CN103233071A - Method for measuring telomere absolute length - Google Patents
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- CN103233071A CN103233071A CN2013101527012A CN201310152701A CN103233071A CN 103233071 A CN103233071 A CN 103233071A CN 2013101527012 A CN2013101527012 A CN 2013101527012A CN 201310152701 A CN201310152701 A CN 201310152701A CN 103233071 A CN103233071 A CN 103233071A
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Abstract
The invention discloses a method for measuring telomere absolute length. By applying oligomer standard, the telomere absolute length instead of relative length can be measured, which is the largest difference compared with the disclosed real-time quantitative PCR (Polymerase Chain Reaction) method. According to the measuring method, the repeatability is good, few DNA (Deoxyribonucleic Acid) substrates are needed, time is saved, accuracy is realized, and the method is applicable to clinical testing the telomere length and monitoring the dynamic variation, thereby predicting one of disease development and aging indices. The method can be further used for researching preimplantation embryo and stem cell quality.
Description
Technical field
The present invention relates to life science, relate in particular to a kind of method of measuring the telomere absolute length.
Background technology
Telomere is the nucleoprotein structure, and hat is at end of chromosome.It is made up of long six aggressiveness sequences (TTAGGG).The degeneration of the six aggressiveness sequence protection ends of chromosome that repeat complete telomere structure and it is to keeping the genomic stable crucial effects that played.The quantity of six aggressiveness sequences (TTAGGG) of telomere can reduce along with the division each time of cell, causes in the whole vital process of organism, and telomere length is ceaselessly shortening.The shortening of telomere length can cause the fusion of telomere end, chromosomal instability, thereby become and comprise lung cancer, mammary cancer, colorectal carcinoma, prostate cancer, the reason of multiple cancer such as leukemia.The shortening of telomere length also is to cause an old and feeble important factor.Studies show that to comprise psychology and physiological pressure that smoke, obesity etc. all can be accelerated the shortening of telomere length.Measure the length of telomere and the dynamic change of monitoring telomere length and in health population and advancing of disease, more and more cause people's attention.Along with stem cell begins to be widely used in clinical practice, studies show that more and more the length of telomere still is used for monitoring the stem cell quality, the prediction stem cell becomes one of important indicator of live time in vivo.
There are a lot of methods can be used for measuring the length of telomere at present, for example (1) utilizes Southern blot technology to carry out end limit fragment analysis (Terminal Restriction Fragment (TRF) analysis) by the hybridization of DNA and telomeric sequence probe; (2) flow cytometer method; (3) in situ hybridization; (4) real-time quantitative PCR etc.Except the TRF method, additive method has a same disadvantage---can only measure the relative length of telomere, and the TRF method needs relatively large DNA.The telomere absolute length is to set up typical curve earlier, and telomere calculates based on this; Relative length is the result by comparing and obtain with gene such as the expression amount of house-keeping gene of contrast.Do not appear in the newspapers about the measuring method of telomere absolute length at present.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide a kind of accurately, save time, the method for the DNA base consumption is few, repeatability is strong measurement telomere absolute length.
Technical scheme: in order to realize the foregoing invention purpose, a kind of method of measuring the telomere absolute length of the present invention comprises:
(1) from blood sample, extract genomic dna, measure DNA concentration with spectrophotometer, be diluted to 20ng, standby in 4 ℃ of preservations;
(2) set up the telomere length typical curve:
The 84mer oligo that will have 14 tumor-necrosis factor glycoproteins TTAGGG dilutes by concentration gradient, obtains the telomere length typical curve by the qPCR reaction,
In the formula (I), c
TThe volumetric molar concentration of expression 84mer oligo, M
OligoThe molecular weight of expression 84mer oligo;
(3) set up the typical curve of diploid gene group number of copies:
Single copy gene 36B4 by the concentration gradient dilution identical with described step (2), is reacted the typical curve that obtains diploid gene group number of copies by qPCR,
In the formula (II), c represents the volumetric molar concentration of 36B4, and M represents the molecular weight of 36B4;
(4)
What the present invention was measured is the absolute length of telomere, but not relative length, these are different with many employing real time quantitative PCR methods of the prior art.Single copy gene is used for controlling in contrast the amplification reaction of each sample, and determines the genome number of copies of each sample.The selection of single copy gene is extremely important for result's reliability, and genomic number of copies slight change all can have influence on the accuracy of telomere length.The present invention selects for use 36B4 as single copy gene, and it is as the P0 of coding acid nuclear protein body, and sequence is:
36B4（75bp）：5’-CAGCAAGTGG GAAGGTGTAA TCCGTCTCCA CAGACAAGGC CAGGACTCGT TTGTACCCGT TGATGATAGA ATGGG-3’。
QPCR reaction system of the present invention is:
The PCR reaction conditions is: 95 ° of pre-sex change 10min of C; 95 ° of C sex change 15sec, 60 ° of C annealing 1min circulate 40 times.
Described upstream primer comprises the upstream primer of the qPCR that is respectively applied to telomere and 36B4:
Tel F:5 '-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3 ', or 36B4F:5 '-CAGCAAGTGGGAAGGTGTAATCC-3 '
Described downstream primer comprises the downstream primer of the qPCR that is respectively applied to telomere and 36B4:
Tel R:5 '-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3 ', or 36B4R:5 '-CCCATTCTATCATCAACGGGTACAA-3 ',
Wherein, tel F and tel R are respectively upstream primer and the downstream primer sequence of telomere, and 36B4F and 36B4R are respectively upstream primer and the downstream primer sequence of single copy gene.
All PCR reaction substrates of the present invention make it remain on 20ng by adding the pBR322 plasmid DNA.
Beneficial effect: a kind of method of measuring the telomere absolute length of the present invention is that a kind of new accurate timesaving real time quantitative PCR method is used for measuring the telomere absolute length.The present invention can measure the absolute length of telomere, rather than relative length.This be with delivered real time quantitative PCR method compare maximum differently, and repeatable strong, required DNA substrate is few, saves time accurately, more accommodates for the clinical detection telomere length, monitors its dynamic change, to be used for prediction disease progression and aging.
Description of drawings
Fig. 1 represents the typical curve of the telomeric sequence length of each PCR reaction;
Fig. 2 represents the typical curve of each PCR reaction diploid gene group number of copies;
Fig. 3 tests the routine figure of contrast as a result for the present invention,
Fig. 4 checks experimental error with 1301 clones as positive control.
Embodiment
Embodiment
Below in conjunction with accompanying drawing the embodiment of the invention is further specified, present embodiment is at having 23 to the chromosomal mankind, so the genome telomere adds up to 92.
1, from blood sample, extract genomic dna, measure DNA concentration with the Nanodrop spectrophotometer, standby in 4 ℃ of preservations;
2, the drafting of telomere length typical curve (as shown in Figure 1):
(concentration gradient is respectively 10 of definition volumetric molar concentration to the 84mer oligo that will have 14 tumor-necrosis factor glycoproteins 5 '-TTAGGG-3 ' by the concentration gradient dilution
-1To 10
-6), obtaining the telomere length typical curve by the qPCR reaction, the PCR primer is as follows:
tel R：5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’，
tel F：5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’。
In the formula (I), c
TThe volumetric molar concentration of expression 84mer oligo is orientated 60pg/L herein as, M
OligoThe molecular weight of expression 84meroligo is 26667.2 as calculated, therefore, and the scalar=26667.2/6.02 of a telomere * 10
23=0.44 * 10
-19Gram.Concentration is that the telomere molecule number under the 60pg/L is 1.36 * 10
9, multiply by oligo length 84 and both got telomeric sequence at the content 1.18 * 10 of this concentration
8Kb.Under above-mentioned concentration gradient, obtain 1.18 * 10 respectively
8Kb, 1.18 * 10
7Kb, 1.18 * 10
6Kb, 1.18 * 10
5Kb, 1.18 * 10
4Kb, 1.18 * 10
3Kb, interpolation pBR322 plasmid DNA remains in the qPCR process it and maintains 20ng.
3, the typical curve of diploid gene group number of copies (as shown in Figure 2):
Single copy gene 36B4 is diluted by the concentration gradient identical with step 2, obtain the typical curve of diploid gene group number of copies by the qPCR reaction, wherein the sequence of 36B4 is: 5 '-CAGCAAGTGG GAAGGTGTAA TCCGTCTCCA CAGACAAGGC CAGGACTCGT TTGTACCCGT TGATGATAGA ATGGG-3 ', and its primer is respectively:
36B4F：5’-CAGCAAGTGGGAAGGTGTAATCC-3’，
36B4R：5’-CCCATTCTATCATCAACGGGTACAA-3’。
In the formula (II), c represents the volumetric molar concentration of 36B4, is defined as 200pg/L herein, and M represents the molecular weight of the 36B4 of 75bp, is 23268.1 as calculated, therefore, and scalar=23268.1/6.02 of a 36B4 * 10
23=0.38 * 10
-19Gram.Concentration is that the 36B4 molecule number under the 200pg/L is 5.26 * 10
9Individual gene copy is equivalent to 2.63 * 10
9The diploid gene group.Under above-mentioned concentration gradient, obtain 2.63 * 10 respectively
9Kb, 2.63 * 10
8Kb, 2.63 * 10
7Kb, 2.63 * 10
6Kb, 2.63 * 10
5Kb, 2.63 * 10
4Kb.Make it remain on 20ng by adding the pBR322 plasmid DNA.
The reaction system of qPCR is in the above step 2,3:
The PCR reaction conditions is: 95 ° of pre-sex change 10min of C, and 95 ° of C sex change annealing 15sec, 60 ° of C annealing 1min circulate 40 times.
4、
In conjunction with Fig. 1 and Fig. 2, the relation conefficient of two typical curves is 0.99.
The test example
Method of the present invention utilizes real-time quantitative PCR can accurately measure the telomere length of different hematopoiesis liver cell population, can be used for instructing and monitor growth in the body after the hematopoietic stem cell transplantation, survival and function.
Please refer to shown in Figure 3, the positive clone of LN+ (lineage positive cells), the negative clone of LN-(lineage negative cells), CD34+CD38+/CD34+CD38-is marrow hemopoietic stem cells, wherein, CD34+CD38-is more original, and the differentiation and proliferation ability is stronger, so have long telomere length.Monitor telomere length and actual data consistent.
Because lymph matricyte system 1301(1301lymphoblastic cell line) therefore the about 80bp of telomere length is used for doing experiment contrast, and sample repeats 3 times, and all qPCR all finish at RotorGene6000.The result that measuring method of the present invention draws is quite accurate, by statistics, please refer to shown in Figure 4, the experiment in error coefficient be 3%, error coefficient is 8% between experiment.As seen the repeatability of present method is very high.
The above only is preferred implementation of the present invention; be noted that for those skilled in the art; under the prerequisite that does not break away from the principle of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
SEQUENCE LISTING
＜110〉the excellent and biotechnology Development Co., Ltd in Nanjing
＜120〉a kind of method of measuring the telomere absolute length
<130> NJYE1301
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> DNA
＜213〉synthetic
<220>
<221> telF
<400> 1
CGGTTTGTTT GGGTTTGGGT TTGGGTTTGG GTTTGGGTT 39
<210> 2
<211> 23
<212> DNA
＜213〉synthetic
<220>
<221> 36B4F
<400> 2
CAGCAAGTGG GAAGGTGTAA TCC 23
<210> 3
<211> 39
<212> DNA
＜213〉synthetic
<220>
<221> telR
<400> 3
GGCTTGCCTT ACCCTTACCC TTACCCTTAC CCTTACCCT 39
<210> 4
<211> 25
<212> DNA
＜213〉synthetic
<220>
<221> 36B4R
<400> 4
<210> 5
<211> 75
<212> DNA
＜213〉synthetic
<220>
<221> 36B4
<400> 5
CAGCAAGTGG GAAGGTGTAA TCCGTCTCCA CAGACAAGGC CAGGACTCGT TTGTACCCGT TGATGATAGA ATGGG 75
Claims (4)
1. method of measuring the telomere absolute length is characterized in that comprising:
(1) from blood sample, extract genomic dna, measure DNA concentration with spectrophotometer, be diluted to 20ng, standby in 4 ℃ of preservations;
(2) set up the telomere length typical curve:
The 84mer oligo that will have 14 tumor-necrosis factor glycoproteins TTAGGG dilutes by concentration gradient, obtains the telomere length typical curve by the qPCR reaction,
In the formula (I), c
TThe volumetric molar concentration of expression 84mer oligo, M
OligoThe molecular weight of expression 84mer oligo;
(3) set up the typical curve of diploid gene group number of copies:
Single copy gene 36B4 by the concentration gradient dilution identical with described step (2), is reacted the typical curve that obtains diploid gene group number of copies by qPCR,
In the formula (II), c represents the volumetric molar concentration of 36B4, and M represents the molecular weight of 36B4;
（4）
2. a kind of method of measuring the telomere absolute length according to claim 1 is characterized in that:
Described qPCR reaction system is:
The PCR reaction conditions is: 95 ° of pre-sex change 10min of C; 95 ° of C sex change 15sec, 60 ° of C annealing 1min circulate 40 times.
3. a kind of method of measuring the telomere absolute length according to claim 2 is characterized in that:
Described upstream primer comprises the upstream primer of the qPCR that is respectively applied to telomere and 36B4
Tel F:5 '-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3 ', or 36B4F:5 '-CAGCAAGTGGGAAGGTGTAATCC-3 '
Described downstream primer comprises the downstream primer of the qPCR that is respectively applied to telomere and 36B4
Tel R:5 '-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3 ', or 36B4R:5 '-CCCATTCTATCATCAACGGGTACAA-3 '.
4. a kind of method of measuring the telomere absolute length according to claim 1 and 2 is characterized in that: all PCR reaction substrates make it remain on 20ng by adding the pBR322 plasmid DNA.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158125A (en) * | 2015-06-11 | 2015-12-16 | 东南大学 | Measuring method for length of telomere |
| CN106755429A (en) * | 2016-12-27 | 2017-05-31 | 上海三誉生物科技有限公司 | A kind of method of accurate measurement crowd telomere length |
| CN109273051A (en) * | 2018-08-30 | 2019-01-25 | 夏茂 | Identity information encoding model based on telomere length |
| CN109943626A (en) * | 2019-04-18 | 2019-06-28 | 潍坊峡山荆卫生物科技有限公司 | A kind of PCR method detecting absolute telomere length |
| CN111705114A (en) * | 2020-06-11 | 2020-09-25 | 湖南圣洲生物科技有限公司 | Method and kit for detecting absolute length of human telomere based on real-time digital PCR |
| CN113957136A (en) * | 2021-10-27 | 2022-01-21 | 中科解码(北京)生物技术有限公司 | Telomere length detection primer composition, kit and application thereof |
| CN116555402A (en) * | 2023-06-27 | 2023-08-08 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
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| CN102660650A (en) * | 2012-05-25 | 2012-09-12 | 云南路易斯中药现代化工程技术研究中心 | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application |
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Patent Citations (1)
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| CN102660650A (en) * | 2012-05-25 | 2012-09-12 | 云南路易斯中药现代化工程技术研究中心 | Reagent kit for measuring absolute value of average length of chromosome telomeres in samples by real-time quantitative PCR (polymerase chain reaction) and reagent kit application |
Non-Patent Citations (4)
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| NATHAN JO ET AL.: "A quantitative real-time PCR method for absolute telomere length", 《BIOTECHNIQUES》, vol. 44, no. 6, 31 May 2008 (2008-05-31), XP001537019, DOI: doi:10.2144/000112761 * |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158125A (en) * | 2015-06-11 | 2015-12-16 | 东南大学 | Measuring method for length of telomere |
| CN105158125B (en) * | 2015-06-11 | 2018-01-05 | 东南大学 | A kind of telomere length measuring method |
| CN106755429A (en) * | 2016-12-27 | 2017-05-31 | 上海三誉生物科技有限公司 | A kind of method of accurate measurement crowd telomere length |
| CN109273051A (en) * | 2018-08-30 | 2019-01-25 | 夏茂 | Identity information encoding model based on telomere length |
| CN109273051B (en) * | 2018-08-30 | 2022-01-18 | 夏茂 | Identity information coding method based on telomere length |
| CN109943626A (en) * | 2019-04-18 | 2019-06-28 | 潍坊峡山荆卫生物科技有限公司 | A kind of PCR method detecting absolute telomere length |
| CN111705114A (en) * | 2020-06-11 | 2020-09-25 | 湖南圣洲生物科技有限公司 | Method and kit for detecting absolute length of human telomere based on real-time digital PCR |
| CN111705114B (en) * | 2020-06-11 | 2023-08-15 | 湖南圣洲生物科技有限公司 | Method and kit for detecting absolute length of human telomeres based on real-time digital PCR |
| CN113957136A (en) * | 2021-10-27 | 2022-01-21 | 中科解码(北京)生物技术有限公司 | Telomere length detection primer composition, kit and application thereof |
| CN116555402A (en) * | 2023-06-27 | 2023-08-08 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
| CN116555402B (en) * | 2023-06-27 | 2023-09-26 | 成都云测医学生物技术有限公司 | Telomere detection kit and telomere length detection method for non-disease diagnosis purpose |
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Application publication date: 20130807 |