects of the prior art are as follows:
[011] 1. It takes a long time and costs a lot. The above sequencing method and chip method are expensive and take a long time, especially the design and customization of chips need high cost and time. In addition, data processing is cumbersome. Biological hybridization method needs a complete growth cycle to produce offspring, and it needs to detect a large number of offspring to obtain statistical data.
[012] 2. Special equipment and devices are required. For example, the Southern Blotting Method needs infrastructure and conditions such as radioisotope operation and darkroom, it also needs a large amount of funds to treat radioactive wastes. Non radioactive methods are difficult to achieve the sensitivity of single gene detection, and they are expensive.
[013] 3. The measured data is not accurate enough, so there are many factors affecting the DNA kinetics in the experimental operation, such as temperature and electrolyte environment, which have certain influence on the accuracy of the results.
[014] 4. Insufficient assumptions. The above real-time PCR method is based on an insufficient assumption that the target gene and the internal reference gene have the same amplification coefficient. However, this hypothesis is generally difficult to hold, and the PCR amplification coefficients of genes with different sequences and lengths are different. Because PCR amplifies genes exponentially, the slight difference in PCR amplification coefficient will be multiplied after multiple cycles of amplification, resulting in huge differences in PCR amplification, which directly affects the calculation of gene copy number.
[015] The existing method of rapid determination of gene copy number by real time PCR is based on TapMan fluorescent probe. This quantitative method takes a long time to design and customize the probe, and it is difficult to design ideal TapMan fluorescent probe for some genes, which is more expensive than ordinary fluorescent quantitative PCR. At the same time, the existing methods for quantitative determination of gene copy number in real time need to mix and dilute the target gene and single copy internal reference gene in different proportions, and the repeated mixing and dilution of small volume liquid will greatly increase the experimental error.
[016] The current method for determining the copy number of multicopy gene in genome by real-time PCR is to quantitatively determine the target genes and single copy internal reference genes by using TapMan probe quantitative method. This quantitative method utilizes TapMan probe, it has high cost, complicated operation and high requirements for primer and probe design. At the same time, the repeated mixing and dilution of target gene and single copy internal reference gene will increase the experimental error and reduce the accuracy of determination results. According to the method, the target gene (multicopy gene) and the internal reference gene (known single copy gene) are respectively subjected to gradient dilution by PCR technology combined with common fluorescence quantification, and a standard curve is made without mixing the target gene and the internal reference gene. In addition, the annealing temperature of each primer can be designed by using the temperature gradient program in the 96 well fluorescence quantitative PCR reaction, and the rapid batch determination of seven multicopy gene Y can be realized at one time.
[017] 1) Extract DNA samples of individual organisms to be detected, and PCR amplification primers are designed for multicopy gene (Xi, X2, X3 ... X.) to be detected and single copy gene (Y) known as internal reference.
[018] 2) Take PCR amplification of each gene (XI, X2, X 3 .. .Xn, Y) with individual DNA samples as templates, and carry out purification of PCR products.
[019] 3) Determine the mass concentration of PCR purified products of each gene, and convert the mass concentration into molar concentration according to its nucleic acid sequence, which is Mi, M2, M3...Mn, and My.
[020] 4) Gradient dilution of PCR purified products of each gene.
[021] 5) Take PCR purified products of each gene after gradient dilution and tissue DNA samples as templates, prepare standard curves of these genes by the same
RT-PCR reaction, and obtain the content (Ct value) of each gene in tissue DNA samples.
[022] 6) According to the standard curve of each gene and the expression amount of each gene in the tissue DNA sample, the molar concentrations mi, m2, m3 mn and my of each gene are calculated.
[023] 7) According to the relationship that the number of multicopy gene in the same tissue DNA sample is multiple of the copy number of single copy gene, the copy number of the tested genes in the genome is obtained by a set of equations.
[024] The attached figure shows the experimental principle diagram of this technology.
[025] Fig. 1: Flow solution of PCR method for rapid batch determination of the copy number of multiple copies of genome.
[026] Fig. 2: Layout design of PT-PCR. Using the temperature gradient program of fluorescence quantitative instrument, eight rows of temperatures from top to bottom are designed as the suitable annealing temperatures for primers of genes Xi, X2, X3, X4, X5, X6, X 7 and Y; Templates added in the first ten wells of each row are gradient dilution templates of PCR purified products of this gene, while DNA samples of organism tissues are used as templates in the last two wells. It should be noted that the amount of templates for each hole must be consistent.
[027] Fig. 3: Standard curve of molar concentration -Ct value of gene X. (multicopy gene to be tested). Convert the converted molar concentration to the dilution multiple in the standard curve. The molar concentration of the gene in DNA samples is calculated by using the relationship between the molar concentration and Ct value Ct= anlog2M+bn.
[028] Fig. 4: Standard curve of molar concentration -Ct value of gene Y (single copy gene Y).
[029] The specific implementation mode of the invention will be described in detail with reference to Fig. 1, wherein the tissue DNA sample in Fig. 1 can be extracted by any kit. For the same tissue DNA sample, some multi copy genes (to be determined gene) and the known single copy gene (internal reference gene) are amplified and purified by simple PCR. Measuring their respective mass concentrations, and converting the mass concentrations into molar concentrations according to their nucleic acid sequences; Then, the standard curves of these genes are made by the same RT PCR reaction with gradient diluted PCR products and tissue DNA samples as templates, and the molar concentration of each gene in DNA samples is obtained at the same time. Under the same extraction efficiency, the copy number of multicopy gene is the mathematical relationship between the number of multicopy gene divided by the number of single copy gene in the same volume, and the equation is established to solve the copy number of multicopy gene.
[030] Xn represents a multicopy gene whose copy number needs to be determined in the genome, and Y represents a single copy gene that has been confirmed (such as al- antitrypsin gene and glucagon receptor gene of human).
[031] Mn and My represent the molar concentrations of gene Xn and gene Y purified by PCR respectively, which are converted from the molar mass calculated by combining the mass concentration measured by a high-precision nucleic acid protein instrument with the nucleotide sequence of the genes.
[032] Ct(Xn) and Ct(Y) are Ct values obtained by real-time fluorescence quantitative reaction with the same tissue DNA sample as the template.
[033] mn and my are the molar concentrations of gene Xn and gene Y obtained by combining Ct(Xn) and Ct(Y) values with standard curves of X and gene Y (abscissa is exchanged by mass concentration for dilution multiples) (the valuesof mnand my can be read directly by the drawings, and they are multiples of Mn and My respectively)( Refer to Fig. 2 and Fig. 3).
[034] N is the copy number of geneXnin genome.
[035] Take one 96 well plate RT-PCR reaction to determine the copy number of 7 genes as the example, the specific operation is as follows:
[036] 1. Carry out PCR amplification on extracted tissue DNA with multicopy
gene.Xi, X2, X3, X4, X5, X 6, X7 and the known single copy gene Y (such as
glucagon receptor gene GCG) to be determined respectively (the amount of templates
can be different). The amplified products are purified (only Xi, X2, X3, X4, X5
, X 6, X 7 and gene Y in the products) are purified, and then the mass concentration is
determined and read directly by nucleic acid protein analyzer and other instruments.
[037] 2. Calculate the molar mass of each gene sequence, and finally convert the mass concentration of PCR purified product of each gene into molar concentration (divided by the mass concentration and the molar mass). They are Mi, M2, M3, M4, M5, M6, M7 and My respectively.
[038] 3. The .Xi, X2, X3, X4, X5, X6, X7 and gene Y after concentration
determination are diluted by gradient, which are used as templates for fluorescence quantification (the amount of templates is 2[d).
[039] 4. At the same time, in the same batch reaction of preparing standard curve for each gene by RT-PCR (primer is the same as PCR primer in step (1)), quantitative reaction with extracted organism tissue DNA as template (template amount of 2[1) is added to ensure the same amplification efficiency of genes with tissue DNA as the template and of purified PCR products as the template. At last, the concentration values
of this gene in tissue DNA are Ct(Xi), Ct(X2), Ct(X3), Ct(X4), Ct(X5), Ct(X),
Ct(X7), Ct(Y).
[040] 5. In light of the standard curve of each gene, replace the logarithmic value of dilution multiple of abscissa with the measured molar concentration of the gene, and obtain the linear function of Ct value to logarithmic value of molar concentration Ct= alog2M + b (wherein a is the slope of the standard curve; b is the intercept of standard curve. They are all calculated according to the known Ct values of each gene at the known molar concentration).The molar concentration of the gene in the DNA sample is obtained by combining the Ct value of the gene in the DNA sample of the organism tissue. They are mi,m2,m3,m4, ms, m, my and my.
[041] 6. According to the same extraction efficiency of each gene in the same tissue DNA extraction, it can be determined that the molar number of a certain multicopy gene and a single copy gene in the same volume of extracted DNA is a fixed integer, that is, the copy number of the multicopy gene. According to this relationship, the equations can be solved.
Ct(Xi) - a,10g2 m'+b 1 Fbi-ct(xi) by-C(Y)7
[042] Ct(Y) - aYlog 2 m 1 a a
m, = NI x my
Ct(X 7 ) - 7 l1g 2 M 7 +b 7 -Ct(X 7 ) b-Ct(Y)
Ct(Y) = - aJlOg2 m+ ±: li N7 =2 "' ay
[043 m, = N, x m
[044] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.
[045] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable
Claims (7)
1. The rapid batch determination method for the copy number of genome
multicopy gene by PCR technology comprises the following steps:
1) Extract DNA samples of individual organisms to be detected, and design
PCR amplification primers for multicopy gene (Xi, X2, X3 ...X.) to be detected and
known single copy gene (Y) as internal reference.
2) Take PCR amplification of each gene(X1, X2, X3 ...Xn, Y) with individual
DNA sample as the templates, and carry out purification of PCR products.
3) Determine the mass concentration of PCR purified products of each gene,
and convert the mass concentration into molar concentration according to its nucleic
acid sequence.
4) Gradient dilution of PCR purified products of each gene.
5) Take PCR purified products of each gene after gradient dilution and tissue
DNA samples as the templates, preparing standard curves of these genes by the same
RT-PCR reaction, and obtain the content (Ct value) of each gene in tissue DNA
samples.
6) Calculate the molar concentration of each gene by the standard curve of
each gene and the expression amount of each gene in the tissue DNA sample.
7) As the relationship that the number of multicopy gene in the same tissue
DNA sample is multiple of the copy number of single copy gene, the copy number of
each tested gene in the genome is obtained by a set of equations.
2. The method according to claim 1, characterized in that the primers of the
multicopy gene and the single copy gene are common PCR primers, and expensive
TaqMan PCR fluorescent probes are not needed which are difficult to design.
3. The method according to claim 1, characterized in that the molar concentration
is determined after the PCR products of multicopy gene and single copy gene are
purified, and the accuracy is high.
4. The method according to claim 1, characterized in that the same RT-PCR
reaction is a reaction in which PCR purified products of each gene after gradient
dilution are used as templates, and DNA samples of organism tissues are used as the
templates respectively in the same fluorescence quantitative reaction, in order to ensure
the same amplification efficiency of each gene.
5. The method according to claim 1, wherein the molar concentration of each
gene in the tissue DNA sample is calculated by the standard curve of the gene and the
Ct value of the gene in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020104121A AU2020104121A4 (en) | 2020-12-16 | 2020-12-16 | rapid batch determination method for the copy number of genome multicopy gene by PCR technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020104121A AU2020104121A4 (en) | 2020-12-16 | 2020-12-16 | rapid batch determination method for the copy number of genome multicopy gene by PCR technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020104121A4 true AU2020104121A4 (en) | 2021-03-04 |
Family
ID=74715959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020104121A Ceased AU2020104121A4 (en) | 2020-12-16 | 2020-12-16 | rapid batch determination method for the copy number of genome multicopy gene by PCR technology |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2020104121A4 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736900A (en) * | 2021-08-30 | 2021-12-03 | 华南农业大学 | Method for screening single-copy T-DNA transgenic plant |
| CN114317693A (en) * | 2021-12-31 | 2022-04-12 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human 5S ribosome DNA copy number detection kit and detection method |
| WO2023179053A1 (en) * | 2022-03-22 | 2023-09-28 | 上海润达榕嘉生物科技有限公司 | Method for detecting number of copies of target gene |
-
2020
- 2020-12-16 AU AU2020104121A patent/AU2020104121A4/en not_active Ceased
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736900A (en) * | 2021-08-30 | 2021-12-03 | 华南农业大学 | Method for screening single-copy T-DNA transgenic plant |
| CN113736900B (en) * | 2021-08-30 | 2023-07-25 | 华南农业大学 | Method for screening single copy T-DNA transgenic plants |
| CN114317693A (en) * | 2021-12-31 | 2022-04-12 | 中国疾病预防控制中心职业卫生与中毒控制所 | Human 5S ribosome DNA copy number detection kit and detection method |
| WO2023179053A1 (en) * | 2022-03-22 | 2023-09-28 | 上海润达榕嘉生物科技有限公司 | Method for detecting number of copies of target gene |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020104121A4 (en) | rapid batch determination method for the copy number of genome multicopy gene by PCR technology | |
| CN102344961B (en) | A kind of polygenic method of application extensive parallel sequencing technology inspection multiple goal of economy | |
| CN107660234A (en) | The method of prediction organ-graft refection is sequenced using two generations | |
| CN110241257A (en) | A kind of while 11 kinds of related microorganisms that spread through sex intercourse of detection and identification methods | |
| CN101956008A (en) | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca | |
| CN115820922A (en) | Primer probe combination and kit for detecting cryptococcus | |
| Zhou et al. | Applying GeoChip analysis to disparate microbial communities | |
| WO2011146942A1 (en) | Methods and kits to analyze microrna by nucleic acid sequencing | |
| CN107287283B (en) | High-throughput detection kit for multiple SNP sites related to children susceptibility diseases and use method thereof | |
| CN110408612B (en) | Protective agent for low-concentration DNA standard substance, preservation method and application | |
| CN110923306A (en) | Primer, probe, kit and method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction) | |
| Li et al. | Development of a novel reference plasmid for accurate quantification of genetically modified Kefeng6 rice DNA in food and feed samples | |
| CN103103280A (en) | Quick and batch test method for copy number of multi-copy genes of genomes | |
| CN109762909A (en) | A kind of 44 site InDels composite amplification detection kits for sample medical jurisprudence individual appreciation of degrading | |
| CN111733287B (en) | Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome | |
| CN113684316A (en) | Novel coronavirus detection kit based on digital PCR and application | |
| CN113403377A (en) | SNP (Single nucleotide polymorphism) site primer composition for detecting drug and nutrient metabolic capability and application | |
| CN109415759B (en) | Method for producing DNA probe and method for analyzing genomic DNA using DNA probe | |
| CN107435064B (en) | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting harmful golden algae in chlorella culture | |
| Rantala-Ylinen et al. | Molecular methods: chip assay and quantitative real-time PCR: in detecting hepatotoxic cyanobacteria | |
| CN111424074B (en) | Nucleic acid detection kit for quantifying trace residue of mouse source tissue | |
| CN108359729A (en) | Nucleic acid sequence, kit and its detection method for detecting risk of obesity site | |
| CN108118097B (en) | Primer probe, kit and method for quantitatively detecting dysentery amoeba | |
| Deleye et al. | Genome-wide copy number alteration detection in preimplantation genetic diagnosis | |
| Jyothy et al. | REAL-TIME PCR OR QUANTITATIVE PCR (QPCR)–A REVOLUTION IN MODERN SCIENCE |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |