CN109371168A - Detect the new method of relaxation cyclic DNA and covalently closed circular DNA mutational site in hepatitis type B virus - Google Patents
Detect the new method of relaxation cyclic DNA and covalently closed circular DNA mutational site in hepatitis type B virus Download PDFInfo
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Abstract
The invention discloses the new methods of relaxation cyclic DNA and covalently closed circular DNA mutational site in a kind of detection hepatitis type B virus, belong to field of biotechnology, are a kind of easy, easy, stable, reliable detection systems.It is applied respectively in wild-type template, saltant type template and actually detected sample, is about 10 with template DNA dosage after optimizing detection condition6Copies or so, annealing temperature show preferable discrimination when being 63 DEG C.However, the also non-perfection of this detection method, in terms of discrimination, seemingly since there are bigger molecular binding affinities compared with A:T between G:C, so saltant type is better than to wild type discrimination.For the rite-directed mutagenesis Locus Analysis in Shoots of extensive sample, since detection case is simple, therefore method designed in present invention research is applied, quick and precisely, therefore a kind of good detection system of can yet be regarded as.
Description
Technical field
The invention belongs to field of biotechnology, it is related to relaxation cyclic DNA in a kind of detection hepatitis type B virus and covalently closes
The new method in cyclization shape DNA mutation site, specifically, being related to allele-specific polymerase chain reaction for detecting second
The new method of relaxation cyclic DNA and covalently closed circular DNA mutational site in Hepatitis virus.
Background technique
It is that chronic hepatitis B is difficult to the reason of eradicating that HBV cccDNA, which cannot be removed efficiently,.Research shows that HBVrcDNA
It is the necessary front body structure that cccDNA is formed, gene order situation of change when converting for research rcDNA structure, the present invention establishes
A kind of easy rapid analysis method of based on PCR technology is converted to detect the HBV rcDNA after rite-directed mutagenesis and structure
Genome sequence situation after HBV cccDNA, so that it is determined that final structure is changed the mechanism.
Usually, it will be appreciated that the sequence situation of privileged site using PCR amplification respective segments and can be sequenced, with determination
The base composition of sequence, but PCR product need to could even be used for sequencing analysis by electroresis appraisal after purification during this.
Summary of the invention
It is an object of the invention to propose relaxation cyclic DNA and virus covalently closed circular in a kind of detection hepatitis type B virus
The new method in DNA mutation site.It is only the base composition situation for knowing corresponding site by the direct electrophoresis of product after PCR.This
The Method And Principle of invention is design upstream primer F1826CAnd F1826A, their 3 ' ends carry corresponding positions before and after rite-directed mutagenesis respectively
The base C (wild type) or A (saltant type) of point.In PCR reaction, since the product of 3 ' end direct relation synthesis reaction of DNA prolongs
It stretches, therefore it requires have stringenter matching in amplification.In conjunction with downstream primer R2043And RrcSame template is expanded respectively, if template
The site nt1826 is wild type, then only wild primers are to can amplify product, and mutant primers can not expand product；
Vice versa.Meanwhile to guarantee that the single base mismatch primer of design can effectively facilitate the progress of PCR reaction, primer sequence is answered
As short as possible and reaction condition should carefully optimize.
Its technical solution is as follows:
A kind of new method detecting relaxation cyclic DNA and covalently closed circular DNA mutational site in hepatitis type B virus, packet
Include following steps:
Stablize with the pch9 plasmid transfection HepG2 cell from C1826A and by the hbv replication that G418 is screened thin
HBV rcDNA and HBV cccDNA in born of the same parents system distinguishes segment of the PCR amplification containing nt1826, is cloned by T-A, detects bacterium solution mould
Nt1826 bit base situation in plate.Bacterium solution culture 3h dilutes 20 times with PBS, and pch9 copy number/ul is the pch9 matter with 10pg
Grain copy number is roughly the same, and about 106Copies or so.63 DEG C of annealing temperature of reaction condition selection, recurring number 33, row bacterium solution PCR,
Electrophoresis loading 10ul.Mutant type identification uses F1826A/R2043Primer pair, it is contemplated that primer size about 200bp；Wild type identification uses
F1826C/RrcPrimer pair, it is contemplated that primer size about 300bp.
F1826A:5 ' ACGCACCATGCAACTTTTTA3 '
F1826C:5 ' ACGCACCATGCAACTTTTTC3 '
R2043:5 ' CAATGCTCAGGAGACTCTAAGGC3 '
Rrc:5 ' ACAAGAGTTGCCTGAACTTTAGGC3 '
Step 1, design mutational site diagnostic primers two are right, and mutational site is located at 3 ' end of forward primer, as this method is surveyed
Primer pair F1826A and F1826C are tried, two pairs of primer (saltant type and wild type) amplified productions there should be apparent length scale area
Point, it can be identified with electrophoresis, such as the 200bp product and 300bp product in this method.Primer dosage, the forward and reverse primer of 10pmol/ul
Each 1ul.
Step 2, amplification template DNA dosage are with 10pg (i.e. about 106Copies it is) best, therefore need to is by template configuration
10pg/ul(106Copies gene number/comparable quality of ul or volume) to PCR amplification, template consumption 1ul.Draw in mutational site
Object and wild type site primer are both needed to augmentation detection specimen dna respectively.
Step 3, PCR optimum reaction condition: 63 DEG C of annealing temperature are chosen, recurring number 33.I.e. reaction condition is set as 95 DEG C
5min；95 DEG C of 20sec, 63 DEG C of 20sec, 72 DEG C of 20sec × 33；72℃3min；4℃Forever.
Step 4 is directed to above-mentioned condition, and reaction system is as follows:
Step 5, by products therefrom 10ul loading, agarose gel electrophoresis is carried out, if detection specimen dna corresponding site is open country
Raw type, then only wild primers are to can amplify product, and mutant primers can not expand product, and vice versa, with this
Identify corresponding site in detection specimen dna and is mutated situation.
Further, to guarantee that the single base mismatch primer of design can effectively facilitate the progress of PCR reaction, primer sequence is answered
As short as possible and reaction condition should carefully optimize.Reaction given above condition and parameter and standard will also run business into particular one fine tuning if necessary
It is whole.
The invention has the benefit that
The present invention is a kind of easy, easy, stable, reliable detection system.In wild-type template, saltant type template and reality
It is applied respectively in border detection sample, is about 10 with template DNA dosage after optimizing detection condition6Copies or so, annealing temperature are
Preferable discrimination is showed at 63 DEG C.However, the also non-perfection of this detection method, in terms of discrimination, like due to G:C
Between compared with A:T have bigger molecular binding affinities, so to wild type discrimination be better than saltant type.For the fixed point of extensive sample
Mutational site analysis since detection case is simple, therefore applies method designed in present invention research, quick and precisely, therefore not
Losing is a kind of good detection system.
Detailed description of the invention
Fig. 1 is PCR identification point mutation schematic illustration and primer sequence；
Fig. 2 is 10ng wild-type template PCR amplification result；1:F1826A/R2043Amplified production；2:F1826A/RrcAmplification produces
Object；3:F1826C/R2043Amplified production；4:F1826C/RrcAmplified production；5: negative control；M:DL2000Marker
Fig. 3 is the optimization of 10pg wild-type template annealing temperature；1,7,13,5,11,17: mutant primers are to F1826A/R2043
Amplified production；2,8,14: mutant primers are to F1826A/RrcAmplified production；3,9,15,6,12,18: wild primers are to F1826C/
R2043Amplified production；4,10,16: wild primers are to F1826C/RrcAmplified production；M:DL2000Marker；
Fig. 4 is C1826A mutant plasmids template reaction condition optimizing；
Fig. 5 is TA clone's bacterium solution template qualification result；A1-A6:TA clones 20 times of dilution templates of bacterium solution；M:DL2000
Marker。
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and detailed description.
Primer sequence and reaction principle are as shown in Figure 1.
Firstly, the present invention is using wild type pch9 plasmid (1.1 times of bodies containing HBV) as template, wild type diagnostic primers pair
F1826C/R2043, F1826C/RrcAnd saltant type diagnostic primers are to F1826A/R2043, F1826A/RrcRear electrophoresis is expanded to test and analyze.For
To best resolving effect, present invention is generally directed to template consumptions and annealing temperature to optimize, and using 10pg, 10ng dosage, move back
Fiery temperature uses 68 DEG C, 63 DEG C, 58 DEG C, PCR reaction system: 2X GoTaq Green Master Mix (Promega) 10ul,
Each 1ul of the forward and reverse primer of 10pmol, template 1ul, ddH2O supplies 20ul.10ng template, reaction condition use touchdown PCR, specifically
It is as follows: 95 DEG C, 5min；95 DEG C of 20sec, 65 DEG C of (- 1 DEG C/Cycle) 20sec, 72 DEG C of 20sec × 10；95 DEG C of 20sec, 55 DEG C
20sec, 72 DEG C of 20sec × 25；72℃5min；4℃Forever.Electrophoresis result such as Fig. 2 shows that mutant primers are to F1826A/
R2043、F1826A/RrcAnd wild primers are to F1826C/R2043、F1826C/RrcCan amplify purpose size product, but saltant type and
Wild type fails to make effective differentiation.10pg template, reaction condition: 95 DEG C of 5min；63 DEG C of or of 95 DEG C of 20sec, 68 DEG C of or
58 DEG C of 20sec, 72 DEG C of 20sec × 35；72℃3min；4℃Forever.Electrophoresis result such as Fig. 3 shows, in 63 DEG C of annealing temperature and
At 58 DEG C, identification mutant primers to identification wild primers to present good discrimination.
Secondly, the present invention utilizes the pch9 plasmid template of saltant type C1826A, 10pg (10pg/ul), 10ng are also used
(10ng/ul) dosage, annealing temperature use 72 DEG C, 68 DEG C, 63 DEG C, and other conditions are identical as wild-type template amplification.Using open country
Raw type diagnostic primers are to F1826C/R2043, F1826C/RrcAnd saltant type diagnostic primers are to F1826A/R2043, F1826A/RrcExpand rear electrophoresis
It tests and analyzes.If Fig. 4 shows, when annealing temperature is 63 DEG C, when template consumption 10pg, identification saltant type and identification wild primers pair
Good discrimination is made to mutant plasmid.
Finally, the present invention is with from C1826A pch9 plasmid transfection HepG2 cell and the HBV that is screened by G418
HBV rcDNA and the HBV cccDNA replicated in stable cell lines distinguishes segment of the PCR amplification containing nt1826, is cloned by T-A,
Detect nt1826 bit base situation in bacterium solution template.Bacterium solution culture 3h, with PBS dilute 20 times, pch9 copy number/ul i.e. with
The pch9 plasmid copy number of 10pg is roughly the same, and about 106Copies or so.Reaction condition chooses 63 DEG C of annealing temperature, recurring number
33, row bacterium solution PCR, electrophoresis loading 10ul.Mutant type identification uses F1826A/R2043Primer pair, it is contemplated that primer size about 200bp；
Wild type identification uses F1826C/RrcPrimer pair, it is contemplated that primer size about 300bp.It is first that 6 TA clone's bacterium solution samples are selected at random
Row PCR identification, electrophoresis loading 10ul, as a result as Fig. 5 shows.
Bacterium solution template is cloned to TA, it is total that the present invention randomly selects part HBV rcDNA and HBV cccDNA sample simultaneously
25 progress sequencing analysis, sequencing result and electrophoresis result are compared, and same sample is using identification wild primers
Pair and identification mutant primers it is completely the same to PCR amplification rear electrophoresis analysis result and sequencing result, saltant type and wild type area
Indexing is good.
Therefore, the method in this based on PCR detection mutational site is a kind of easy, easy, stable, reliable detection system
System.It applies in wild-type template, saltant type template and actually detected sample, after optimizing detection condition, is used with template DNA respectively
Amount is about 106Copies or so, annealing temperature show preferable discrimination when being 63 DEG C.However, this detection method is also non-
Perfection, in terms of discrimination, seemingly since there are bigger molecular binding affinities compared with A:T between G:C, so to wild type discrimination
Better than saltant type.For the rite-directed mutagenesis Locus Analysis in Shoots of extensive sample, since detection case is simple, therefore present invention research is applied
In designed method, quick and precisely, therefore a kind of good detection system of can yet be regarded as.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Sequence table
<110>Chuanbei Medical College
<120>new method of relaxation cyclic DNA and covalently closed circular DNA mutational site in hepatitis type B virus is detected
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
acgcaccatg caacttttta 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acgcaccatg caactttttc 20
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caatgctcag gagactctaa ggc 23
<210> 4
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
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acaagagttg cctgaacttt aggc 24
Claims (2)
1. the new method of relaxation cyclic DNA and covalently closed circular DNA mutational site, special in a kind of detection hepatitis type B virus
Sign is, comprising the following steps:
Step 1, design mutational site diagnostic primers two are right, and mutational site is located at 3 ' end of forward primer, as this method test is drawn
For object to F1826A and F1826C, two pairs of primer extension products should have apparent length scale to distinguish, and be identified with electrophoresis, such as this method
In 200bp product and 300bp product；Primer dosage, each 1ul of the forward and reverse primer of 10pmol/ul；
Step 2, amplification template DNA dosage are 10pg, therefore need to be 10pg/ul by template configuration, to PCR amplification, template consumption
1ul；Mutational site primer and wild type site primer are both needed to augmentation detection specimen dna respectively；
Step 3, PCR reaction condition: 63 DEG C of annealing temperature are chosen, recurring number 33；I.e. reaction condition is set as 95 DEG C of 5min；95℃
20sec, 63 DEG C of 20sec, 72 DEG C of 20sec × 33；72℃3min；4℃Forever；
Step 4 is directed to above-mentioned condition, and reaction system is as follows:
Step 5, by products therefrom 10ul loading, agarose gel electrophoresis is carried out, if detection specimen dna corresponding site is wild
Type, then only wild primers are to can amplify product, and mutant primers can not expand product, and vice versa, is reflected with this
Corresponding site situation Jian Ce not be mutated in specimen dna.
2. relaxation cyclic DNA and covalently closed circular DNA are mutated position in detection hepatitis type B virus according to claim 1
The new method of point, which is characterized in that two pairs of primers are saltant type and wild type described in step 1, and nucleotide sequence is such as
Shown in SEQ:ID:NO:1-SEQ:ID:NO:4.
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Cited By (1)
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| CN112980928A (en) * | 2021-04-02 | 2021-06-18 | 川北医学院附属医院 | Stem loop primer-assisted isothermal nucleic acid amplification method |
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