CN106978484A - The unbalanced method of amplified allele is solved by PCR additives - Google Patents
The unbalanced method of amplified allele is solved by PCR additives Download PDFInfo
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Abstract
The unbalanced method of amplified allele is solved by PCR additives the invention discloses one kind.This method for due to " part allele to be measured unwind in PCR amplification procedures to form complicated secondary structure or/and DNA double chain unwind it is insufficient " caused by the unbalanced situation of amplified allele, comprise the following steps：By adding PCR additives into original PCR reaction systems, to reduce the stability of duplex DNA, so as to solve the unbalanced problem of amplified allele；Original PCR reaction systems enter the PCR reaction systems used when performing PCR amplifies existing amplified allele imbalance situation to allele to be measured by described.It is demonstrated experimentally that this method can effectively solve really due to " part allele to be measured unwind in PCR amplification procedures to form complicated secondary structure or/and DNA double chain unwind it is insufficient " caused by amplified allele it is uneven.
Description
Technical field
The invention belongs to gene magnification field, it is related to a kind of solution unbalanced method of amplified allele, and in particular to
It is a kind of that the unbalanced method of amplified allele is solved by PCR additives.
Background technology
Round pcr is based on PCR, is a kind of technology that can specifically amplify and expand specific gene fragment,
The increase of tens thousand of times of genetic fragment can be made.The core of its principle is Oligonucleolide primers with one section in single-stranded DNA templates mutually
Complementary series combines hybridization, forms partially double stranded, DNA synthesis is carried out in the presence of archaeal dna polymerase.And Oligonucleolide primers and list
The combination of chain DNA template is to be based on basepairing rule：Base pairing follows G (guanine)：C (cytimidine), A (adenine)：T
(thymidine)/U (uracil) Watson-Crick basepairing rules.
Each intracellular in human body to have 23 pairs of chromosomes, usual gene is diallele.When carrying out regular-PCR amplification,
Two allele generally difference without amplification efficiency.But in complicated gene, influence the factor of PCR amplification efficiencies
The amplification efficiency of two allele can be caused to there is substantially amplification difference, i.e. amplified allele imbalance, can be gone out when serious
An existing amplified allele yield is few (amplification failure), and the allele of heterozygosis is detected as homozygosis equipotential base by mistake
Cause.Such as HLA genes are exactly typical complicated gene, with high GC content, high homology, the features such as high polymorphism.Mesh
Before, HLA gene numbers already exceed 10,000, and effective amplification of these genes is the important foundation of HLA high-resolution partings.But with
Upper feature determines that the amplimer design of HLA genes is especially difficult and it is very difficult to expand, and the non-spy of HLA genes easily occurs
The problems such as different amplification, allelic loss or amplified allele are uneven, i.e. corresponding gene amplification efficiency are low or failure (is expanded
It is uneven) heterozygous genes site is lost an allele, parting mistake is homozygous gene site.
During PCR, the DNA double chain of two allele can unwind.When one of allele unwinds, due to
Base pairing, single stranded DNA may form complicated secondary structure because of its own sequence base feature；Or due to series feature
DNA double chain is set to unwind insufficient, these produce influence to archaeal dna polymerase extension, so as to influence PCR amplification efficiency；But in addition
One amplified allele efficiency is normal.Expand which results in the difference of two amplified allele efficiency, i.e. allele
Increase uneven.For the amplified allele energy imbalance appeared above, prior art is solved using following strategy：Pin
The allele design specific primer low to amplification efficiency, is solved using the mode of specific amplification.
But existing solution has following drawback：The single allele of specific amplification, it is multiple facing height
Miscellaneous gene, such as HLA genes, there is substantial amounts of allele.Some allele can not fill because of complicated secondary structure or unwind
Grading causes amplification inequality.If using an amplified allele imbalance is found, just designing a specific primer plan
Slightly, will soon face needs to design a large amount of primers, the extremely difficult situation of design of primers.
The content of the invention
In order to effectively solve the unbalanced problem of amplified allele, solve allele the invention provides one kind and expand
Increase unbalanced method.
The solution unbalanced method of amplified allele provided by the present invention, is directed to due to " part equipotential base to be measured
Complicated secondary structure is formed because being unwind in PCR amplification procedures or/and DNA double chain is unwind insufficient, and remaining equipotential base to be measured
Gene-amplification efficiency is normal " caused by amplified allele unbalanced situation, specifically may include following steps：By to original
There are addition PCR additives in PCR reaction systems, to reduce the stability of duplex DNA, make secondary structure easily upon opening, DNA gathers
Synthase is more prone to by secondary structure area when extending, so as to solve the unbalanced problem of amplified allele to be measured；Institute
Stating original PCR reaction systems is：Enter performing PCR amplification to the allele to be measured, the amplified allele to be measured occur not
The PCR reaction systems used during balance.
Wherein, the PCR additives are a kind of synergist in PCR reactions, and raising is traditionally played in PCR reactions
Yield and prevent the beneficial effects such as invalid amplification that the specificity of PCR reactions, increase PCR are expanded.
In the process, the PCR additives can for any of any PCR additives or appoint it is several, for example：Sweet tea
Dish alkali, formamide, DMSO etc..
Wherein, the amount of the PCR additives added into original PCR reaction systems is to reach the equipotential to be measured
Gene magnification is balanced, i.e., amplified allele efficiency is close or consistent for degree.
In methods described, in PCR amplification system, the PCR additives can be stepped up in concentration gradient form
Amount, the amplified allele balance to be measured can be made always by increasing to, i.e. amplified allele efficiency is close or consistent.
In the process, the allele to be measured can be any gene, particularly highly complex gene, for example
HLA genes, it is specific such as HLA-DQB1 genes.
In the present invention, the allele to be measured is specially genotype respectively DQB1*03:03:02(IMGT/HLA
Acc No:) and DQB1*06 HLA00629:01:01(IMGT/HLA Acc No:HLA00643 HLA-DQB1 genes).
It is the sweet tea by adding 0.4-0.6M into original PCR reaction systems in first example of the present invention
To solve the allele to be measured, (genotype is respectively DQB1*03 to dish alkali:03:02 and DQB1*06:01:01 HLA-DQB1
Gene) amplification inequality the problem of.
It is by adding 1%-2.5% volumes into original PCR reaction systems in second example of the present invention
To solve the allele to be measured, (genotype is respectively DQB1*03 to the formamide of percentage composition:03:02 and DQB1*06:01:
01 HLA-DQB1 genes) amplification inequality the problem of.
It is by adding 1%-5% volumes hundred into original PCR reaction systems in the 3rd example of the present invention
To solve the allele to be measured, (genotype is respectively DQB1*03 to the DMSO of point content:03:02 and DQB1*06:01:01
HLA-DQB1 genes) amplification inequality the problem of.
It is respectively DQB1*03 to the genotype:03:02 and DQB1*06:01:01 HLA-DQB1 genes enter performing PCR expansion
During increasing, the primer pair used is the single-stranded primer pair constituted of two DNA shown in the sequence 1 in sequence table and sequence 2.
Accordingly, original PCR reaction systems are referring specifically to embodiment 1.
It is respectively DQB1*03 to the genotype:03:02 and DQB1*06:01:01 HLA-DQB1 genes enter performing PCR expansion
During increasing, amplification program is as follows：96℃3min；96 DEG C of 25s, 60 DEG C of 45s, 72 DEG C of 45s, 35 circulations；72℃5min；12 DEG C of guarantors
Deposit.
The present invention be directed to due to " part allele to be measured unwind in PCR amplification procedures to be formed complicated secondary structure or/
Unwind with DNA double chain insufficient, and remaining amplified allele efficiency to be measured is normal " caused by amplified allele it is uneven
Situation there is provided solve amplified allele not by adding certain density PCR additives into original PCR reaction systems
The method of balance.It is demonstrated experimentally that this method can be solved effectively because part allele to be measured is in PCR amplification procedures really
In unwind to form complicated secondary structure or/and DNA double chain unwind it is insufficient, and remaining amplified allele efficiency to be measured it is normal and
Caused amplified allele is uneven.
Brief description of the drawings
Fig. 1 is to DQB1*03:03:02/DQB1*06:01:Added when 01 combined sample enters performing PCR amplification in reaction system
The sequencing result of various concentrations betaine group.
Fig. 2 is to DQB1*03:03:02/DQB1*06:01:Added when 01 combined sample enters performing PCR amplification in reaction system
The sequencing result of various concentrations formamide group.
Fig. 3 is to DQB1*03:03:02/DQB1*06:01:Added when 01 combined sample enters performing PCR amplification in reaction system
The sequencing result of various concentrations DMSO groups.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment, passing through PCR additives, to solve amplified allele uneven
PCR additives are a kind of synergist in PCR reactions, and frequent species has formamide, glycine betaine, DMSO etc..This hair
Bright research finds that PCR additives can reduce the stability of duplex DNA, make secondary structure easily upon opening, contributes to DNA to gather
Synthase extends through secondary structure area.During PCR, the single-stranded formation of an allele in two allele is answered
Miscellaneous secondary structure or DNA double chain are unwind insufficient, cause asking for two inconsistent i.e. amplification inequalities of amplified allele efficiency
Topic, can add the PCR additives of certain density similar glycine betaine to solve problems.
This method for solving amplified allele imbalance problem by PCR additives is carried out with instantiation below
Illustrate.
Allele：Genotype is respectively DQB1*03:03:02(IMGT/HLA Acc No:) and DQB1* HLA00629
06:01:01(IMGT/HLA Acc No:HLA00643 HLA-DQB1 genes).
1st, original PCR primer, reaction system and response procedures
(1) PCR primer
For expanding DQB1*03:03:02 and DQB1*06:01:The primer of 01 allele：
DQB-F：5 '-TGATTCCCCGCAGAGGATTTCG-3 ' (sequence 1)；
DQB-R：5 '-AGGGGCGACGACGCTCACCTC-3 ' (sequence 2).
(2) reaction system：
(3) response procedures：
96℃3min；96 DEG C of 25s, 60 DEG C of 45s, 72 DEG C of 45s, 35 circulations；72℃5min；12 DEG C of preservations.
As a result show：Use as above original PCR primer, reaction system and response procedures to genotype for DQB1*03:03:
02/DQB1*06:01:When the HLA-DQB1 genes of 01 heterozygous enter performing PCR amplification, amplified allele can be produced unbalanced
Situation, DQB1*03:03:02/DQB1*06:01:DQB1*03 in 01 combination:03:02 sequencing peak is very low, that is, occurs in that amplification
Unbalanced situation.As shown in Figure 1, Figure 2, in Fig. 3 shown in " 0M "/" 0% ".
2nd, the PCR amplification conditions after changing
Amplimer and pcr amplification reaction program Complete Synchronization rapid 1.
Reaction system is changed to (1) as follows or (2) or (3)：
(1) on the basis of the reaction system of step 1, add final concentration be respectively 0M, 0.2M, 0.4M, 0.6M, 0.8M,
1.0M glycine betaine.
(2) on the basis of the reaction system of step 1, it is respectively 0%, 1%, 1.25%, 2.5%, 5% to add final concentration
Formamide.Wherein, % represents volumn concentration.
(3) on the basis of the reaction system of step 1, it is respectively 0%, 1%, 5%, 10%, 12% to add final concentration
DMSO.Wherein, % represents volumn concentration.
As a result show：Use the PCR amplification conditions after change to genotype for DQB1*03:03:02/DQB1*06:01:01
The HLA-DQB1 genes of heterozygous enter performing PCR amplification, in the case where being added without PCR additives (0M/0% concentration), heterozygosis position
Point DQB1*03:03:02 sequencing peak is very low；With the increase of PCR additive concentrations, DQB1*03:03:02 sequencing peak is relatively high
Degree gradually rises, DQB1*06:01:01 sequencing peak relative altitude is gradually reduced.It is 0.4M-0.6M scopes, first in beet alkali concn
Amide concentration be 1%-2.5%, DMSO concentration in 1%-5%, peak height no significant differences are sequenced in 2 allele.Respectively such as
Shown in Fig. 1, Fig. 2, Fig. 3.
<110>Beijing Bo Aojing allusion quotations Bioisystech Co., Ltd；Chengdu Bo Aojing cores bio tech ltd
<120>The unbalanced method of amplified allele is solved by PCR additives
<130> GNCLN170605
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
tgattccccg cagaggattt cg 22
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
aggggcgacg acgctcacct c 21
Claims (10)
1. one kind solve due to " part allele to be measured unwind in PCR amplification procedures to be formed complicated secondary structure or/and
DNA double chain is unwind insufficient " caused by the unbalanced method of amplified allele, comprise the following steps：By to original PCR
PCR additives are added in reaction system, to reduce the stability of duplex DNA, so as to solve the amplified allele to be measured
Unbalanced problem；Original PCR reaction systems are：It is described to enter performing PCR amplification to allele to be measured, occur described to be measured
The PCR reaction systems used during amplified allele imbalance situation.
2. according to the method described in claim 1, it is characterised in that：The PCR additives be glycine betaine, formamide, DMSO or
Any of other PCR additives are appointed several.
3. method according to claim 1 or 2, it is characterised in that：Described in being added into original PCR reaction systems
The amount of PCR additives is degree to reach the amplified allele balance to be measured.
4. according to any described method in claim 1-3, it is characterised in that：The allele to be measured be HLA genes or
Other any genes.
5. method according to claim 4, it is characterised in that：The allele to be measured is that genotype is respectively DQB1*
03:03:02 and DQB1*06:01:01 HLA-DQB1 genes.
6. method according to claim 5, it is characterised in that：It is by original PCR reactants in methods described
0.4-0.6M glycine betaine is added in system to solve the unbalanced problem of amplified allele to be measured.
7. method according to claim 5, it is characterised in that：It is by original PCR reactants in methods described
The formamide of 1%-2.5% volumn concentrations is added in system to solve the unbalanced problem of amplified allele to be measured
's.
8. method according to claim 5, it is characterised in that：It is by original PCR reactants in methods described
The DMSO of 1%-5% volumn concentrations is added in system to solve the unbalanced problem of amplified allele to be measured.
9. according to any described method in claim 5-8, it is characterised in that：It is respectively DQB1*03 to the genotype:
03:02 and DQB1*06:01:When 01 HLA-DQB1 genes enter performing PCR amplification, the primer pair used is sequence 1 in sequence table
With the primer pair of two single-stranded compositions of DNA shown in sequence 2.
10. according to any described method in claim 5-8, it is characterised in that：It is respectively DQB1*03 to the genotype:
03:02 and DQB1*06:01:When 01 HLA-DQB1 genes enter performing PCR amplification, the amplification program of use is as follows：96℃3min；
96 DEG C of 25s, 60 DEG C of 45s, 72 DEG C of 45s, 35 circulations；72℃5min；12 DEG C of preservations.
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Inventor after: Liu Xiang Inventor after: Lan Gengxin Inventor after: Li Hong Inventor after: Zhao Xiaodan Inventor after: Wang Lei Inventor after: Li Li Inventor after: Chen Lu Inventor after: Qin Xuefei Inventor after: Li Jinxiu Inventor before: Liu Xiang Inventor before: Lan Gengxin Inventor before: Zhao Xiaodan Inventor before: Wang Lei Inventor before: Chen Lu Inventor before: Qin Xuefei Inventor before: Li Jinxiu |
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Application publication date: 20170725 |