CN110184344A - Detect the method and primer pair of HTT gene C AG trinucleotide repeats sequence - Google Patents
Detect the method and primer pair of HTT gene C AG trinucleotide repeats sequence Download PDFInfo
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Abstract
The present invention provides the methods and primer pair of detection HTT gene C AG trinucleotide repeats sequence, this method comprises: synthetic primer pair, 5 ' ends of the forward primer of primer pair are marked at least one end in 5 ' ends of reverse primer by fluorophor；Genomic DNA is extracted from sample to be tested, as DNA profiling；PCR amplification system of the configuration comprising primer pair and amplification template；PCR amplification system is carried out amplification reaction, the PCR product containing CAG trinucleotide repeats sequence is obtained；Capillary electrophoresis detection PCR product, and according to the HTT gene C AG Trinucleotide repeats number of electrophoresis detection result calculating sample to be tested.This programme can be detected trinucleotide repeats sequence without sequencing.
Description
Technical field
The present invention relates to technical field of biological, in particular to detect the side of HTT gene C AG trinucleotide repeats sequence
Method and primer pair.
Background technique
Huntington's chorea (Huntington ' s disease, HD) is also known as Huntington disease, is a kind of concealment onset, with dance
Sample involuntary movement, phrenoblabia and the dull-witted hereditary neurological degenerative disease being characterized are stepped, is autosomal dominant inheritance.
It is by the CAG on the huntingtin gene IT15 (interesting transcript 15) of No. 4 the short arm of a chromosome that it is pathogenic
Caused by trinucleotide amplification mutation extremely.
HD is autosomal dominant inherited disease, and the expression product of IT15 gene (also referred to as HD gene or HTT gene) is big
Polypeptide Huntingdon (HTT) albumen of small about 3144 amino acid.The Huntington protein of mutation contains the glutamine residue of amplification
Chain, pathological change are mainly limited to central nervous system, the most prominent with caudate nucleus and shell core (neostriatum) atrophy.Although mesh
Before think HD morbidity with mutation the toxicity of HTT albumen it is related, but the disease specifically pathophysiological mechanism it is not yet clear.Full generation
The illness rate of boundary HD is about 2.7/100 000, and disease incidence is about annual 0.38/100 000.Europe, North America, Australia trouble
Sick rate is about 5.7/100 000, and the illness rate in Asia is about 0.4/100 000.
HTT genetic test is most important auxiliary examination, and the threshold value that the CAG of Disease-causing gene HTT repeats copy number is 36: small
It is not pathogenic in 36,36~39 incomplete penetrances；Greater than 39 complete penetrances.Genetic test sensibility 98.8%, specificity
100%.CAG repeats the main determining factor that copy number is age of onset in HTT gene, and repetition copy number is higher, age of onset
It is more early.HTT gene detects before also can be used as the symptom of risky family member, and the patient for carrying pathogenic HTT gene is feasible
Antenatal exaination.
The method of domestic detection HD Trinucleotide repeats number is at present, PCR (Polymerase Chain Reaction,
Polymerase chain reaction) product direct Sequencing or gel extraction PCR product segment direct Sequencing.But since HTT gene C AG is repeated
Segment G/C content is high, and secondary structure is complicated, is often difficult to obtain ideal sequencer map, cause to clinical diagnosis Huntington's chorea
Difficulty.
Summary of the invention
The present invention provides the methods and primer pair of detection HTT gene C AG trinucleotide repeats sequence, without sequencing
Detect trinucleotide repeats sequence.
In order to achieve the above object, the present invention is achieved through the following technical solutions:
Firstly, may include following step the present invention provides the method for detection HTT gene C AG trinucleotide repeats sequence
Rapid: at least one end in synthetic primer pair, 5 ' ends of the forward primer of the primer pair and 5 ' ends of reverse primer is by fluorescent base
Group's label；Genomic DNA is extracted from sample to be tested, as DNA profiling；Configuration includes the primer pair and the amplification template
PCR amplification system；The PCR amplification system is carried out amplification reaction, the PCR containing CAG trinucleotide repeats sequence is obtained
Product；PCR product described in capillary electrophoresis detection, and calculate according to electrophoresis detection result the HTT gene C AG of the sample to be tested
Trinucleotide repeats number.
In detail, when carrying out PCR amplification using the primer of fluorescent marker, can make equally to mark on the chain band of PCR product
There is fluorophor.This DNA fragmentation with fluorescent molecule can be inhaled in carrying out Capillary Electrophoresis by laser scanning with fluorescence
The mode for receiving peak is recorded.And then the HTT gene C AG Trinucleotide repeats number of sample to be tested can be calculated based on this.
As it can be seen that when detecting Trinucleotide repeats catastrophe using fluorescent marker str locus analytical technology, without pair
PCR product, which carries out sequencing, can be obtained Trinucleotide repeats number, therefore method is easy, reliable, at low cost and be easy to automate.
Preferably, the nucleotide sequence of the forward primer of the primer pair is as shown in the SEQ ID NO.1 and reverse primer
Nucleotide sequence is as shown in SEQ ID NO.2.That is:
HTT F:5 '-TGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3 '
HTT R:5 '-CGGCTGAGGAAGCTGAGGAG-3 '.
Preferably, the fluorophor includes any one in FAM, HEX, TAMARA, ROX.Wherein, FAM, that is, carboxyl
Fluorescein, as blue-fluorescence labeling dye；HEX, that is, chlordene fluorescein, as green fluorescence labeling dye；TAMARA, that is, tetramethyl
- 6 carboxyrhodamine of base, as yellow flag dyestuff；ROX, that is, rhodamine, as red fluorescence labeling dye.
Preferably, the PCR amplification system includes: 8~14 μ L, 2 × PCR μ of buffer20~30 L, 2.0mM of distilled water
The forward primer 0.5~3 the μ L, 0.5~3 μ of the reverse primer of 0.1~2 μm of ol of dNTP 8~12 μ L, 0.1~2 μm of ol
Archaeal dna polymerase 0.5-2 the μ L, the 5~500ng of DNA profiling of L, 0.5~2U/ μ L.
For example, for 8~14 μ L this value range, specific value can be 8,9,10,11,12,13 or 14；It is right
In 20~30 μ L this value range, specific value can be 20,22,24,26,28 or 30；For this value model of 8~12 μ L
It encloses, specific value can be 8,9,10,11 or 12；For 0.1~2 μm of ol this value range, specific value can for 0.1,
0.3,0.5,1,1.4,1.6,1.8 or 2；For 0.5~3 μ L this value range, specific value can for 0.5,1,1.5,2,
2.5 or 3；For 0.1~2 μm of ol this value range, specific value can be 0.1,0.3,0.5,1,1.5,1.8 or 2；For
0.5~3 μ L this value range, specific value can be 0.5,1,1.5,2,2.5 or 3；For this value of 0.5~2U/ μ L
Range, specific value can be 0.5,1,1.5 or 2；For this value range of 0.5-2 μ L, specific value can for 0.5,1,
1.5 or 2；For this value range of 5~500ng, specific value can be 5,10,30,50,100,200,300,400,450
Or 500.
In detail, archaeal dna polymerase may include e. coli dna polymerase, Klenowfragment, T7DNA polymerase,
T4DNA polymerase, the T7DNA polymerase of modified, Taq DNA polymerase, KOD FX Neo, KOD FX, KOD Plus, LA
Taq, r Taq, EX Taq, FastPfu Fly DNA polymerase, One Taq archaeal dna polymerase, phusion High-
Fidelity DNA Polymerase、Hot Start Taq DNA Polymerase、ThermoSequenase DNA
Any one or more in Polymerase.
Preferably, the reaction condition of the amplified reaction are as follows: 90~98 DEG C of 0.5~10min；90~95 DEG C 0.17~
1min, 50~70 DEG C of 0.17~1min, 68~72 DEG C of 0.5~10min, totally 25~45 recycle；68~72 DEG C of 0~10min.
For example, for 90~98 DEG C of this value ranges, specific value can be 90,91,93,95,97 or 98；It is right
In this value range of 0.5~10min, specific value can be 0.5,1,3,5,7,9 or 10；For 90~95 DEG C of this values
Range, specific value can be 90,91,92,93,94 or 95；For this value range of 0.17~1min, specific value can be with
It is 0.17,0.2,0.3,0.5,0.7,0.8,0.9 or 1；For 50~70 DEG C of this value ranges, specific value can for 50,
55,60,65 or 70；For 68~72 DEG C of this value ranges, specific value can be 68,69,70,71 or 72；For 0.5~
This value range of 10min, specific value can be 0.5,1,2,4,7,9 or 10；For 25~45 this value ranges, tool
Body value can be 25,30,35,40 or 45；For this value range of 0~10min, specific value can be 0,1,3,6,9
Or 10.
Specifically, using PCR instrument by executing following step, Lai Shixian PCR amplification:
Step A1: at 90~98 DEG C, 0.5~10min is preheated to PCR amplification system；
Step A2: at 90~95 DEG C, 0.17~1min of reaction of degeneration (RD) is carried out to the PCR amplification system after preheating；
Step A3: at 50~70 DEG C, 0.17~1min of annealing reaction is carried out to the PCR amplification system after reaction of degeneration (RD)；
Step A4: at 68~72 DEG C, 0.5~10min of extension is carried out to the PCR amplification system after annealing reaction；
Step A5: step A2-A4 is recycled 25~45 times, the PCR amplification system after each secondary extension is recycled
Reaction；
Step A6: at 68~72 DEG C, carrying out 0~10min of whole extension to the PCR amplification system after circular response,
Obtain the PCR product containing CAG trinucleotide repeats sequence.
Later, PCR product is kept in dark place at 2~15 DEG C.
Preferably, PCR product described in the capillary electrophoresis detection, and it is described to test sample according to the calculating of electrophoresis detection result
This HTT gene C AG Trinucleotide repeats number, comprising: detect the PCR product using Capillary Electrophoresis order-checking instrument；Utilize hair
Cons electrophoresis analyzes software and carries out data analysis to electrophoresis detection result, with the clip size of the determination PCR product；Utilize public affairs
Formula one calculates the HTT gene C AG Trinucleotide repeats number of the sample to be tested；
Wherein, the formula one includes: N=(L-83)/3；
Wherein, N is the HTT gene C AG Trinucleotide repeats number of the sample to be tested；L is that the segment of the PCR product is big
It is small, unit bp；83 be the sum of primer length and 5 ' and 3 ' end flanking bases numbers, unit bp；3 be the weight of CAG trinucleotide
Multiple unit base number, unit bp.
In detail, the upstream and downstream alkali of HTT gene C AG trinucleotide repeats sequence can be known from public gene pool in advance
Basic sequence, and the upstream and downstream primer of design is combined, so that it is determined that the sum of above-mentioned primer length and 5 ' and 3 ' end flanking bases numbers out
It is 83.In this way, clip size subtracts 83 value usually be HTT gene C AG trinucleotide repeats sequence base number.
Preferably, the sample to be tested includes blood, serum, blood plasma, buccal swab, any one in tissue.
In addition, the present invention also provides the primer pair of detection HTT gene C AG trinucleotide repeats sequence, the primer pair
Forward primer nucleotide sequence as shown in SEQ ID NO.1, the nucleotide sequence of the reverse primer of the primer pair is by SEQ
Shown in ID NO.2；At least one end in 5 ' ends of forward primer and 5 ' ends of reverse primer is marked by fluorophor.
In detail, above-mentioned specificity amplification primer can be designed according to HTT gene C AG trinucleotide repeats sequence.Then
Fluorophor is added in designing primer pair, specifically there can be fluorescent marker for only forward primer or upstream primer, or
Only reverse primer or downstream primer have fluorescent marker or forward and reverse primer to have fluorescent marker.
Preferably, the fluorophor includes any one in FAM, HEX, TAMARA, ROX.
In addition, the present invention also provides above-mentioned primer pair in preparation for detecting HTT gene C AG trinucleotide repeats sequence
Reagent or kit in application.
In addition, the answering in the detection of HTT gene C AG trinucleotide repeats sequence the present invention also provides above-mentioned primer pair
With.
Compared with the existing technology, the present invention at least can have it is following the utility model has the advantages that
The present invention provides the methods and primer pair of detection HTT gene C AG trinucleotide repeats sequence, by expanding PCR
Product after increasing carries out capillary electrophoresis detection, to calculate the HTT gene C AG Trinucleotide repeats number of sample to be tested.This inspection
Survey method can be detected trinucleotide repeats sequence without sequencing, therefore with spy that is easy, reliable, at low cost and being easy to automate
Point.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the agarose gel electrophoresis results that one embodiment of the invention provides；
Fig. 2 is the CAG repeating part of this sequencer map as one embodiment of the invention provides；
Fig. 3 is the CAG repeating part of the sequencer map for another sample that one embodiment of the invention provides；
Fig. 4 is another agarose gel electrophoresis results that one embodiment of the invention provides；
Fig. 5 is the another agarose gel electrophoresis results that one embodiment of the invention provides；
Fig. 6 is the fluorescent absorption peak figure of this PCR product as one embodiment of the invention provides；
Fig. 7 is the fluorescent absorption peak figure of the PCR product for another sample that one embodiment of the invention provides.
Specific embodiment
No special explanation, reagent used by the present invention is implemented are commercial goods, data used by the present invention is implemented
Library is disclosed online database.Following embodiment is exemplary, and for explaining only the invention, and should not be understood as to this
The limitation of invention.
Embodiment 1
Design and synthesize primer sets, comprising the following steps:
Step 1.1: according to HTT gene C AG trinucleotide repeats sequence and pertinent literature, specificity amplification primer is designed,
And forward primer is marked by fluorophor FAM.
FAM, that is, Fluoresceincarboxylic acid, as blue-fluorescence labeling dye, by Hua Da gene, Co., Ltd is synthesized.
The primer sequence designed are as follows:
HTT F:5 '-FAM-TGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3 '
HTT R:5 '-CGGCTGAGGAAGCTGAGGAG-3 '
Wherein, F refers to forward primer, and R refers to reverse primer.
Step 1.2: designed primer in synthesis 1.1.
Embodiment 2
Genomic DNA is extracted from sample to be tested, is included the following steps:
Step 2.1: reagent prepares.Including preparing dehydrated alcohol and kit.
The kit of preparation is poba gene group DNA extraction kit (Tiangeng): being added in rinsing liquid PW, GD using preceding elder generation
Enter dehydrated alcohol, volume is added referring to the label on bottle.
Step 2.2: genomic DNA is extracted from blood.It can specifically include following step (1)~(11):
(1) blood of 200 μ L is added in 1.5mL centrifuge tube.
(2) 20 μ L Proteinase K solution are added, mix.
(3) 200 μ L buffer GB are added, are sufficiently mixed by inversion, 70 DEG C of water-bath 10min (solution strain is limpid), briefly from
The heart.
(4) 200 μ L dehydrated alcohols are added, sufficiently oscillation mixes 15s (being likely to occur flocculent deposit at this time), brief centrifugation.
(5) previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12000rpm (13400 × g) is centrifuged 1min, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
(6) 500 μ L buffer GD (whether preoperation inspection has been added dehydrated alcohol) are added,
12000rpm (13400 × g) is centrifuged 1min, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
(7) 600 μ L rinsing liquid PW (whether preoperation inspection has been added dehydrated alcohol) are added,
12000rpm (13400 × g) is centrifuged 1min, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
(8) repetitive operation previous step.
(9) 12000rpm (13400 × g) is centrifuged 2min, outwells waste liquid；Adsorption column CB3 is placed in and is placed at room temperature for several minutes
(5~10min), thoroughly to dry rinsing liquid remaining in adsorbent material.
(10) adsorption column CB3 is placed in 1.5mL sterile centrifugation tube and (cuts the lid of centrifuge tube in advance), to adsorbed film
Intermediate position 100 μ L elution buffer TE are vacantly added dropwise, be placed at room temperature for 5min, 12000rpm (13400 × g) is centrifuged 2min,
Solution is collected into centrifuge tube.
(11) solution is transferred in a new 1.5mL sterile centrifugation tube.
In embodiment 2, through step 2.2, the genomic DNA of 3 random normal controls samples is obtained, details please refer to
Following table 1.
Table 1
| Number | Concentration (ng/ μ L) | A260/A280 | Template source details |
| FW | 35.1 | 1.897 | Normal human blood genomic DNA |
| LY | 28.35 | 1.903 | Normal human blood genomic DNA |
| LH | 31.35 | 1.923 | Normal human blood genomic DNA |
In table 1, A260 and A280 are respectively represented in 260nm and the absorbance at 280nm.
As shown in Table 1, the value of the A260/A280 of 3 samples is in 1.8~2.0 this critical field (indicating that DNA is purer)
It is interior.
Embodiment 3
PCR product amplification method, may comprise steps of:
Step 3.1: being with 15 CAP samples of genomic DNA obtained in step 2.2 and 2016-2018 respectively
DNA profiling, and using the primer synthesized in step 1.2, to configure PCR reaction system.The concrete composition of this reaction system is such as
Shown in following table 2.
Table 2
| Agent formulations | Volume |
| 2×KOD FX Buffer | 25μL |
| 2.0mM dNTP | 10μL |
| HTT-F(10uM) | 1.5μL |
| HTT-R(10uM) | 1.5μL |
| KOD FX enzyme | 1μL |
| DNA | 1μL |
| DI water | 11μL |
CAP (COLLEGE OF AMERICAN PATHOLOGISTS, College of American Pathologists) laboratory is in nineteen forty-six
It establishes, is the leader that the Good Laboratory being widely acknowledged guarantees.The execution method that the room CAP interstitial is commented is organizer to participation
Laboratory provides a set of sample to be measured, participates in laboratory and completes the detection of the set sample under normal conditions, and result is returned
To organizer, organizer evaluates the consistency of reported result and expected results, and is determined by the comparison of laboratory monitoring
The calibration in laboratory, detectability and monitor its continuous capability.
Step 3.2: according to PCR reaction condition shown in following Table 3, the program of PCR instrument is set.
Table 3
Step 3.3: the PCR instrument set using program carries out PCR expansion to the PCR reaction system configured in step 3.1
Increase reaction, obtains PCR product.
In embodiment 3, through step 3.3, a batch has expanded 15 CAP samples of 2016-2018 and 3 random normal persons
The genomic DNA of check sample, and obtain corresponding PCR product.
Embodiment 4
Sequencing is carried out to the amplified production of above-mentioned 18 samples simultaneously in the same time and fluorescent marker STR is analyzed.
PCR product after the detection amplification of 4.1 PCR sequencing PCRs, may comprise steps of:
Step 4.1.1: PCR product obtained in agarose gel electrophoresis detecting step 3.3, to verify PCR product segment
Size.The electrophoretogram of 18 samples is as shown in Figure 1.
Step 4.1.2: electrophoresis detection result obtained in step 4.1.1 is sent to sequencing company, gel extraction PCR is carried out
Product segment carrys out direct Sequencing, and software analyzes sequencing result.Specifically, using the above-mentioned reverse primer without fluorophor as
Sequencing primer.
The analysis result of PCR sequencing PCR is as described in Table 4.
Wherein, as an example, the sequencer map of a sample CAG repeating part as shown in Fig. 2, another sample sequencing
The CAG repeating part of figure is as shown in Figure 3.
The discovery of PCR sequencing PCR test result:
(1) it please refers to table 4 and combines Fig. 1, find the sequencing biobelt of part heterozygosis sample from that must not separate closely and, so that phase
Answer CAG repeat number unknowable；
(2) table 4 is please referred to, in the CAG repeat number that PCR sequencing PCR measures, a part is not within the scope of CAP gives；
(3) Fig. 2 and Fig. 3 are please referred to, sequencer map tends to have miscellaneous peak, the bad interpretation of beginning that CAG is counted.
As it can be seen that the CAG repeat number testing result of PCR sequencing PCR allows of no optimist.
PCR product after the amplification of 4.2 capillary electrophoresis detections, may comprise steps of:
Step 4.2.1: using GS500LIZ as internal standard, resulting using ABI 3730XL sequenator detecting step 3.3
Amplified production.
Step 4.2.2: data analysis is carried out with GeneMarker software, determines product clip size.
Step 4.2.3: the product clip size determined with GeneMarker software according to step 4.2 utilizes above-mentioned formula
One calculates the HTT gene C AG Trinucleotide repeats number of sample to be tested.
The result of STR analysis please refers to following table 5.In table 5, number 1~15 respectively indicates 2016-MGL2-04,2016-
MGL2-05、2016-MGL2-06、2016-MGL2-13、2016-MGL2-14、2016-MGL2-15、2017-MGL2-04、
2017-MGL2-05、2017-MGL2-06、2017-MGL2-13、2017-MGL2-14、2017-MGL2-15、2018-MGL2-
04、2018-MGL2-05、2018-MGL2-06。
Embodiment 5
PCR product obtained in 5.1 agarose gel electrophoresis detecting steps 3.3, to verify the size of PCR product segment.
It is executed respectively twice in different time.
Two electrophoretogram difference are as shown in Figure 4 and Figure 5.
The not homogeneous agarose gel electrophoresis results that Fig. 3 to Fig. 5 can be seen that each sample by contrast are consistent.
5.2 methods for using above-mentioned 4.2 carry out fluorescent marker STR analysis to the amplified production of above-mentioned each sample.In not
It is executed respectively twice with the time.
The result of STR analysis twice is as described in Table 5.Wherein, as an example, sample number LY, when experiment
Between for 2018.9.11 fluorescent absorption peak figure it is as shown in Figure 6；Sample number is 2017-MGL2-15, and experimental period is
2018.9.13 fluorescent absorption peak figure it is as shown in Figure 7.
In fluorescent absorption peak figure, in the front of each main peak, theoretically can a miscellaneous peak, can be described as shadow peak
(stutter peak) is then known as shadow band/ghost band (stutter band) in gel electrophoresis.
Shadow peak is as caused by the sliding mispairing (polymerase slippage) of Taq enzyme, this is the spy of Taq enzyme
Property (other archaeal dna polymerases also have this problem), is difficult to be overcome by optimized expansion condition at present.Existing research table
It is bright, by change/optimized expansion condition, the formula of buffer and selection of polymerase type etc., to attempt to overcome shadow peak
The problem of, but it is ineffective.
(in the present embodiment, recurring unit is the length of the clip size at a shadow peak recurring unit usually smaller than main peak
CAG, therefore this length is 3bp), more rare, the case where also will appear small 2~3 recurring units.Usually compare at shadow peak
Main peak is low, and peak area also can be smaller.
Although being difficult to overcome shadow peak problem by optimized expansion condition at present, based on above-mentioned analysis, can lead to
Experience is crossed to judge main peak and shadow peak.Stripe size is pressed, is greatly main peak.
As shown in FIG. 6 and 7, appearance situation is consistent with foregoing description.
As shown in fig. 6, record has a main peak in fluorescent absorption peak figure shown in Fig. 6, phase can get according to peak position out
Answer the clip size of DNA fragmentation.Due to only having recorded a main peak, therefore Fig. 6 is usually that a CAG repeats detection STR analysis homozygosis
As a result.Referring to FIG. 6, the clip size of amplified fragments is 127.6bp, and then the CAG repeat number of the amplified fragments can be calculated
It is 15.
As shown in fig. 7, in fluorescent absorption peak figure shown in Fig. 7 record there are two main peak, according to it is each go out peak position can obtain
Obtain the clip size of corresponding DNA fragments.Since there are two main peaks for record, therefore Fig. 7 is usually that CAG repetition detection STR analysis is miscellaneous
Close result.Referring to FIG. 7, the clip size of an amplified fragments is 136.7bp, and then can calculate this in two amplified fragments
The CAG repeat number of amplified fragments is 18, and the clip size of another amplified fragments is 164.1bp, and then can calculate the amplification piece
The CAG repeat number of section is 27.
As shown in FIG. 6 and 7, the specific value of the clip size and repeat number determined, can be in fluorescent absorption peak figure
It is identified.
The discovery of capillary electrophoresis test result:
(1) table 5 is please referred to, two fluorescent absorption peaks separation of discovery heterozygosis sample is obvious easily to be distinguished；
(2) fluorescent marker STR analysis can provide the complete result of each sample, and the CAG repeat number measured is in CAP
In the range of giving；
(3) it please refers to table 5 and combines Fig. 6 and Fig. 7, it is found that the fluorescent absorption spike shape in fluorescent absorption peak figure is obvious, it is miscellaneous
Peak is few and easily distinguishable miscellaneous peak；
(4) by the comparing result listed in table 5 it is found that coincidence rate is 100%, show that the embodiment of the present invention is practical.
As it can be seen that the CAG repeat number testing result of capillary electrophoresis is more optimistic and ideal.
In conclusion the embodiment of the present invention establishes a kind of fluorescent marker STR detection tri- nucleosides of mankind HTT gene C AG
The primer and method of sour repetitive sequence, and through 15 CAP Inter-laboratory comparisons samples of detection and 3 normal control samples, show this
Primer and method it is practical.The embodiment of the present invention uses fluorescent marker str locus analysis method, is not necessarily to gel extraction PCR
Product segment and direct Sequencing reduce the difficulty as caused by sequencing link, make HTT gene C AG trinucleotide repeats sequence
Detection become analysis it is easy, it is accurate, be easy to automate, and it is horizontal to help to improve Huntington's chorea clinical diagnosis.
Table 4
Table 5
It should be noted that, in this document, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment for including a series of elements not only includes those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or equipment institute it is intrinsic
Element.In the absence of more restrictions, the element limited by sentence " including one ", it is not excluded that
There is also other identical factors in the process, method, article or apparatus that includes the element.Finally need to illustrate
Be: the foregoing is merely presently preferred embodiments of the present invention, is only used to illustrate the technical scheme of the present invention, and is not intended to limit this hair
Bright protection scope.Any modification, equivalent substitution, improvement and etc. done all within the spirits and principles of the present invention include
Within the scope of the present invention.
SEQUENCE LISTING
<110>Beijing and conjunction medical diagnostic techniqu limited liability company
<120>method and primer pair of HTT gene C AG trinucleotide repeats sequence are detected
<130> 2019.6.6
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213>artificial sequence (Artificial sequence)
<220>
<223>for detecting the forward primer of HTT gene C AG trinucleotide repeats sequence
<400> 1
tgaaggcctt cgagtccctc aagtccttc 29
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<220>
<223>for detecting the reverse primer of HTT gene C AG trinucleotide repeats sequence
<400> 2
cggctgagga agctgaggag 20
Claims (10)
1. the method for detecting HTT gene C AG trinucleotide repeats sequence characterized by comprising
At least one end in synthetic primer pair, 5 ' ends of the forward primer of the primer pair and 5 ' ends of reverse primer is by fluorescent base
Group's label；
Genomic DNA is extracted from sample to be tested, as DNA profiling；
PCR amplification system of the configuration comprising the primer pair and the amplification template；
The PCR amplification system is carried out amplification reaction, the PCR product containing CAG trinucleotide repeats sequence is obtained；
PCR product described in capillary electrophoresis detection, and calculate according to electrophoresis detection result the HTT gene C AG of the sample to be tested
Trinucleotide repeats number.
2. the method according to claim 1, wherein
The nucleotide sequence of the forward primer of the primer pair as shown in SEQ ID NO.1 and the nucleotide sequence of reverse primer by
Shown in SEQ ID NO.2；
And/or
The fluorophor includes any one in FAM, HEX, TAMARA, ROX.
3. the method according to claim 1, wherein
The PCR amplification system includes: 8~14 μ L, 2 × PCR μ of buffer20~30 L, 2.0mM dNTP8~12 μ of distilled water
0.5~3 μ L, 0.5~2U/ μ of the reverse primer of the forward primer 0.5~3 the μ L, 0.1~2 μm of ol of L, 0.1~2 μm of ol
Archaeal dna polymerase 0.5-2 the μ L, the 5~500ng of DNA profiling of L.
4. the method according to claim 1, wherein
The reaction condition of the amplified reaction are as follows: 90~98 DEG C of 0.5~10min；90~95 DEG C of 0.17~1min, 50~70 DEG C
0.17~1min, 68~72 DEG C of 0.5~10min, totally 25~45 recycle；68~72 DEG C of 0~10min.
5. the method according to claim 1, wherein
PCR product described in the capillary electrophoresis detection, and calculate according to electrophoresis detection result the HTT gene of the sample to be tested
CAG Trinucleotide repeats number, comprising:
The PCR product is detected using Capillary Electrophoresis order-checking instrument；
Data analysis is carried out to electrophoresis detection result using capillary electrophoresis analysis software, with the segment of the determination PCR product
Size；
The HTT gene C AG Trinucleotide repeats number of the sample to be tested is calculated using formula one；
Wherein, the formula one includes: N=(L-83)/3；
Wherein, N is the HTT gene C AG Trinucleotide repeats number of the sample to be tested；L is the clip size of the PCR product,
Unit is bp；83 be the sum of primer length and 5 ' and 3 ' end flanking bases numbers, unit bp；3 be the repetition list of CAG trinucleotide
Bit base number, unit bp.
6. according to claim 1 to any method in 5, which is characterized in that
The sample to be tested includes blood, serum, blood plasma, buccal swab, any one in tissue.
7. detecting the primer pair of HTT gene C AG trinucleotide repeats sequence, which is characterized in that
The nucleotide sequence of the forward primer of the primer pair as shown in SEQ ID NO.1, the reverse primer of the primer pair
Nucleotide sequence is as shown in SEQ ID NO.2；
At least one end in 5 ' ends of forward primer and 5 ' ends of reverse primer is marked by fluorophor.
8. primer pair according to claim 7, which is characterized in that
The fluorophor includes any one in FAM, HEX, TAMARA, ROX.
9. primer pair described in claim 7 or 8 is preparing the reagent for detecting HTT gene C AG trinucleotide repeats sequence
Or the application in kit.
10. application of the primer pair described in claim 7 or 8 in the detection of HTT gene C AG trinucleotide repeats sequence.
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