CN103045726B - Multiple hybrid dna or RNA sequence are carried out to method and the device of gene sequencing - Google Patents
Multiple hybrid dna or RNA sequence are carried out to method and the device of gene sequencing Download PDFInfo
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Abstract
The invention discloses method and device that a kind of DNA or RNA sequence to multiple mixing carries out gene sequencing, add random tags mainly to before building storehouse each DNA or RNA template sequence two ends.Method comprises: mixed with the first downstream primer and DNA sequence dna by the first upstream primer containing random tags and joint, carries out the multiplexed PCR amplification of two PCR circulations, this PCR primer of purifying, obtains a DNA product; One DNA product is mixed with the second upstream primer containing sample index sequence and joint and the second downstream primer, carries out PCR reaction, obtain the second PCR primer; The 2nd DNA product is obtained after purifying; 2nd DNA product is checked order, obtains the sequencing result of each DNA sequence dna.The present invention, by introducing random tags to each DNA molecular, can improve the accuracy of order-checking, significantly reduce the error rate of order-checking, and Accurate Determining often plants the copy number of DNA.
Description
Technical field
The present invention relates to technical field of gene detection, particularly relate to method and device that a kind of DNA or RNA sequence to multiple mixing carries out gene sequencing.
Background technology
In immunocyte group, the diversity of t lymphocyte receptor molecule (TCR, Tcellreceptor) and b lymphocyte receptor molecule (BCR, Bcellreceptor) is in 10 ten thousand to hundred ten thousand.α and the β chain of composition TCR or BCR, not only each self-structure is different, and the formation of each chain also experienced by the combination of numerous gene fragment in V, J or V, J, D different zones.
When studying the diversity of TCR and BCR, numerous gene fragment in V, J or V to TCR and BCR, J, D different zones is needed to check order.In general, existing sequencing technologies can adapt to the requirement of general gene sequencing.But for the diversity of research TCR and BCR, the error rate of existing sequencing technologies is too high, to such an extent as to cannot according to the sequencing result obtained, the technicality between the numerous gene fragments distinguishing out.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of method and the device that multiple hybrid dna or RNA sequence are carried out to gene sequencing, existing sequencing technologies can be utilized to improve the accuracy of gene sequencing, significantly reduce the error rate of gene sequencing.
For solving the problems of the technologies described above, the present invention is directed to different starting templates, have employed two kinds of technical schemes respectively, for with the DNA sequence dna of multiple mixing for template to carry out gene sequencing, the technical scheme adopted is: comprising: mixed the solution of the first upstream primer sequence with N number of DNA sequence dna of t lymphocyte receptor TCR and/or b lymphocyte receptor BCR in the first downstream primer sequence and immunocyte group by M, and carry out the PCR reaction of two circulations, obtain the first PCR primer, wherein, described M and N is natural number, M is more than or equal to the N of 100 times, described first upstream primer sequence or the first downstream primer sequence comprise joint sequence from 5 ' end successively to 3 ' end, sequence label and the first upstream base primer sequence or the first downstream base primer sequence, the sequence label of often pair of first upstream primer sequence and the first downstream primer sequence makes each DNA sequence dna after PCR reacts, have the sequence label being different from other DNA sequence dna, described joint sequence is the sequence making each DNA sequence dna carry out PCR reaction comparably, described first upstream base primer sequence and the first downstream base primer sequence respectively with 3 ' termini-complementary of described DNA sequence dna two strands, separation and purification is carried out to described first PCR primer, obtain a DNA product, the two ends of every article of strand of a described DNA product all comprise the complementary sequence of described first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of described first upstream primer sequence, the solution of one DNA product of described acquisition with the second upstream primer sequence and the second downstream primer sequence is mixed, and carry out the PCR reaction of 20 five to four ten circulations, obtain the second PCR primer, wherein, described second upstream primer sequence comprises the joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the joint sequence in described first downstream primer sequence, separation and purification is carried out to described second PCR primer, obtains the 2nd DNA product, gene sequencing is carried out to described 2nd DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna.
Wherein, described joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, described sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence are linked in sequence, described sequencing primer sequence connects the sequence label of described first upstream primer sequence or the first downstream primer sequence, and described order-checking logo sequence is for distinguishing the multiple DNA sequence dnas from different sample.
Wherein, described second upstream primer sequence comprises the order-checking company joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the order-checking company joint sequence in described first downstream primer sequence.
Wherein, the base number of described sequence label is eight, or seven, or six.
Wherein, described gene sequencing is carried out to the 2nd DNA product, and analyze sequencing result, obtain the step of the sequencing result of each DNA sequence dna, comprising: gene sequencing is carried out to described 2nd DNA product, obtain DNA sequencing result; Described DNA sequencing result is carried out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna; Belong to all DNA sequencing results of same DNA sequence dna relatively, and add up each position in described all DNA sequencing results and occur the frequency of identical base; Judge that described each position occurs whether the frequency of identical base reaches predetermined threshold value; If reach predetermined threshold value, then belong to the base of the described position of same DNA sequence dna described in determining, according to described method, described in can determining, belong to the base of all positions of same DNA sequence dna.
For solving the problems of the technologies described above, another technical solution used in the present invention is: provide a kind of DNA sequence dna to multiple mixing to carry out the device of gene sequencing, described device comprises: first obtains module, for M is mixed the solution of N number of DNA sequence dna of the first upstream primer sequence and t lymphocyte receptor in the first downstream primer sequence and immunocyte group and/or b lymphocyte receptor, and carry out the PCR reaction of two circulations, obtain the first PCR primer, wherein, described M and N is natural number, M is more than or equal to the N of 100 times, described first upstream primer sequence or the first downstream primer sequence comprise joint sequence from 5 ' end successively to 3 ' end, sequence label and the first upstream base primer sequence or the first downstream base primer sequence, the sequence label of often pair of first upstream primer sequence and the first downstream primer sequence makes each DNA sequence dna after PCR reacts, have the sequence label being different from other DNA sequence dna, described joint sequence is the sequence making each DNA sequence dna carry out PCR reaction comparably, described first upstream base primer sequence and the first downstream base primer sequence respectively with 3 ' termini-complementary of described DNA sequence dna two strands, second obtains module, for carrying out separation and purification to described first PCR primer, obtain a DNA product, the two ends of every article of strand of a described DNA product all comprise the complementary sequence of described first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of described first upstream primer sequence, 3rd obtains module, for the solution of a DNA product of described acquisition with the second upstream primer sequence and the second downstream primer sequence is mixed, and carry out the PCR reaction of 20 five to four ten circulations, obtain the second PCR primer, wherein, described second upstream primer sequence comprises the joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the joint sequence in described first downstream primer sequence, 4th obtains module, for carrying out separation and purification to described second PCR primer, obtains the 2nd DNA product, sequencing result obtains module, for carrying out gene sequencing to described 2nd DNA product, and analyzing sequencing result, obtaining the sequencing result of each DNA sequence dna.
Wherein, described joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, described sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence are linked in sequence, described sequencing primer sequence connects the sequence label of described first upstream primer sequence or the first downstream primer sequence, and described order-checking logo sequence is for distinguishing the multiple DNA sequence dnas from different sample.
Wherein, described second upstream primer sequence comprises the order-checking company joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the order-checking company joint sequence in described first downstream primer sequence.
Wherein, the base number of described sequence label is eight, or seven, or six.
Wherein, described sequencing result obtains module and comprises: first obtains unit, for carrying out gene sequencing to described 2nd DNA product, obtains DNA sequencing result; Second obtains unit, for described DNA sequencing result is carried out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna; Statistic unit, for belonging to all DNA sequencing results of same DNA sequence dna described in relatively, and adds up each position in described all DNA sequencing results and occurs the frequency of identical base; Judging unit, for judging that described each position occurs whether the frequency of identical base reaches predetermined threshold value; Determining unit, for when reaching predetermined threshold value, belongs to the base of the described position of same DNA sequence dna described in determining, until belong to the base of all positions of same DNA sequence dna described in determining.
For with multiple mixing RNA for template, carry out checking order, another technical solution used in the present invention is: provide a kind of RNA sequence to multiple mixing to carry out the method for gene sequencing, comprise: the solution of M reverse transcription primer sequence with N number of RNA sequence of t lymphocyte receptor TCR in immunocyte group and/or b lymphocyte receptor BCR is mixed, carry out reverse transcription reaction, obtain the product of cDNA sequence, wherein, described M and N is natural number, M is more than or equal to the N of 100 times, described reverse transcription primer sequence comprises joint sequence from 5 ' end successively to 3 ' end, sequence label and reverse transcription basis primer sequence, 3 ' termini-complementary of described reverse transcription basis primer sequence and described RNA sequence, after the solution hybrid reaction of the product of the cDNA sequence of described acquisition and M front end primer sequence, carry out the PCR reaction of a circulation again, obtain the 3rd PCR primer, wherein, described front end primer sequence comprises joint sequence, sequence label and basis, front end primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of described front end basis primer sequence and described cDNA sequence, separation and purification is carried out to described 3rd PCR primer, obtain the 3rd DNA product, the two ends of every article of strand of described 3rd DNA product all comprise the complementary sequence of described reverse transcription primer sequence and front end primer sequence, or comprise complementary sequence and the front end primer sequence of described reverse transcription primer sequence, the solution of 3rd DNA product of described acquisition with the 3rd upstream primer sequence and the 3rd downstream primer sequence is mixed, and carry out the PCR reaction of 20 five to four ten circulations, obtain the 4th PCR primer, wherein, described 3rd upstream primer sequence comprises the joint sequence in the primer sequence of described front end, and described 3rd downstream primer sequence comprises the joint sequence in described reverse transcription primer sequence, separation and purification is carried out to described 4th PCR primer, obtains the 4th DNA product, gene sequencing is carried out to described 4th DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna, wherein, the sequence label of reverse transcription primer sequence and front end primer sequence makes the DNA sequence dna of each 3rd DNA product have the sequence label being different from other DNA sequence dna, and described joint sequence is the sequence making the DNA sequence dna of each 3rd DNA product carry out PCR reaction comparably.
Wherein, described 3rd upstream primer sequence or the 3rd downstream primer sequence comprise sequencing primer sequence, order-checking logo sequence and order-checking company binding sequence, the sequencing primer sequence of described 3rd upstream primer sequence comprises the joint sequence in the primer sequence of front end, and the sequencing primer sequence of described 3rd downstream primer sequence comprises the joint sequence in reverse transcription primer sequence.
The invention has the beneficial effects as follows: the situation being different from prior art, the present invention mainly make use of a pair first upstream primer sequences and the first downstream primer sequence, this to primer sequence except comprising basic primer sequence, also comprise joint sequence and sequence label, sequence label makes each DNA sequence dna have the sequence label being different from other DNA sequence dna after PCR reaction, after PCR reaction and order-checking, multiple sequencing results of each DNA sequence dna can be obtained, by analyzing multiple sequencing results of each DNA sequence dna, whether the base that can distinguish each position of each DNA sequence dna is the transgenation that other outside empirical factor causes, thus the accuracy of gene sequencing can be improved, the error rate of remarkable minimizing gene sequencing, in addition owing to adding a sequence label to each DNA, can often plant the copy number of DNA fragmentation in initiate dna mixture by Accurate Determining by calculating sequence label.
Accompanying drawing explanation
Fig. 1 is the present invention carries out method one embodiment of gene sequencing schema to the DNA sequence dna of multiple mixing;
Fig. 2 is the present invention carries out another embodiment of method of gene sequencing schema to the DNA sequence dna of multiple mixing;
Fig. 3 is that the present invention carries out the principle schematic of twice PCR reaction in the method for gene sequencing to the DNA sequence dna of multiple mixing;
Fig. 4 is that the present invention carries out another principle schematic of twice PCR reaction in the method for gene sequencing to the DNA sequence dna of multiple mixing;
Fig. 5 is the present invention carries out device one embodiment of gene sequencing structural representation to the DNA sequence dna of multiple mixing;
Fig. 6 is the schema that the RNA sequence of the present invention to multiple mixing carries out method one embodiment of gene sequencing;
Fig. 7 is the principle schematic that the RNA sequence of the present invention to multiple mixing carries out reverse transcription and twice PCR reaction in the method for gene sequencing;
Fig. 8 is another principle schematic that the RNA sequence of the present invention to multiple mixing carries out reverse transcription and twice PCR reaction in the method for gene sequencing.
Embodiment
Below in conjunction with drawings and embodiments, the present invention is described in detail.
Consult Fig. 1, Fig. 1 is the present invention carries out method two embodiments of gene sequencing schema to the DNA sequence dna of multiple mixing, comprising:
Step S101: M is mixed the solution of the first upstream primer sequence with N number of DNA sequence dna of TCR and/or BCR in the first downstream primer sequence and immunocyte group, and carry out the PCR reaction of two circulations, obtain the first PCR primer, wherein, M and N is natural number, M is more than or equal to the N of 100 times, first upstream primer sequence or the first downstream primer sequence comprise joint sequence from 5 ' end successively to 3 ' end, sequence label and the first upstream base primer sequence or the first downstream base primer sequence, the sequence label of often pair of first upstream primer sequence and the first downstream primer sequence makes each DNA sequence dna after PCR reacts, have the sequence label being different from other DNA sequence dna, joint sequence is the sequence making each DNA sequence dna carry out PCR reaction comparably, first upstream base primer sequence and the first downstream base primer sequence respectively with 3 ' termini-complementary of DNA sequence dna two strands.
First upstream base primer sequence of the first upstream primer sequence and 3 ' termini-complementary of a DNA sequence dna strand, first downstream base primer sequence of the first downstream primer sequence and 3 ' termini-complementary of another strand of DNA sequence dna, therefore, first upstream base primer sequence and the first downstream base primer sequence are combined with two strands of DNA respectively, so that carry out PCR reaction.
The sequence label be connected with the first upstream base primer sequence and the first downstream base primer sequence is in order to mark unique on each DNA sequence dna band, and this mark makes each DNA sequence dna have the sequence label being different from other DNA sequence dna after PCR reaction.
The joint sequence be connected with sequence label mainly plays two and acts on, one is to make each DNA sequence dna carry out PCR reaction comparably, two is when the PCR reaction of follow-up carrying out second time, the primer of second time PCR is owing to comprising this joint sequence, therefore, the PCR primer of amplification, except original DNA sequence dna, also comprises sequence label.
M is mixed the solution of the first upstream primer sequence with N number of DNA sequence dna of TCR and/or BCR in the first downstream primer sequence and immunocyte group, and carries out the PCR reaction of two circulations, obtain the first PCR primer.Wherein, M is more than or equal to the N of 100 times, is that in the mixing solutions in order to ensure N number of DNA sequence dna, each DNA sequence dna, after the PCR reaction of two circulations, obtains the sequence label being different from other DNA sequence dna.
In immunocyte group, the diversity of TCR reaches 10
16, the diversity of BCR reaches 10
14, and in reality, in immunocyte group, the diversity of the gene of TCR and BCR reaches 10
5as long as the number of combinations of sequence label at least reaches 10
7, be enough to allow each DNA sequence dna obtain unique mark.In immunocyte group, the size of the gene fragment of TCR and BCR is at about 400bp.
Wherein, the base number of sequence label is eight, or seven, or six.Such as, when the base number of sequence label is eight, can 10 be obtained
9individual different sequence labels combination; When the base number of sequence label is seven, can 10 be obtained
8individual different sequence labels combination; When the base number of sequence label is six, can 10 be obtained
7individual different sequence labels combination.
Wherein, joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence are linked in sequence, sequencing primer sequence connects the sequence label of the first upstream primer sequence or the first downstream primer sequence, and order-checking logo sequence is for distinguishing the multiple DNA sequence dnas from different sample.
In order to better obtain the best effect that can reach of sequenator when checking order, the joint sequence adopting order-checking company to provide is good selection, generally comprise sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, it is helpful that sequencing primer sequence pair obtains good sequencing result, order-checking logo sequence contributes to distinguishing the multiple DNA sequence dnas from different sample, and namely multiple DNA sequence dnas of same sample adopt the joint sequence of same order-checking logo sequence.
Step S102: separation and purification is carried out to the first PCR primer, obtain a DNA product, the two ends of every article of strand of the one DNA product all comprise the complementary sequence of the first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of the first upstream primer sequence.
First PCR primer is the mixture of the different size fragments comprising a DNA product, after separation and purification, obtain the mixture of the basically identical DNA product of clip size, namely the two ends of every bar strand all comprise the complementary sequence of the first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of the first upstream primer sequence.
Step S103: the DNA product obtained is mixed with the solution of the second upstream primer sequence and the second downstream primer sequence, and carry out the PCR reaction of 20 five to four ten circulations, obtain the second PCR primer, wherein, second upstream primer sequence comprises the joint sequence in the first upstream primer sequence, and the second downstream primer sequence comprises the joint sequence in the first downstream primer sequence.
One DNA product is obtaining after the PCR reaction of two circulations, and quality and quantity is all little, cannot meet subsequent request, therefore, obtain a large amount of DNA products met the demands, need to carry out secondary PCR reaction.
Second upstream primer sequence comprises the joint sequence in the first upstream primer sequence, second downstream primer sequence comprises the joint sequence in the first downstream primer sequence, therefore, by the PCR reaction of 20 five to four ten circulations, a large amount of DNA sequence dnas comprising a DNA product can be obtained, i.e. the second PCR primer.
Wherein, in step S101, when joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company's joint sequence, then the second upstream primer sequence comprises the order-checking company joint sequence in the first upstream primer sequence, and the second downstream primer sequence comprises the order-checking company joint sequence in the first downstream primer sequence.
Step S104: carry out separation and purification to the second PCR primer, obtains the 2nd DNA product.
Second PCR primer contains the impurity component of other different size fragments, and after separation and purification, obtain the mixture of the 2nd basically identical DNA product of clip size, the 2nd DNA product is the DNA sequence dna comprising a DNA product of a large amount of purifying.
Step S105: gene sequencing is carried out to the 2nd DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna.
Utilize existing sequencing technologies, such as the sequenator of Illumina company, gene sequencing can be carried out to the 2nd DNA product, and analyze sequencing result, obtain the sequencing result of each DNA sequence dna.
The detailed process of step S105, refers to Fig. 2, comprising:
Step S201: carry out gene sequencing to the 2nd DNA product, obtains DNA sequencing result.
Step S202: DNA sequencing result is carried out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna.
Because each DNA sequence dna has sequence label, what sequence label was identical is same DNA sequence dna.After all DNA sequencing results of acquisition, carry out taxonomic revision according to sequence label, all DNA sequencing results belonging to same DNA sequence dna can be obtained.
Step S203: compare all DNA sequencing results belonging to same DNA sequence dna, and in adding up all DNA sequencing result there is the frequency of identical base in each position.
Such as, same DNA sequence dna, always has 10 DNA sequencing results, 5th position has 7 sequencing results to be A, and two sequencing results are G, and a sequencing result is T, then the 5th position occurs that the frequency of A is 0.7, occurs that the frequency of G is 0.2, occurs that the frequency of T is 0.1.
Step S204: judge that each position occurs whether the frequency of identical base reaches predetermined threshold value.
Step S205: if reach predetermined threshold value, then determine the base of the described position belonging to same DNA sequence dna, according to method, can determine the base of all positions belonging to same DNA sequence dna.
If each position of setting occurs that the predetermined threshold value of the frequency of identical base is 0.67, according to above-mentioned example, the 5th position occurs that the frequency of A is 0.7, and therefore the base of the 5th position determines it is A.The rest may be inferred, can determine the base of all positions belonging to same DNA sequence dna.
Consult Fig. 3, Fig. 3 is that the present invention carries out the principle schematic of twice PCR reaction in the method for gene sequencing to the DNA sequence dna of multiple mixing, as shown in the figure, first upstream primer sequence 21 comprises joint sequence 213, sequence label 212 and the first upstream base primer sequence 211, first downstream primer sequence 22 and comprises joint sequence 223, sequence label 222 and the first downstream base primer sequence 221.
This principle comprises:
Step S301: the first upstream primer sequence 21 mixes with the solution of the first downstream primer sequence 22 and DNA sequence dna 11, carries out the PCR reaction of two circulations, obtains a DNA product 41.
The two ends of every article of strand of the one DNA product 41 all comprise the complementary sequence of the first upstream primer sequence 21 and the first downstream primer sequence 22, or comprise complementary sequence and the first downstream primer sequence 22 of the first upstream primer sequence 21.
Step S302: mixed by the solution of a DNA product 41 with the second upstream primer sequence 31 and the second downstream primer sequence 32, carries out the PCR reaction of 20 five to four ten circulations, obtains the 2nd DNA product 42.
Second upstream primer sequence 31 joint sequence 213, the second downstream primer sequence 32 comprised in the first upstream primer sequence 21 comprises the joint sequence 223 in the first downstream primer sequence 22.
Consult Fig. 4, Fig. 4 is that the present invention carries out another principle schematic of twice PCR reaction in the method for gene sequencing to the DNA sequence dna of multiple mixing, as shown in the figure, first upstream primer sequence 61 comprises joint sequence 613, sequence label 612 and the first upstream base primer sequence 611, and joint sequence 613 comprises sequencing primer sequence 6131, order-checking logo sequence 6132 and order-checking company joint sequence 6133; First downstream primer sequence 62 comprises joint sequence 623, sequence label 622 and the first downstream base primer sequence 621, and joint sequence 623 comprises sequencing primer sequence 6231, order-checking logo sequence 6232 and order-checking company joint sequence 6233.
This principle comprises:
Step S401: the first upstream primer sequence 61 mixes with the solution of the first downstream primer sequence 62 and DNA sequence dna 51, carries out the PCR reaction of two circulations, obtains a DNA product 81.
The two ends of every article of strand of the one DNA product 81 all comprise the complementary sequence of the first upstream primer sequence 61 and the first downstream primer sequence 62, or comprise complementary sequence and the first downstream primer sequence 62 of the first upstream primer sequence 61.
Step S402: mixed by the solution of a DNA product 81 with the second upstream primer sequence 71 and the second downstream primer sequence 72, carries out the PCR reaction of 20 five to four ten circulations, obtains the 2nd DNA product 82.
Second upstream primer sequence 71 order-checking company joint sequence 6133, the second downstream primer sequence 72 comprised in the joint sequence 613 in the first upstream primer sequence 61 comprises the order-checking company joint sequence 6233 in the joint sequence 623 in the first downstream primer sequence 62.
The present invention mainly make use of a pair first upstream primer sequences and the first downstream primer sequence, this to primer sequence except comprising basic primer sequence, also comprise joint sequence and sequence label, sequence label makes each DNA sequence dna have the sequence label being different from other DNA sequence dna after PCR reaction, after PCR reaction and order-checking, multiple sequencing results of each DNA sequence dna can be obtained, by analyzing multiple sequencing results of each DNA sequence dna, whether the base that can distinguish each position of each DNA sequence dna is the transgenation that other outside empirical factor causes, thus the accuracy of gene sequencing can be improved, the error rate of remarkable minimizing gene sequencing, can the copy number of each DNA fragmentation of Accurate Determining additionally by calculating sequence label.
Consult Fig. 5, Fig. 5 is the present invention carries out device one embodiment of gene sequencing structural representation to the DNA sequence dna of multiple mixing, and this device comprises: first obtains module 101, second obtains module 102, the 3rd acquisition module 103, the 4th acquisition module 104 and sequencing result acquisition module 105.
First obtains module 101 for being mixed the solution of N number of DNA sequence dna of the first upstream primer sequence and t lymphocyte receptor in the first downstream primer sequence and immunocyte group and/or b lymphocyte receptor by M, and carry out the PCR reaction of two circulations, obtain the first PCR primer, wherein, M and N is natural number, M is more than or equal to the N of 100 times, first upstream primer sequence or the first downstream primer sequence comprise joint sequence from 5 ' end successively to 3 ' end, sequence label and the first upstream base primer sequence or the first downstream base primer sequence, the sequence label of often pair of first upstream primer sequence and the first downstream primer sequence makes each DNA sequence dna after PCR reacts, have the sequence label being different from other DNA sequence dna, joint sequence is the sequence making each DNA sequence dna carry out PCR reaction comparably, first upstream base primer sequence and the first downstream base primer sequence respectively with 3 ' termini-complementary of DNA sequence dna two strands.
Wherein, joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence are linked in sequence, sequencing primer sequence connects the sequence label of the first upstream primer sequence or the first downstream primer sequence, and order-checking logo sequence is for distinguishing the multiple DNA sequence dnas from different sample.
Wherein, the base number of sequence label is eight, or seven, or six.
Second obtains module 102 for carrying out separation and purification to the first PCR primer, obtain a DNA product, the two ends of every article of strand of the one DNA product all comprise the complementary sequence of the first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of the first upstream primer sequence.
3rd obtains module 103 for being mixed with the solution of the second upstream primer sequence and the second downstream primer sequence by the DNA product obtained, and carry out the PCR reaction of 20 five to four ten circulations, obtain the second PCR primer, wherein, second upstream primer sequence comprises the joint sequence in the first upstream primer sequence, and the second downstream primer sequence comprises the joint sequence in the first downstream primer sequence.
Wherein, the second upstream primer sequence comprises the order-checking company joint sequence in the first upstream primer sequence, and the second downstream primer sequence comprises the order-checking company joint sequence in the first downstream primer sequence.
4th obtains module 104 for carrying out separation and purification to the second PCR primer, obtains the 2nd DNA product.
Sequencing result obtains module 105 for carrying out gene sequencing to the 2nd DNA product, and analyzes sequencing result, obtains the sequencing result of each DNA sequence dna.
Wherein, sequencing result acquisition module comprises: first obtains unit, second obtains unit, statistic unit, judging unit and determining unit.
First obtains unit is used for carrying out gene sequencing to the 2nd DNA product, obtains DNA sequencing result;
Second obtains unit is used for DNA sequencing result to carry out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna;
Statistic unit belongs to all DNA sequencing results of same DNA sequence dna for comparing, and in adding up all DNA sequencing result, the frequency of identical base appears in each position;
Judging unit is for judging that each position occurs whether the frequency of identical base reaches predetermined threshold value;
Determining unit is used for when reaching predetermined threshold value, determines the base of the position belonging to same DNA sequence dna, until determine the base of all positions belonging to same DNA sequence dna.
The present invention mainly make use of a pair first upstream primer sequences and the first downstream primer sequence, this to primer sequence except comprising basic primer sequence, also comprise joint sequence and sequence label, sequence label makes each DNA sequence dna have the sequence label being different from other DNA sequence dna after PCR reaction, after PCR reaction and order-checking, multiple sequencing results of each DNA sequence dna can be obtained, by analyzing multiple sequencing results of each DNA sequence dna, whether the base that can distinguish each position of each DNA sequence dna is the transgenation that other outside empirical factor causes, thus the accuracy of gene sequencing can be improved, the error rate of remarkable minimizing gene sequencing, can the copy number of each DNA fragmentation of Accurate Determining additionally by calculating sequence label.
Consult Fig. 6, Fig. 6 is the schema that the RNA sequence of the present invention to multiple mixing carries out method one embodiment of gene sequencing.If in actual applications, in the immunocyte group of acquisition, the template sequence of t lymphocyte receptor TCR and/or b lymphocyte receptor BCR is RNA sequence, still can obtain final sequencing result by the process of reverse transcription and custom-designed primer sequence.It is the situation of RNA sequence that this implementation method is for template sequence, and the method comprises:
Step S501: the solution of M reverse transcription primer sequence with N number of RNA sequence of t lymphocyte receptor TCR in immunocyte group and/or b lymphocyte receptor BCR is mixed, carry out reverse transcription reaction, obtain the product of cDNA sequence, wherein, M and N is natural number, M is more than or equal to the N of 100 times, and reverse transcription primer sequence comprises joint sequence, sequence label and reverse transcription basis primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of reverse transcription basis primer sequence and RNA sequence.
CDNA sequence is the single-stranded DNA sequence with RNA complementary.This step S501 is transcriptive process,reversed, and in the process of reverse transcription, ThermoScript II is also indispensable.By reverse transcription reaction, the sequence label of reverse transcription primer sequence is connected in cDNA sequence.
M is more than or equal to the N of 100 times, is to ensure that reverse transcription every bar strand cDNA sequence out can obtain unique sequence label being different from other reverse transcription cDNA sequence out.
Reverse transcription primer sequence comprises joint sequence, sequence label and reverse transcription basis primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of reverse transcription basis primer sequence and RNA sequence, thus, after reverse transcription basis primer sequence is combined with 3 ' end of RNA sequence, there is reverse transcription reaction.Joint sequence mainly plays two effects: one is to make each DNA sequence dna carry out PCR reaction comparably, two is when the PCR reaction of follow-up carrying out second time, the primer of second time PCR is owing to comprising this joint sequence, therefore, the PCR primer of amplification, except original DNA sequence dna, also comprises sequence label.
Step S502: after the product of cDNA sequence of acquisition and the solution hybrid reaction of M front end primer sequence, carry out the PCR reaction of a circulation again, obtain the 3rd PCR primer, wherein, front end primer sequence comprises joint sequence, sequence label and basis, front end primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of front end basis primer sequence and cDNA sequence.
The cDNA sequence that step S501 obtains is single-stranded DNA sequence, front end primer sequence is added in step S502, the front end basis primer sequence of front end primer sequence and 3 ' termini-complementary of cDNA sequence, under the effect of front end primer sequence and relevant enzyme, synthesize one and comprise the single-stranded DNA sequence of front end primer sequence and reverse transcription primer complementary respectively with the two ends of cDNA sequence complementation.Then, when reverse transcription primer sequence and front end primer sequence are primer, by the PCR of a circulation, thus can obtain complete and two ends comprise sequence and the front end primer sequence of reverse transcription primer complementary respectively, or comprise the DNA double chain of sequence of reverse transcription primer sequence and front end primer sequence complementation.
Wherein, the base number of sequence label is eight, or seven, or six.Such as, when the base number of sequence label is eight, can 10 be obtained
9individual different sequence labels combination; When the base number of sequence label is seven, can 10 be obtained
8individual different sequence labels combination; When the base number of sequence label is six, can 10 be obtained
7individual different sequence labels combination.
Step S503: separation and purification is carried out to the 3rd PCR primer, obtain the 3rd DNA product, the two ends of every article of strand of the 3rd DNA product all comprise the complementary sequence of reverse transcription primer sequence and front end primer sequence, or comprise complementary sequence and the front end primer sequence of reverse transcription primer sequence;
3rd PCR primer is the mixture comprising the 3rd DNA product, after separation and purification, obtains the mixture of basically identical the 3rd DNA product of clip size, and the 3rd DNA product is the mixture comprising many articles of different DNA double chain-orderings.And the two ends of every article of strand of the 3rd DNA product all comprise the complementary sequence of reverse transcription primer sequence and front end primer sequence, or comprise complementary sequence and the front end primer sequence of reverse transcription primer sequence.
Step S504: the 3rd DNA product obtained is mixed with the solution of the 3rd upstream primer sequence and the 3rd downstream primer sequence, and carry out the PCR reaction of 20 five to four ten circulations, obtain the 4th PCR primer, wherein, 3rd upstream primer sequence comprises the joint sequence in the primer sequence of front end, and the 3rd downstream primer sequence comprises the joint sequence in reverse transcription primer sequence;
3rd DNA product obtains after the PCR reaction through reverse transcription and a circulation, and quality and quantity is all little, cannot meet subsequent request, therefore, obtain a large amount of the 3rd DNA products met the demands, need to carry out secondary PCR reaction.
3rd upstream primer sequence comprises the joint sequence in the primer sequence of front end, 3rd downstream primer sequence comprises the joint sequence in reverse transcription primer sequence, therefore, by the PCR reaction of 20 five to four ten circulations, a large amount of DNA sequence dnas comprising the 3rd DNA product can be obtained, i.e. the 4th PCR primer.
Wherein, 3rd upstream primer sequence or the 3rd downstream primer sequence comprise sequencing primer sequence, order-checking logo sequence and order-checking company binding sequence, the sequencing primer sequence of the 3rd upstream primer sequence comprises the joint sequence in the primer sequence of front end, and the sequencing primer sequence of the 3rd downstream primer sequence comprises the joint sequence in reverse transcription primer sequence.
Step S505: carry out separation and purification to the 4th PCR primer, obtains the 4th DNA product;
Step S506: gene sequencing is carried out to the 4th DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna;
Wherein, the sequence label of reverse transcription primer sequence and front end primer sequence makes the DNA sequence dna of each 3rd DNA product have the sequence label being different from other DNA sequence dna, and joint sequence is the sequence making the DNA sequence dna of each 3rd DNA product carry out PCR reaction comparably.
Wherein, step S506 specifically comprises:
A: carry out gene sequencing to the 4th DNA product, obtains DNA sequencing result.
B: DNA sequencing result is carried out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna.
C: compare all DNA sequencing results belonging to same DNA sequence dna, and in adding up all DNA sequencing result there is the frequency of identical base in each position.
Such as, same DNA sequence dna, always has 10 DNA sequencing results, 5th position has 7 sequencing results to be A, and two sequencing results are G, and a sequencing result is T, then the 5th position occurs that the frequency of A is 0.7, occurs that the frequency of G is 0.2, occurs that the frequency of T is 0.1.
D: judge that each position occurs whether the frequency of identical base reaches predetermined threshold value.
E: if reach predetermined threshold value, then determine the base of the described position belonging to same DNA sequence dna, according to method, can determine the base of all positions belonging to same DNA sequence dna.
If each position of setting occurs that the predetermined threshold value of the frequency of identical base is 0.67, according to above-mentioned example, the 5th position occurs that the frequency of A is 0.7, and therefore the base of the 5th position determines it is A.The rest may be inferred, can determine the base of all positions belonging to same DNA sequence dna.
The present invention mainly make use of a pair reverse transcription primer sequence and front end primer sequence, this to primer sequence except comprising basic primer sequence, also comprise joint sequence and sequence label, sequence label makes each DNA sequence dna obtained through reverse transcription and PCR reaction have the sequence label being different from other DNA sequence dna, after PCR reaction and order-checking, multiple sequencing results of each DNA sequence dna can be obtained, by analyzing multiple sequencing results of each DNA sequence dna, whether the base that can distinguish each position of each DNA sequence dna is the transgenation that other outside empirical factor causes, thus the accuracy of gene sequencing can be improved, the error rate of remarkable minimizing gene sequencing, can the copy number of each DNA fragmentation of Accurate Determining additionally by calculating sequence label.
Consult Fig. 7, Fig. 7 is the principle schematic that the RNA sequence of the present invention to multiple mixing carries out reverse transcription and twice PCR reaction in the method for gene sequencing, as shown in the figure, front end primer sequence 21 ' comprises joint sequence 213 ', sequence label 212 ' and basis, front end primer sequence 211 ', and reverse transcription primer sequence 22 ' comprises joint sequence 223 ', sequence label 222 ' and reverse transcription basis primer sequence 221 '.
This principle comprises:
Step S701: reverse transcription primer sequence 22 ' mixes with the solution of RNA sequence 11 ', carries out reverse transcription reaction, obtains cDNA product 12 '.
The reverse transcription basis primer sequence 221 ' of reverse transcription primer sequence 22 ' is combined with 3 ' end of RNA sequence 11 ', reverse transcription reaction is carried out under the effect of ThermoScript II, generate cDNA product 12 ', thus make cDNA product 12 ' bring joint sequence 223 ' and the sequence label 222 ' of reverse transcription primer sequence 22 '.
Step S702: by cDNA product 12 ' and front end primer sequence 21 ' hybrid reaction, then the PCR reaction carrying out a circulation, obtain the 3rd DNA product 41 '.
Step S703: mixed by the solution of the 3rd DNA product 41 ' with the 3rd upstream primer sequence 31 ' and the 3rd downstream primer sequence 32 ', carries out the PCR reaction of 20 five to four ten circulations, obtains the 4th DNA product 42 '.
3rd upstream primer sequence 31 ' comprises the joint sequence 213 ' of front end primer sequence 21 ', and the 3rd downstream primer sequence 32 ' comprises the joint sequence 223 ' in reverse transcription primer sequence 22 '.
Consult Fig. 8, Fig. 8 is another principle schematic that the RNA sequence of the present invention to multiple mixing carries out reverse transcription and twice PCR reaction in the method for gene sequencing, as shown in the figure, front end primer sequence 61 ' comprises joint sequence 613 ', sequence label 612 ' and basis, front end primer sequence 611 ', and reverse transcription primer sequence 62 ' comprises joint sequence 623 ', sequence label 622 ' and reverse transcription basis primer sequence 621 '.
This principle comprises:
Step S801: reverse transcription primer sequence 62 ' mixes with the solution of RNA sequence 51 ', carries out reverse transcription reaction, obtains cDNA product 52 '.
The reverse transcription basis primer sequence 621 ' of reverse transcription primer sequence 62 ' is combined with 3 ' end of RNA sequence 51 ', reverse transcription reaction is carried out under the effect of ThermoScript II, generate cDNA product 52 ', thus make cDNA product 52 ' bring joint sequence 623 ' and the sequence label 622 ' of reverse transcription primer sequence 62 '.
Step S802: after cDNA product 52 ' and front end primer sequence 61 ' hybrid reaction, then the PCR reaction carrying out a circulation, obtain the 3rd DNA product 81 '.
Step S803: by the 3rd DNA product 81 ' and the 3rd upstream primer sequence 71 ' and the solution of the 3rd downstream primer sequence 72 ' mix, and carry out the PCR reaction of 20 five to four ten circulations, obtain the 4th DNA product 82 '.
3rd upstream primer sequence 71 ' comprise sequencing primer sequence 71 1 ', order-checking logo sequence 71 2 ' and order-checking company binding sequence 713 ', sequencing primer sequence 71 1 ' comprises the joint sequence 613 ' of front end primer sequence 61 '; 3rd downstream primer sequence 72 ' comprises sequencing primer sequence 721 ', order-checking logo sequence 722 ' and order-checking company binding sequence 723 ', and sequencing primer sequence 721 ' comprises the joint sequence 623 ' of reverse transcription primer sequence 62 '.
The foregoing is only embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. the DNA sequence dna of multiple mixing is carried out to a method for gene sequencing, it is characterized in that, comprising:
M is mixed the solution of the first upstream primer sequence with N number of DNA sequence dna of t lymphocyte receptor TCR and/or b lymphocyte receptor BCR in the first downstream primer sequence and immunocyte group, and carry out the PCR reaction of two circulations, obtain the first PCR primer, wherein, described M and N is natural number, M is more than or equal to the N of 100 times, described first upstream primer sequence or the first downstream primer sequence comprise joint sequence from 5 ' end successively to 3 ' end, sequence label and the first upstream base primer sequence or the first downstream base primer sequence, the sequence label of often pair of first upstream primer sequence and the first downstream primer sequence makes each DNA sequence dna after PCR reacts, have the sequence label being different from other DNA sequence dna, described joint sequence is the sequence making each DNA sequence dna carry out PCR reaction comparably, described first upstream base primer sequence and the first downstream base primer sequence respectively with 3 ' termini-complementary of described DNA sequence dna two strands,
Separation and purification is carried out to described first PCR primer, obtain a DNA product, the two ends of every article of strand of a described DNA product all comprise the complementary sequence of described first upstream primer sequence and the first downstream primer sequence, or comprise complementary sequence and the first downstream primer sequence of described first upstream primer sequence;
The solution of one DNA product of described acquisition with the second upstream primer sequence and the second downstream primer sequence is mixed, and carry out the PCR reaction of 20 five to four ten circulations, obtain the second PCR primer, wherein, described second upstream primer sequence comprises the joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the joint sequence in described first downstream primer sequence;
Separation and purification is carried out to described second PCR primer, obtains the 2nd DNA product;
Gene sequencing is carried out to described 2nd DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna;
Wherein, described joint sequence comprises sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence, described sequencing primer sequence, order-checking logo sequence and order-checking company joint sequence are linked in sequence, described sequencing primer sequence connects the sequence label of described first upstream primer sequence or the first downstream primer sequence, and described order-checking logo sequence is for distinguishing the multiple DNA sequence dnas from different sample;
Wherein, the base number of described sequence label is eight, or seven, or six.
2. method according to claim 1, it is characterized in that, described second upstream primer sequence comprises the order-checking company joint sequence in described first upstream primer sequence, and described second downstream primer sequence comprises the order-checking company joint sequence in described first downstream primer sequence.
3. the method according to any one of claim 1 to 2, is characterized in that, describedly carries out gene sequencing to the 2nd DNA product, and analyzes sequencing result, obtains the step of the sequencing result of each DNA sequence dna, comprising:
Gene sequencing is carried out to described 2nd DNA product, obtains DNA sequencing result;
Described DNA sequencing result is carried out taxonomic revision according to sequence label, obtains all DNA sequencing results belonging to same DNA sequence dna;
Belong to all DNA sequencing results of same DNA sequence dna relatively, and add up each position in described all DNA sequencing results and occur the frequency of identical base;
Judge that described each position occurs whether the frequency of identical base reaches predetermined threshold value;
If reach predetermined threshold value, then belong to the base of the described position of same DNA sequence dna described in determining, according to described method, described in can determining, belong to the base of all positions of same DNA sequence dna.
4. the RNA sequence of multiple mixing is carried out to a method for gene sequencing, it is characterized in that, comprising:
The solution of M reverse transcription primer sequence with N number of RNA sequence of t lymphocyte receptor TCR in immunocyte group and/or b lymphocyte receptor BCR is mixed, carry out reverse transcription reaction, obtain the product of cDNA sequence, wherein, described M and N is natural number, M is more than or equal to the N of 100 times, described reverse transcription primer sequence comprises joint sequence, sequence label and reverse transcription basis primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of described reverse transcription basis primer sequence and described RNA sequence;
After the solution hybrid reaction of the product of the cDNA sequence of described acquisition and M front end primer sequence, carry out the PCR reaction of a circulation again, obtain the 3rd PCR primer, wherein, described front end primer sequence comprises joint sequence, sequence label and basis, front end primer sequence successively from 5 ' end to 3 ' end, 3 ' termini-complementary of described front end basis primer sequence and described cDNA sequence;
Separation and purification is carried out to described 3rd PCR primer, obtain the 3rd DNA product, the two ends of every article of strand of described 3rd DNA product all comprise the complementary sequence of described reverse transcription primer sequence and front end primer sequence, or comprise complementary sequence and the front end primer sequence of described reverse transcription primer sequence;
The solution of 3rd DNA product of described acquisition with the 3rd upstream primer sequence and the 3rd downstream primer sequence is mixed, and carry out the PCR reaction of 20 five to four ten circulations, obtain the 4th PCR primer, wherein, described 3rd upstream primer sequence comprises the joint sequence in the primer sequence of described front end, and described 3rd downstream primer sequence comprises the joint sequence in described reverse transcription primer sequence;
Separation and purification is carried out to described 4th PCR primer, obtains the 4th DNA product;
Gene sequencing is carried out to described 4th DNA product, and analyzes sequencing result, obtain the sequencing result of each DNA sequence dna;
Wherein, the sequence label of reverse transcription primer sequence and front end primer sequence makes the DNA sequence dna of each 3rd DNA product have the sequence label being different from other DNA sequence dna, and described joint sequence is the sequence making the DNA sequence dna of each 3rd DNA product carry out PCR reaction comparably;
Wherein, described 3rd upstream primer sequence or the 3rd downstream primer sequence comprise sequencing primer sequence, order-checking logo sequence and order-checking company binding sequence, the sequencing primer sequence of described 3rd upstream primer sequence comprises the joint sequence in the primer sequence of front end, and the sequencing primer sequence of described 3rd downstream primer sequence comprises the joint sequence in reverse transcription primer sequence;
Wherein, the base number of described sequence label is eight, or seven, or six.
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