CN109943626A - A kind of PCR method detecting absolute telomere length - Google Patents
A kind of PCR method detecting absolute telomere length Download PDFInfo
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- CN109943626A CN109943626A CN201910312065.2A CN201910312065A CN109943626A CN 109943626 A CN109943626 A CN 109943626A CN 201910312065 A CN201910312065 A CN 201910312065A CN 109943626 A CN109943626 A CN 109943626A
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Abstract
The present invention provides a kind of PCR methods of absolute telomere length (absolute telomere length) of detection, can easily and accurately detect the absolute growth of people's telomere, belong to molecular biotechnology and medical science.The method that the present invention uses gene chemical synthesis, is respectively synthesized the oligonucleotide sequence of telomere and single copy gene 36B4, and be connected on plasmid vector and construct standard items, use Escherichia coli amplification in vitro plasmid standard, Plasmid DNA is extracted, concentration is measured, calculates copy number and telomere total length.Determine standard items initial amount, carry out gradient dilution, telomere and the standard curve of 36B4 are respectively obtained using fluorescence quantitative PCR method (Q-PCR method), by standard curve to the telomere total length of sample to be tested DNA and the copy number absolute quantitation of 36B4, to calculate the average absolute telomere length of sample to be tested DNA.Telomere length is detected using absolute telomere length PCR method, has the advantages that at low cost, the period is short, accuracy is high, is suitble to high-throughput detection, is easy to clinical promotion and application, practicability is stronger.
Description
Technical field
The present invention relates to molecular biotechnology and medical science, more particularly to one kind are long for detecting the absolute telomere of people
The method of degree makes standard by the telomere and single copy gene 36B4 plasmid standard of building by fluorescence quantifying PCR method
Curve, realizes the detection of the absolute telomere length of people, and this method is known as absolute telomere length PCR method (aTL PCR).
Background technique
Telomere is a kind of special construction of eukaryocyte end of chromosome, is made of DNA and telomere protein, telomeric dna
It is the highly conserved repeated nucleotide sequences rich in G.The telomeric sequence of different plant species is not consistent, human chromosomal telomere by
Simple duplicate (TTAGGG) the sequence composition in 5 ' → 3 ' directions, telomere length 0.5-20kb.Telomere is nonstructural gene, no
Have the function of coding protein, but there are other important biological functions, such as guarantee the complete copy of DNA, maintains
Chromosome structure is stablized, and cell division cycle is controlled, and can be used as the splitting ability of " division clock " reflection cell of cell.Normally
In the case of, due to the end of DNA polymerase not can completely duplicated chromosome, telomeric dna can be with each division of cell
And shorten, when it shortens to certain length, chromosome is then unable to complete normal replication, and cell mitogen can also stop therewith
It is stagnant, then enter ageing phase, therefore the variation of telomere length decides the destiny of cell.
Because telomere length is negatively correlated with the age, telomere assesses aging state initially as a kind of aging biomarker.
But Recent study shows that telomere is also related to many health status, such as tumour generation, cardiovascular and cerebrovascular disease, nerve are moved back
Row disease etc. or even social environment, living habit, Psychology and behavior also will affect the length of telomere.Telomere is in assessment physiology, the heart
Practicability in reason and biobehavioral research increasingly increases, and especially has great meaning to the risk assessment aspect of health status
Justice, therefore it is badly in need of a kind of accuracy height and economical and practical telomere detection method.
Method of a variety of methods for telomere length detection has now been developed, commonly mainly there is telomerase limitation
Property fragments analysis method (TRF), fluorescence quantitative PCR method (Q-PCR), quantitative fluorescence hybridization in situ (Q-FISH), streaming fluorescence
Hybridization in situ (Flow- FISH) etc., but each detection method has the advantages that its uniqueness, but have certain limitation and
The scope of application.TRF method is the method for the most classic telomere length detection of most original, is that telomere length detects " goldstandard ", this
Kind method has been widely used for basic research, clinical research and epidemiological study, but this kind of method DNA dosage is big and real
It is long to test the period, is not suitable for extensive pattern detection.And Q-FISH method needs metaphase cell, Flow- FISH method needs fresh
Blood, it is high and expensive to sample requirement, limit their popularization and application.Q-PCR method have it is easy, economical, high-throughput,
The advantages such as DNA dosage is few, are suitble to extensive sample high throughput to detect, but the Q-PCR method being widely used at present can only pass through end
The ratio of the amount (S) of the amount (T) and single copy gene of grain amplified production to quantify telomere length, can only obtain the opposite of telomere
Length cannot obtain the absolute growth of telomere.
Summary of the invention
Goal of the invention: the present invention detects telomere length to solve Q-PCR method, can only obtain telomere relative length, cannot
The problem of obtaining telomere absolute growth provides a kind of PCR method for detecting absolute telomere length, realizes that Q-PCR method is absolutely held
The detection of grain length.
Technical solution: the method that the present invention uses gene chemical synthesis is respectively synthesized telomere and the few core of single copy gene 36B4
Nucleotide sequence, and be connected on plasmid vector and construct standard items, using Escherichia coli amplification in vitro plasmid standard, extract plasmid
DNA measures concentration, calculates copy number and telomere total length.It determines standard items initial amount, carries out gradient dilution, it is fixed using fluorescence
Amount PCR method (Q-PCR) respectively obtains telomere and the standard curve of 36B4, total by telomere of the standard curve to sample to be tested DNA
The copy number absolute quantitation of length and 36B4, to calculate the average absolute telomere length of sample to be tested DNA.
Above-mentioned absolute telomere length PCR method comprising the steps of:
(1) extracting genome DNA: taking appropriate whole blood or cell, extracts genomic DNA with genome DNA extracting reagent kit, uses
Microplate reader measures its concentration and purity；
(2) primer of telomere and single copy gene 36B4 is designed and synthesized；
(3) plasmid vector is selected, the gene of design and artificial synthesized telomere and single copy gene 36B4 is connected to plasmid vector
On, construct plasmid standard；
(4) plasmid standard is expanded using Escherichia coli；
(5) extraction of plasmid DNA: extracting Plasmid DNA using small amount plasmid extraction kit, measures its concentration and pure using microplate reader
Degree；
(6) production of standard curve:
1. telomere standard curve making: according to gene chemical synthesis report, obtaining the total length of telomere plasmid standard, calculate plasmid
Average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；According to formula plasmid copy number copies/ul
=(6.02× 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW g/mol), formula telomere length (kb/
Ul)=copies/ul × 84bp, calculate the corresponding telomere total length of standard items starting template amount 20ng Plasmid DNA be 6.96 ×
1085 points, the corresponding telomere total length of the minimum template quantity of standard items is arranged in kb, proportional diluted, extension rate 10, standard items
It is 6.96 × 104Kb quantifies sample to be tested DNA telomere total length by telomere standard curve, obtains specific value.
2. single copy gene 36B4 standard curve making: according to gene chemical synthesis report, obtaining single copy gene 36B4 matter
The total length of grain standard items, calculates plasmid average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；Root
According to formula plasmid copy number copies/ul=(6.02 × 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW
G/mol), the copy number for calculating the corresponding single copy gene 36B4 of standard items starting template amount 0.2ng Plasmid DNA is 6.56 ×
107, proportional diluted, extension rate 10, standard items 5 points of setting, the corresponding single copy gene of the minimum template quantity of standard items
36B4 copy number is 6.56 × 103, sample to be tested DNA monoploid copy number is quantified by 36B4 standard curve, is obtained
Specific value.
(7) absolutely telomere length PCR detects sample to be tested DNA telomere length:
1. telomere reaction system and reaction condition: PowerUp SYBR Green Master Mix (2 ×) 10ul, Tel-F
(10uM) 0.54ul, Tel-R(10uM) 1.8ul, DNA2ul, Rnase-free H2O 5.66ul；By 50 DEG C of 2min, 95 DEG C
2min, 95 DEG C of 15S, 60 DEG C of 1min, 40 circulations are reacted.
2. single copy gene 36B4 reaction system and reaction condition: PowerUp SYBR Green Master Mix (2 ×)
10ul, 36B4-F(10uM) 0.6ul, 36B4-R(10uM) 1ul, DNA2ul, Rnase-free H2O 6.4ul；By 50 DEG C
2min, 95 DEG C of 2min, 95 DEG C of 15S, 52 DEG C of 15S, 72 DEG C of 1min, 40 circulations are reacted.
(8) data are analyzed: by sample to be tested DNA telomere total length and monoploid copy number, calculating average absolute telomere
Length.
Further, telomere and single copy gene 36B4 primer sequence are as follows in step (2):
Tel-FCGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
TEL-RGGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT
36B4-FCAGCAAGTGGGAAGGTGTAATCC
36B4-R CCCATTCTATCATCAACGGGTACAA
Further, step (3) telomere and single copy gene 36B4 standard items sequence are as follows:
Telomere pMV261 plasmid vector
TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGT
TAGGGTTAGGG
36B4 pUC57 plasmid vector
CAGCAAGTGGGAAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTGTACCCGTTGATGATAGAATG
GG
The beneficial effects of the present invention are: the present invention is added telomere and singly copies on the basis of Q-PCR method detects telomere relative length
Shellfish gene 36B4 standard curve, telomere and single copy gene 36B4 to sample to be tested DNA carry out absolute quantitation, to be put down
Absolute telomere length.Present invention sample DNA to be detected, including human lymphocyte genomic DNA, people's Whole Blood Genomic DNA and
Human archeocyte system genomic DNA, it is only necessary to which 10ngDNA can be obtained absolute telomere length.Detection method, detection at
This is low, flux is high, and testing result is accurate, reliable, and extensive sample high throughput is suitble to detect.
Detailed description of the invention
It, below will be to needed in specific embodiment in order to illustrate more clearly of the specific embodiment of the invention
Attached drawing is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as
Restriction to range for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other relevant attached drawings；
Fig. 1 is that specific embodiment of the invention telomere plasmid standard constructs map；
Fig. 2 is that specific embodiment of the invention single copy gene 36B4 plasmid standard constructs map；
Fig. 3 is specific embodiment of the invention telomere amplification curve graph；
Fig. 4 is specific embodiment of the invention telomere melting curve figure；
Fig. 5 is specific embodiment of the invention telomere canonical plotting；
Fig. 6 is specific embodiment of the invention single copy gene 36B4 amplification curve diagram；
Fig. 7 is specific embodiment of the invention single copy gene 36B4 melting curve figure；
Fig. 8 is specific embodiment of the invention single copy gene 36B4 canonical plotting.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The present invention detection absolute telomere length of human lymphocyte DNA is specifically described below:
(1) human lymphocyte separates: fresh anticoagulation cirumferential blood 2ml is acquired, with GE Ficoll-Paque PLUS lymphocyte point
Chaotropic separates lymphocyte；
(2) extracting genome DNA: taking the lymphocyte of separation, is tried with TIANGEN blood/cell/tissue extracting genome DNA
Agent box extracts lymphocyte genomic DNA, measures its concentration and purity using microplate reader, A260/A280 value between 1.8-2.1 it
Between DNA purity it is qualified, can be used for detecting.
(3) telomere and single copy gene 36B4 primer are designed and synthesized；
| Title | Sequence 5 ' → 3 ' | Base number | Way of purification |
| Tel-F | CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT | 39 | HPLC |
| TEL-R | GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT | 39 | HPLC |
| 36B4-F | CAGCAAGTGGGAAGGTGTAATCC | 23 | HPLC |
| 36B4-R | CCCATTCTATCATCAACGGGTACAA | 25 | HPLC |
(4) plasmid vector is selected, the gene of design and artificial synthesized telomere and single copy gene 36B4 is connected to plasmid vector
On, construct plasmid standard；
| Standard items | Plasmid vector | Sequence 5 ' → 3 ' | Length |
| TEL | pMV261 | TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG | 84bp |
| 36B4 | pUC57 | CAGCAAGTGGGAAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTGTACCCGTTGATGATAGAATGGG | 75bp |
(5) plasmid standard is expanded using Escherichia coli: the plasmid standard of building being imported in Escherichia coli, is cultivated using LB
Base culture Escherichia coli expand plasmid standard；
(6) extraction of plasmid DNA: Plasmid DNA is extracted using Axygen small amount plasmid extraction kit, measures it using microplate reader
Concentration and purity, A260/A280 value DNA purity between 1.8-2.1 is qualified, can be used for detecting；
(7) production of standard curve:
1. telomere standard curve making: according to gene chemical synthesis report, obtaining the total length of telomere plasmid standard, calculate plasmid
Average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；According to formula plasmid copy number copies/ul
=(6.02× 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW g/mol), formula telomere length (kb/
Ul)=copies/ul × 84bp, calculate the corresponding telomere total length of standard items starting template amount 20ng Plasmid DNA be 6.96 ×
1085 points, the corresponding telomere total length of the minimum template quantity of standard items is arranged in kb, proportional diluted, extension rate 10, standard items
It is 6.96 × 104Kb quantifies sample to be tested DNA telomere total length by telomere standard curve, obtains specific value.
2. single copy gene 36B4 standard curve making: according to gene chemical synthesis report, obtaining single copy gene 36B4 matter
The total length of grain standard items, calculates plasmid average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；Root
According to formula plasmid copy number copies/ul=(6.02 × 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW
G/mol), the copy number for calculating the corresponding single copy gene 36B4 of standard items starting template amount 0.2ng Plasmid DNA is 6.56 ×
107, proportional diluted, extension rate 10, standard items 5 points of setting, the corresponding single copy gene of the minimum template quantity of standard items
36B4 copy number is 6.56 × 103, sample to be tested DNA monoploid copy number is quantified by 36B4 standard curve, is obtained
Specific value.
(8) absolutely telomere length PCR method detects human lymphocyte DNA telomere length:
1. primer is prepared: the DNA synthesis report respective items of 10 times of volumes are added or manage the RT-PCR of the additional amount of upper label prompt
Grade Water is configured to the working solution of 10uM.
2. PCR reaction system:
Telomere reaction system:
| Reagent | Volume |
| ABI PowerUp SYBR Green Master Mix(2×) | 10ul |
| Tel-F(10uM) | 0.54ul |
| Tel-R(10uM) | 1.8ul |
| Rnase-free H2O | 5.66ul |
| DNA | 2ul |
36B4 reaction system:
| Reagent | Volume |
| ABI PowerUp SYBR Green Master Mix(2×) | 10ul |
| 36B4-F(10uM) | 0.6ul |
| 36B4-R(10uM) | 1ul |
| Rnase-free H2O | 6.4ul |
| DNA | 2ul |
3. PCR reaction condition
| Program TEL | 36B4 |
| Activate 50 DEG C of 2min of UDG enzyme | 50℃ 2min |
| 95 DEG C of 2min of initial denaturation | 95℃ 2min |
| It is denaturalized 95 DEG C of 15s | 95℃ 15s |
| Anneal 60 DEG C of 1min | 52℃ 15s |
| Extend | 72℃ 1min |
| Recurring number 40 | 40 |
PCR is divided to for two partly, and first part is the detection total telomere length of sample DNA, and second part is detection sample DNA list copy
Gene 36B4 copy number, each reaction all include standard curve.This two parts carries out on two 96 orifice plates respectively, to ensure
The stability and accuracy of experimental data, position of each sample on two plates are mutual corresponding (including row and columns), often
Three multiple holes are arranged in a sample can reduce detection error to greatest extent.
To avoid polluting in experiment, experimental result false positive is caused, experiment must be provided with one without template every time
DNA but containing ingredient every other in PCR reaction system blank control reaction.And to ensure between each piece of plate sample value
There is comparativity and reduce experimental error, needs that Positive control wells (sample of known telomere length) is added in each block of plate.
(9) data are analyzed:
After reaction, blank control is first checked for, it is ensured that experiment is pollution-free；The amplification of observation caliber product and sample to be tested, really
Each sample to be tested is protected to fall in the linear extent of standard curve generation；Observe melting curve, it is ensured that without nonspecific products
Amplification；The standard error of the Ct value of three secondary orifices of each sample must be at 0.5Ct (CV > 5%).
Fluorescence quantitative PCR instrument system establishing criteria curve calculates two quantitative values of each sample DNA to be detected, i.e. telomere
Total degree (kb/ reaction) and haploid genome copy number (haploid genome copy number/reaction), the ratio of the two
Value is sample to be tested DNA monoploid average absolute telomere length.Because in mankind monoploid contain 23 chromosomes, 46 telomeres,
So monoploid average absolute telomere length/46=average absolute telomere length.
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein (including technology art
Language and scientific term) there is meaning identical with the general understanding of those of ordinary skill in fields of the present invention.Should also
Understand, those terms such as defined in the general dictionary, which should be understood that, to be had and the meaning in the context of the prior art
The consistent meaning of justice, and unless defined as here, it will not be explained in an idealized or overly formal meaning.
It should be noted last that: the above embodiments are only used to illustrate and not limit the technical solutions of the present invention, although ginseng
It is described the invention in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that: it still can be to this
Invention is modified or replaced equivalently, without departing from the spirit or scope of the invention, or any substitutions,
It is intended to be within the scope of the claims of the invention.
Claims (3)
1. a kind of PCR method for detecting absolute telomere length, which is characterized in that using the method for gene chemical synthesis, be respectively synthesized end
The oligonucleotide sequence of grain and single copy gene 36B4, and be connected on plasmid vector and construct standard items, use Escherichia coli body
Outer amplification plasmid standard extracts Plasmid DNA, measures concentration, calculates copy number and telomere total length；Determine that standard items originate
Amount carries out gradient dilution, respectively obtains telomere and the standard curve of 36B4 using fluorescence quantitative PCR method (Q-PCR method), pass through mark
Directrix curve is to the telomere total length of sample to be tested DNA and the copy number absolute quantitation of 36B4, to calculate sample to be tested DNA's
Average absolute telomere length, step include:
(1) extracting genome DNA: taking appropriate whole blood or cell, extracts genomic DNA with genome DNA extracting reagent kit, uses
Microplate reader measures its concentration and purity；
(2) primer of telomere and single copy gene 36B4 is designed and synthesized；
(3) plasmid vector is selected, the gene of design and artificial synthesized telomere and single copy gene 36B4 is connected to plasmid vector
On, construct plasmid standard；
(4) plasmid standard is expanded using Escherichia coli；
(5) extraction of plasmid DNA: extracting Plasmid DNA using small amount plasmid extraction kit, measures its concentration and pure using microplate reader
Degree；
(6) production of standard curve:
1. telomere standard curve making: according to gene chemical synthesis report, obtaining the total length of telomere plasmid standard, calculate plasmid
Average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；According to formula plasmid copy number copies/ul
=(6.02× 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW g/mol), formula telomere length (kb/
Ul)=copies/ul × 84bp, calculate the corresponding telomere total length of standard items starting template amount 20ng Plasmid DNA be 6.96 ×
1085 points, the corresponding telomere total length of the minimum template quantity of standard items is arranged in kb, proportional diluted, extension rate 10, standard items
It is 6.96 × 104Kb quantifies sample to be tested DNA telomere total length by telomere standard curve, obtains specific value；
2. single copy gene 36B4 standard curve making: according to gene chemical synthesis report, obtaining single copy gene 36B4 plasmid mark
The total length of quasi- product calculates plasmid average molecular weight (MW)=(base logarithm) × (660 dalton/base) g/mol；According to public affairs
Formula plasmid copy number copies/ul=(6.02 × 1023Copies/mol) × (concentration ng/ul × 10-9)/(MW g/
Mol), the corresponding single copy gene 36B4 copy number of standard items starting template amount 0.2ng Plasmid DNA is 6.56 × 107, wait ratios
5 points, the corresponding single copy gene 36B4 copy number of the minimum template quantity of standard items is arranged in dilution, extension rate 10, standard items
It is 6.56 × 103, sample to be tested DNA monoploid copy number is quantified by 36B4 standard curve, obtains specific value；
(7) absolutely telomere length PCR detects sample to be tested DNA telomere length:
1. telomere reaction system and reaction condition: PowerUp SYBR Green Master Mix (2 ×) 10ul, Tel-F
(10uM) 0.54ul, Tel-R(10uM) 1.8ul, DNA2ul, Rnase-free H2O 5.66ul；By 50 DEG C of 2min, 95 DEG C
2min, 95 DEG C of 15S, 60 DEG C of 1min, 40 circulations are reacted；
2. single copy gene 36B4 reaction system and reaction condition: PowerUp SYBR Green Master Mix (2 ×)
10ul, 36B4-F(10uM) 0.6ul, 36B4-R(10uM) 1ul, DNA2ul, Rnase-free H2O 6.4ul；By 50 DEG C
2min, 95 DEG C of 2min, 95 DEG C of 15S, 52 DEG C of 15S, 72 DEG C of 1min, 40 circulations are reacted；
(8) data are analyzed: by sample to be tested DNA telomere total length and monoploid copy number, calculating average absolute telomere length.
2. the method according to claim 1, which is characterized in that telomere and single copy gene 36B4 primer in step (2)
Sequence is as follows:
Tel-FCGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
TEL-RGGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT
36B4-FCAGCAAGTGGGAAGGTGTAATCC
36B4-R CCCATTCTATCATCAACGGGTACAA。
3. the method according to claim 1, which is characterized in that step (3) telomere and single copy gene 36B4 plasmid carry
Body and standard items sequence are as follows:
Telomere pMV261 plasmid vector TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG
GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
36B4 pUC57 plasmid vector
CAGCAAGTGGGAAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTGTACCCGTTGATGATAGAAT
GGG。
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| 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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