CN103602658A - Novel capture and enrichment technology for targeting nucleic acid molecules - Google Patents
Novel capture and enrichment technology for targeting nucleic acid molecules Download PDFInfo
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Abstract
The invention discloses a novel capture and enrichment technology for targeting nucleic acid molecules. The targeting nucleic acid molecule fragments are hybridized through special capture probes; and probe molecules marked with acrylamide groups are fixed in acrylamide glue, so that the targeting nucleic acid molecule fragments are captured. The technology can be used for functional gene capture and high-throughput sequencing analysis of microbial community, fishing and high-throughput sequencing analysis of genes related to diseases in human genome, and fishing and analysis of signature sequences of environment pathogenic microorganisms. Besides, the technology can be used for development of diagnostic kit for a plurality of molecular diseases and pathogenic microorganisms. The technology is relatively low in cost. Glue efficiency of acrylamide molecules is far higher than connection efficiency of magnetic beads and probes, so that capture efficiency is greatly increased.
Description
Technical field
The present invention relates to biological technical field, particularly relate to catching and beneficiation technologies of a kind of nucleic acid molecule.
Background technology
The appearance of a new generation's high-flux sequence instrument greatly reduces nucleic acid sequencing cost, adopts high-flux sequence instrument, a large amount of nucleotide sequence that checked order in a few years, and the full genome of multiple biology is sequenced, and a lot of human genomes are also checked order.Mankind's genome sequencing is the important application of high-flux sequence instrument.But, most research and a certain specific fragment of diagnosing target only need to check order in great amount of samples.For example, for human genome, exon is protein coding region, and exon only accounts for complete genomic about 1%.In most of situation, particularly for the screening of disease-related exons mutation, only survey exon region just enough.In addition, increasing research is found, the analysis of the grand genome functions gene of microflora and multiple mankind's major disease, environmental pollution, atmosphere Greenhouse effect etc. are closely related, and grand genome is very huge data, the grand genome of human intestine microflora is 100 times of human genome, different people, the different physiological periods of same person all has different microfloras and forms, but in these huge microfloras, what play a key effect often only has partial function gene, for Yi Ge microflora, only need to check order to a small amount of functional gene and just can reach the object of analysis.
A large amount of molecules carry out target parallel analysis, need to develop trapping nucleic acids technology, and existing report and the marketization at present caught product and mainly contained two kinds, and the first is catching and enrichment based on high-throughput hybridization analysis chip.First this method is that cost is higher.High-throughput hybridization chip itself is a very expensive technology, some chip hybridization analysis cost is higher than current high-flux sequence analysis cost, if the chip with this costliness carries out enrichment and target acquisition molecule, and then carries out sequencing analysis, cost is higher; Adopting high-throughput chip to catch with second drawback of enrichment target molecule is that capture rate is lower, and chip is solid-phase hybridization with the mode of catching fragment hybridization, and the hybridization efficiency of solid phase molecular hybridization is lower.The second catching method is based on liquid phase molecular hybridization and magnetic bead separation method in the market, first the method carries out a step solution hybridization, then a special group on group of mark on probe and magnetic bead is covalently bound, capture molecules and magnetic bead are linked together, magnetic bead is by the effect in magnetic field, will catch fragment and non-specific fragment is separated.The method is compared with first method, and capture rate and cost are all minimized.But because the method needs a step covalently bound, joint efficiency directly affects catches and enrichment productive rate, in addition, because probe mark price is higher, so it is still higher to catch cost.
Summary of the invention
For above-mentioned problems of the prior art, the invention provides catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule.
The technical scheme that the present invention adopts is:
Catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule is provided, comprises following operation steps:
1) by nucleic acid molecule fragment to be captured, cut the nucleic acid molecule fragment that glue reclaims 200bp-500bp, connection universal joint, forms target nucleic acid molecule fragment;
2) the target nucleic acid molecule fragment of step 1) is mixed with capture probe and hybridization solution, complete hybridization, described capture probe is modified with acrylamide chemical group;
3) to step 2) add acrylamide stock solution, ammonium persulphate and TEMED in the mixing solutions that forms, form acrylamide soln, and described acrylamide soln is placed in to one end of acrylamide gel shaped device, after solidifying completely, form acrylamide gel, and be filled with conductive rubber at the two ends of described acrylamide gel shaped device;
4) acrylamide gel after step 3) is placed in to the TBE electrophoresis liquid of electrophoresis chamber, electrophoresis is removed non-specific fragment wherein;
5) acrylamide gel after step 4) reaction is placed in to sex change electrophoresis solution, sex change electrophoresis is separated with capture probe by target nucleic acid molecule fragment, and described target nucleic acid molecule fragment is collected in one section of conductive rubber with electric current;
6) collect the target nucleic acid molecule fragment in conductive rubber in step 5), carry out high-flux sequence analysis.
In a preferred embodiment of the present invention, described nucleic acid molecule to be captured is single double-stranded linear or single double-stranded annular DNA or RNA.
In a preferred embodiment of the present invention, described capture probe is single stranded DNA, RNA, PNA or LNA.
In a preferred embodiment of the present invention, in described capture probe 5 ' end, 3 ' end or any base, modify the acrylamide chemical group of any number.
In a preferred embodiment of the present invention, acrylamide and N in the stock solution of acrylamide described in step 3), the mass ratio of N ＇-methylene bisacrylamide is 19:1 or 29:1.
In a preferred embodiment of the present invention, described acrylamide gel shaped device is that non-conducting material is made, and comprises plastic hose, glass groove or Glass tubing.
In a preferred embodiment of the present invention, described acrylamide soln mass volume ratio concentration is 2%-6%.
In a preferred embodiment of the present invention, that the two ends of described acrylamide gel shaped device are filled is that the conductive rubber put is conduction arbitrarily, nucleic acid molecule is had and filtered or adsorbing material.
In a preferred embodiment of the present invention, described conductive rubber is agarose gel.
In a preferred embodiment of the present invention, the described sex change electrophoresis solution in step 5) is basic solution, comprises NaOH solution, KOH solution or Ca (OH)
2solution.
The invention has the beneficial effects as follows:
(1) a kind of novel targeted nucleic acid molecule of the present invention catches and beneficiation technologies, adopt a large amount of capture probes to be combined with target nucleic acid molecule fragment, and capture probe is fixed in acrylamide gel, by running glue, remove non-specific fragment wherein, target nucleic acid molecule fragment can be collected by sex change electrophoresis method again, make target nucleic acid molecule fragment be able to enrichment, then enter next step sequencing analysis.The grand genome of microflora is huge, and pin of the present invention can carry out enrichment to interested gene or fragment, and further analyzes.
(2) catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule of the present invention, the crossbred of employing is hybridization in solution, then hybrid molecule is fixed, to improve the hybridization efficiency of capture probe and target nucleic acid molecule fragment.Research report in the past, nucleic acid molecule is caught the solid liquid phase molecular hybridizations that adopt more, namely probe molecule is fixed on solid phase carrier, then molecule and probe hybridization will be hunted down, by the molecule of hybrid capture, stayed further check analysis, the molecule not got on by hybridization is rinsed removal.With respect to solid liquid phase molecular hybridization, liquid phase molecular hybridization has improved the collision opportunity between molecule greatly, improves the hybridization efficiency between molecule, has further improved the detection sensitivity of nucleic acid molecule, has also improved the detection sensitivity of pathogenic micro-organism.
(3) catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule of the present invention, the crossbred of employing acrylamide gel immobilized capture probes and target nucleic acid molecule fragment, colloid is larger to the fixed amount of nucleic acid molecule, so capture rate is high.Probe fixed amount in colloid can reach 8 μ M, and our multiplex PCR primer concentration is 0.4 μ M, and colloid has so high fixed capacity, and most probes and crossbred are all fixed, and therefore has higher fixed efficiency.At present, the capture technique of unique employing solution hybridization, is to adopt magnetic bead stationary probe and crossbred.Magnetic bead connects affinity element, on probe, connect vitamin H, though the joint efficiency of this avidin and vitamin H is high, but joint efficiency between the vitamin H (liquid phase) on affinity on magnetic bead element (solid phase carrier) and probe is the highest, also only has 30% efficiency, most crossbreds cannot be because of connecting less than on magnetic bead, and cannot catch.
(4) catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule of the present invention, can adopt the method for original position solid single molecular cloning to expand the copy number of nucleic acid molecule to be detected, to have improved nucleic acid molecule quantity of the catch.When in grand genome, target nucleic acid molecule fragment is less, the present invention can adopt the method for original position amplification to increase target fragment quantity.Amplified nucleic acid molecule efficiency and be amplified molecule and primer molecule between collision opportunity be proportionate, in theory, pcr amplification reaction can increase out by the individual molecule in solution, but finds in actual experiment operating process, and the sensitivity that PCR detects is to be greater than 10000 molecules.The present invention is before carrying out unit molecule amplification, first by technology such as electrophoresis or precipitations, non-target fragment is removed, remaining fragment only has primer and target molecule, has so greatly increased target molecule and primer collision opportunity before, has also greatly improved the amplification efficiency of target molecule.
(5) a kind of novel targeted nucleic acid molecule of the present invention catches and beneficiation technologies, adopt original position solid single molecular cloning method to increase target nucleic acid molecules quantity of the catch, reduce the bias of amplified nucleic acid molecule, be more suitable for the content of high-flux sequence quantitative analysis target fragment.Conventional universal primer PCR amplification, difference due to template molecule sequence and higher structure, segment as low in GC content is easier to be amplified, therefore in a large amount of segment amplification procedures, the segment that in final reaction solution, small pieces and GC content are low is more, and the high segment of large segment and GC content is less.The nucleic acid template molecules that amplification efficiency is high can get more and more, and the low people's template molecule of amplification efficiency rate of growth can be more and more less, and while finally adopting gene chip hybridization technology for detection, the molecule of low amplification efficiency almost can't detect.The present invention adopts the amplification of original position unit molecule, in some amplification region, only has a template molecule, this template is not subject to the impact of other template molecules in amplification procedure, therefore, the amplification efficiency difference of each template molecule is less, when adopting nucleic acid molecule chip hybridization to detect, can react really stoste amplifying nucleic acid molecular template quantity, namely microbe population.
(6) catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule of the present invention, adopts universal primer amplification, to improve amplification efficiency.Most report employing multiplex PCRs or the repeatedly method of PCR detect multiple-microorganism, and the bias of multiplex PCR technology amplification is larger, and some molecule is easier to amplification, and some molecule can increase out substantially; Even if the quantity of the nucleic acid template molecules that can increase is out also very limited like this, simultaneously.Repeatedly PCR method is comparatively loaded down with trivial details, has therefore limited the kind that nucleic acid molecule detects.The present invention adopts universal primer amplification, namely template molecule to be amplified two ends is connected to joint, and different templates molecule has identical joint, and the joint sequence of then usining carries out pcr amplification as primer sequence, has so just improved the amplification efficiency of various template molecules.
(7) a kind of novel targeted nucleic acid molecule of the present invention catch with beneficiation technologies in target nucleic acid molecule fragment enrichment cost relatively low.The present invention adopts the method for acrylamide gel polymerization that probe is fixed in acrylamide gel, acrylamide molecule of probe modification, and the modification group of compare other fixing meanss and probe, cost is relatively cheap.Do not need expensive antibody or other proteic substances, the probe of use can single sintering, repeatedly use.Add that fixed efficiency of the present invention is higher, the enriching method of other nucleic acid molecule of comparing, cost is lower.
(8) catching and beneficiation technologies of a kind of novel targeted nucleic acid molecule of the present invention, reduces sample analysis cost greatly.Most grand genome sample genomic informations are doted on greatly, and our interested target fragment often only accounts for grand genomic seldom ratio (be less than ten thousand/), most information for we analyze grand genome and disease or other proterties anticipate discuss less.Yet current order-checking cost is still higher, adopt the present invention target fragment can be caught out and carry out sequencing analysis, greatly reduce analysis cost.Because grand genomic information amount is large, target fragment content is relatively little, so have relatively high expectations for capture technique.The present invention is based on glue technique for fixing fixed efficiency high, realized catching and enrichment of grand genome target fragment.The present invention has not only reduced the order-checking cost of DNA, has also reduced the manual analysis expense of sample simultaneously.
Accompanying drawing explanation
Fig. 1 is fundamental diagram of the present invention;
A wherein: the non-targeted nucleic acid molecule fragment that is broken into 200-500bp;
B: the target nucleic acid molecule fragment that is broken into 200-500bp;
C: the non-targeted nucleic acid molecule fragment that is connected with joint;
D: the target nucleic acid molecule fragment that is connected with joint;
PCR product after e:Linker-PCR amplification;
F: the probe that is marked with chemical group;
G: the crossbred of probe and target nucleic acid molecule fragment;
H: the colloid that is fixedly connected with probe and target nucleic acid molecule fragment crossbred and non-targeted nucleic acid molecule fragment;
I: removed the non-targeted nucleic acid molecule fragment colloid that is fixedly connected with probe and target nucleic acid molecule fragment crossbred afterwards;
J: sequence of nucleic acid molecules analyser.
Embodiment
Below in conjunction with accompanying drawing, preferred embodiment of the present invention is described in detail, thereby so that advantages and features of the invention can be easier to be it will be appreciated by those skilled in the art that, protection scope of the present invention is made to more explicit defining.
Consult Fig. 1, wherein step 1 is target nucleic acid molecule fragment and non-targeted nucleic acid molecule fragment connection universal joint, step 2 is Linker-PCR, step 3 is hybridized for probe and target nucleic acid molecule fragment in hybridization solution, step 4 is for to be fastened on the crossbred of target nucleic acid molecule fragment and probe in solid phase colloid by the chemical group on probe, step 5 is for adopting the technology such as electrophoresis that non-targeted nucleic acid molecule fragment is removed, and step 6 is acquisition target to nucleic acid molecule fragment the high-flux sequence analysis that enters next step.
embodiment 1 adopts polyacrylamide gel capture technique and high-flux sequence instrument target to analyze several functions gene distribution in the grand genome of enteric microorganism:
(1) Design & preparation of probe
Enteric microorganism functional gene DNA sequencing fragment is selected: adopt the softwares such as Mega or Clustal W, carry out information biology comparison, complete the selection of 20,000 functional gene nucleotide sequences.
The design of probe: adopt the softwares such as primer5.0, for microorganism specific fragment designing probe.
Synthetic and the mark of probe: probe adopts DNA synthesizer to synthesize, after all probes are synthetic, carries out acrylamide group of 5 ' end mark.
(2) the grand genome extraction of intestinal microflora, genomic fragment and both sides connect joint
Intestinal microflora macro genome DNA extracts: gather the fresh stool of 200mg, and the enteric microorganism in PBS suspension stool, then centrifugal collection microorganism, further adopts the reagent such as N,O-Diacetylmuramidase to extract the macro genome DNA in microflora.
The ultrasonication of genomic dna: choose the suitable ultrasonoscope of power, genomic dna is broken at random to the gene fragment of 200bp-500bp size.
Genomic DNA fragment two ends are connected (treatment process is with number of patent application CN200610098379.X) with general connexon: the random segment purification process that ultrasonic wave is obtained, remove some little and broken segments, then adopt respectively T4 polynueleotide kinase, T4 archaeal dna polymerase, Klenow large fragment DNA polysaccharase I and Taq polysaccharase are processed, make random fragment produce a TA sticky end, then be connected with a general connexon (linker1:5 '-GTCGGAGGCCAAGGCGGCCGT 3 ' linker2:5 '-CGCCTTGGCCTCCGACT-3 '), make genomic fragment two ends connect two general connexons.Connect product stand-by after QIAquick purification kit (QIAGEN) purifying.
(3) Linker-PCR amplification
In order to avoid easy amplified fragments to be increased in a large number as far as possible, the cycle number of PCR is 20-26.Reaction system is 100 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation, the Taq archaeal dna polymerase of 4U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again.Standby after PCR product purification.
(4) two ends are connected with genome and the probe hybridization of joint and are fixed on polyacrylamide gel
The PCR product with universal primer of preparation is mixed by 3:1 with hybridization solution (Telechem), and 95 ℃ of sex change 5 minutes, are then cooled fast to room temperature.Hybridization solution is mixed with acrylamide soln, wherein, acrylamide monomer (29:1, w/w acrylamide: bisacrylamide) final concentration is 2% (w/v), ammonium persulphate (APS) is 1% (w/v), TEMED(N, N, N ', N '-tetramethylethylene diamine) be 0.5%.Above-mentioned mixed solution is poured into transparent plastic hose (diameter is about 2mm-10mm, long for 15cm-20cm) one end, after isocolloid solidifies, by the agarose solution of the other end perfusion upper 2%, between acrylamide gel and agarose gel, can not have bubble.
(5) electrophoresis is removed non-specific fragment, and sex change electrophoresis is collected target fragment
Remove non-specific fragment: will be equipped with in the plastic hose horizontalization plate electrophoresis groove of gel, the two ends of flexible pipe are inserted in the electrophoresis liquid (1 * TBE) of the two poles of the earth groove, the electric current only gel from flexible pipe passes through.Electric current is 100 mA electrophoresis time 10 minutes.
Target fragment is collected: the electrophoresis liquid in the groove of electrophoresis chamber two ends is replaced by 0.1M NaOH solution, the plastic hose of having removed non-specific fragment is placed in to both sides NaOH solution, carry out electrophoresis, electric current is 100 mA, electrophoresis 10 minutes.According to DNA indicator, cut target fragment agarose gel, extract target fragment, frozen standby.
(6) universal primer PCR amplification
Reaction system is 50 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation, the Taq archaeal dna polymerase of 2U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again, PCR cycle number is standby after 35, PCR product purification.
(7) high-flux sequence instrument sequencing analysis target fragment
Above-mentioned target fragment is carried out respectively to high-flux sequence instrument sequencing analysis, and carry out bioinformatic analysis.
embodiment 2 adopts polyacrylamide gel to catch the target analysis of carrying out cancer related gene exons mutation in human genome with high-flux sequence instrument:
(1) Design & preparation of probe
Cancer related gene exon nucleotide sequence is selected: adopt the softwares such as Mega or Clustal W, carry out information biology comparison, complete the selection of 1000 functional gene nucleotide sequences.
The design of probe: adopt the softwares such as primer5.0, for microorganism specific fragment designing probe.
Synthetic and the mark of probe: probe adopts DNA synthesizer to synthesize, after all probes are synthetic, carries out acrylamide group of 5 ' end mark.
(2) the grand genome extraction of intestinal microflora, genomic fragment and both sides connect joint
Human genome DNA extracts: gather 2ml venous blood, adopt erythrocyte splitting and protease K digesting, phenol-chloroform method extracting human genome DNA.
The ultrasonication of genomic dna: choose the suitable ultrasonoscope of power, genomic dna is broken at random to the gene fragment of 200bp-500bp size.
Genomic DNA fragment two ends are connected (treatment process is with number of patent application CN200610098379.X) with general connexon: the random segment purification process that ultrasonic wave is obtained, remove some little and broken segments, then adopt respectively T4 polynueleotide kinase, T4 DNA polysaccharase, Klenow large fragment DNA polysaccharase I and Taq polysaccharase are processed, make random fragment produce a TA sticky end, then be connected with a general connexon (linker1:5 '-GTCGGAGGCCAAGGCGGCCGT 3 ' linker2:5 '-CGCCTTGGCCTCCGACT-3 '), make genomic fragment two ends connect two general connexons.Connect product stand-by after QIAquick purification kit (QIAGEN) purifying.
(3) Linker-PCR amplification
In order to avoid easy amplified fragments to be increased in a large number as far as possible, the cycle number of PCR is 20-26.Reaction system is 100 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation, the Taq archaeal dna polymerase of 4U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again.Standby after PCR product purification.
(4) two ends are connected with genome and the probe hybridization of joint and are fixed on polyacrylamide gel
The PCR product with universal primer of preparation is mixed by 3:1 with hybridization solution (Telechem), and 95 ℃ of sex change 5 minutes, are then cooled fast to room temperature.Hybridization solution is mixed with acrylamide soln, wherein, acrylamide monomer (19:1, w/w acrylamide: bisacrylamide) final concentration is 6% (w/v), ammonium persulphate (APS) is 1% (w/v), TEMED(N, N, N ', N '-tetramethylethylene diamine) be 0.5%.Above-mentioned mixed solution is poured into transparent plastic hose (diameter is about 2mm-10mm, long for 15cm-20cm) one end, after isocolloid solidifies, by the agarose solution of the other end perfusion upper 2%, between acrylamide gel and agarose gel, can not have bubble.
(5) electrophoresis is removed non-specific fragment, and sex change electrophoresis is collected target fragment
Remove non-specific fragment: will be equipped with in the plastic hose horizontalization plate electrophoresis groove of gel, the two ends of flexible pipe are inserted in the electrophoresis liquid (1 * TBE) of the two poles of the earth groove, the electric current only gel from flexible pipe passes through.Electric current is 100 mA electrophoresis time 10 minutes.
Target fragment is collected: the electrophoresis liquid in the groove of electrophoresis chamber two ends is replaced by 0.1M NaOH solution, the plastic hose of having removed non-specific fragment is placed in to both sides NaOH solution, carry out electrophoresis, electric current is 100 mA, electrophoresis 10 minutes.According to DNA indicator, cut target fragment agarose gel, extract target fragment, frozen standby.
(6) universal primer PCR amplification
Reaction system is 50 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation, the Taq archaeal dna polymerase of 2U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again.PCR cycle number is 35.Standby after PCR product purification.
(7) high-flux sequence instrument sequencing analysis target fragment
Above-mentioned target fragment is carried out respectively to high-flux sequence instrument sequencing analysis, and carry out bioinformatic analysis.
embodiment 3 employing polyacrylamide gels are caught with high-flux sequence instrument target and are carried out micro-pathogenic micro-organism floristic analysing in food:
(1) Design & preparation of probe
Bacterial classification specific DNA sequence Piece Selection: adopt the softwares such as Mega or Clustal W, carry out information biology comparison, complete the selection of 100 kinds of above food pathogenic micro-organism specific genes.
The design of probe: adopt the softwares such as primer5.0, for microorganism specific fragment designing probe.
Synthetic and the mark of probe: probe adopts DNA synthesizer to synthesize, simultaneously at acrylamide group of 5 ' end mark.
(2) pathogenic micro-organism genome extraction, genomic fragment and both sides connect joint
Pathogenic micro-organism extracting genome DNA: gather cooling meat and the duck series product after butchering processing, carry out six 50cm with sterile cotton swab
2sampling, then rinses the microorganism on cotton swab get off with PBS centrifugal 10 minutes of 14000rpm, separate microorganism.Adopt the microbial DNA method for extracting after test kit Huo Zhe research department optimizes, carry out the extracting of microorganism complete genome DNA.
The ultrasonication of genomic dna: choose the suitable ultrasonoscope of power, genomic dna is broken at random to the gene fragment of 200bp-500bp size.
Genomic DNA fragment two ends are connected (treatment process is with number of patent application CN200610098379.X) with general connexon: the random segment purification process that ultrasonic wave is obtained, remove some little and broken segments, then adopt respectively T4 polynueleotide kinase, T4 DNA polysaccharase, Klenow large fragment DNA polysaccharase I and Taq polysaccharase are processed, make random fragment produce a TA sticky end, then be connected with a general connexon (linker1:5 '-GTCGGAGGCCAAGGCGGCCGT 3 ' linker2:5 '-CGCCTTGGCCTCCGACT-3 '), make genomic fragment two ends connect two general connexons.Connect product stand-by after QIAquick purification kit (QIAGEN) purifying.
(3) universal primer PCR amplification
In order to avoid easy amplified fragments to be increased in a large number as far as possible, the cycle number of PCR is 20-26.Reaction system is 100 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation, the Taq archaeal dna polymerase of 4U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again.Standby after PCR product purification.
(4) two ends are connected with genome and the probe hybridization of joint and are fixed on polyacrylamide gel
The PCR product with universal primer of preparation is mixed by 3:1 with hybridization solution (Telechem), and 95 ℃ of sex change 5 minutes, are then cooled fast to room temperature.Hybridization solution is mixed with acrylamide soln, wherein, acrylamide monomer (29:1, w/w acrylamide: bisacrylamide) final concentration is 2% (w/v), ammonium persulphate (APS)) be 1% (w/v), TEMED(N, N, N ', N '-tetramethylethylene diamine) be 0.5%.Above-mentioned mixed solution is poured into transparent plastic hose (diameter is about 2mm-10mm, long for 15cm-20cm) one end, after isocolloid solidifies, by the agarose solution of the other end perfusion upper 2%, between acrylamide gel and agarose gel, can not have bubble.
(5) electrophoresis is removed non-specific fragment, and sex change electrophoresis is collected target fragment
Remove non-specific fragment: will be equipped with in the plastic hose horizontalization plate electrophoresis groove of gel, the two ends of flexible pipe are inserted in the electrophoresis liquid (1 * TBE) of the two poles of the earth groove, the electric current only gel from flexible pipe passes through.Electric current is 100 mA electrophoresis time 10 minutes.
Target fragment is collected: the electrophoresis liquid in the groove of electrophoresis chamber two ends is replaced by 0.1M NaOH solution, the plastic hose of having removed non-specific fragment is placed in to both sides NaOH solution, carry out electrophoresis, electric current is 100 mA, electrophoresis 10 minutes.According to DNA indicator, cut target fragment sepharose, extract target fragment, frozen standby.
(6) universal primer PCR amplification
Reaction system is 50 μ l, wherein contains 1 * reaction buffer, the Mg of 2.5 mM
2+, 0.4 μ M and primer connexon complementation (a wherein terminal modified fluorescence molecule of primer 5 '), the Taq archaeal dna polymerase of 2U, the dNTP of 5% DMSO and 200 μ M.On Gene Amp 2400 PCR System, carry out following pcr amplification: 95 ℃ of denaturations 4 minutes, then 95 ℃ of sex change are 1 minute, 70 ℃ of renaturation 2 minutes and 30 seconds, 72 ℃ are extended 30 seconds, and last, 72 ℃ are extended 10 minutes again.PCR cycle number is 35.Standby after PCR product purification.
(7) gene chips is carried out Pathogenic Microorganisms On Tropical
The preparation of gene chip: 1. probe preparation: for analyzing gene to be detected, design gene chip probes, a 5 ' terminal modified amino chemical group of probe.2. aldehyde slide preparation: select the total reflection slide of sizeable low fluorescence background, rinse surface.The slide rinsing is placed in to the beaker that fills deionized water, ultrasonic cleaning 1-2 hour.Slide after supersound washing is placed in the container of the sealing that contains 10% NaOH solution and soaks 30 minutes.Finally with deionized-distilled water again thoroughly clean, dry for standby; The slide glass of washes clean is placed in and contains 10% Alfa Aesar(3-methacryloxypropyltrimethoxysilane) acetone soln soak 1 hour, with acetone and deionized-distilled water thoroughly clean each twice, nitrogen dries up, dry for standby.3. probe is fixed on aldehyde slide: amido modified probe point sample, in slide, is then carried out to the condensation reaction of amino aldehyde radical, and covalent linkage is connected to probe on slide.
Gene chips is carried out Pathogenic Microorganisms On Tropical: above-mentioned PCR product is reacted with chip hybridization, and the slide after hybridization carries out scanner scanning and detects fluorescent signal, analyzes pathogenic micro-organism kind.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes specification sheets of the present invention and accompanying drawing content to do; 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 (10)
1. catching and a beneficiation technologies of novel targeted nucleic acid molecule, is characterized in that, comprises following operation steps:
1) by nucleic acid molecule fragment to be captured, cut the nucleic acid molecule fragment that glue reclaims 200bp-500bp, connection universal joint, forms target nucleic acid molecule fragment;
2) the target nucleic acid molecule fragment of step 1) is mixed with capture probe and hybridization solution, complete hybridization, described capture probe is modified with acrylamide chemical group;
3) to step 2) add acrylamide stock solution, ammonium persulphate and TEMED in the mixing solutions that forms, form acrylamide soln, and described acrylamide soln is placed in to one end of acrylamide gel shaped device, after solidifying completely, form acrylamide gel, and be filled with conductive rubber at the two ends of described acrylamide gel shaped device;
4) acrylamide gel after step 3) is placed in to the TBE electrophoresis liquid of electrophoresis chamber, electrophoresis is removed non-specific fragment wherein;
5) acrylamide gel after step 4) reaction is placed in to sex change electrophoresis solution, sex change electrophoresis is separated with capture probe by target nucleic acid molecule fragment, and described target nucleic acid molecule fragment is collected in one section of conductive rubber with electric current;
6) collect the target nucleic acid molecule fragment in conductive rubber in step 5), carry out high-flux sequence analysis.
2. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, described nucleic acid molecule to be captured is single double-stranded linear or single double-stranded annular DNA or RNA.
3. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, described capture probe is single stranded DNA, RNA, PNA or LNA.
4. according to the catching and beneficiation technologies of the novel targeted nucleic acid molecule described in claim 1 or 3, it is characterized in that, in described capture probe 5 ' end, 3 ' end or arbitrarily base, modify the acrylamide chemical group of any number.
5. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, acrylamide and N in the stock solution of acrylamide described in step 3), and the mass ratio of N ＇-methylene bisacrylamide is 19:1 or 29:1.
6. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, described acrylamide gel shaped device is that non-conducting material is made, and comprises plastic hose, glass groove or Glass tubing.
7. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, described acrylamide soln mass volume ratio concentration is 2%-6%.
8. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, that the two ends of described acrylamide gel shaped device are filled is that the conductive rubber put is conduction arbitrarily, nucleic acid molecule is had and filtered or adsorbing material.
9. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 8, is characterized in that, described conductive rubber is agarose gel.
10. catching and beneficiation technologies of novel targeted nucleic acid molecule according to claim 1, is characterized in that, the described sex change electrophoresis solution in step 5) is basic solution, comprises NaOH solution, KOH solution or Ca (OH)
2solution.
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| CN106086013B (en) * | 2016-06-30 | 2018-10-19 | 厦门艾德生物医药科技股份有限公司 | A kind of probe and design method for nucleic acid enriching capture |
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| CN106191256A (en) * | 2016-07-15 | 2016-12-07 | 艾吉泰康生物科技(北京)有限公司 | A kind of method carrying out DNA methylation order-checking for target area |
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