CN1766123A - Nonspecific bonding probe removal method through electrophoresis in biological chip hybridization - Google Patents

Abstract

The electrophoresis removing method for non-special bonding probe in biochip hybridization for cleaning chip comprises: dipping the hybridized biochip into buffer liquid in electric field; removing the free nucleic acid molecule with negative charge to positive electrode by the electric field to force objective probe off chip and enter buffer liquid for removing. This invention can remove efficiently the non-special bonding probe, increases SNR and recognition ability to wrong match of single basic group, and make obtained information more precise. This method uses electrophoresis instead of traditional cleaning, first, removing residual label target sequence as well as the target material as the dynamic balance between target for forming dual-chain structure and free target.

Description

The electrophoresis removing method of the non-special bonding probe in the biochip hybridization

Technical field

The present invention relates to a kind of method of removing the non-special bonding probe in the biochip hybridization process, relate in particular to the electrophoresis removing method of the non-special bonding probe in a kind of biochip hybridization.

Background technology

Gene chip is a new and high technology that develops rapidly in life science in recent years.So-called gene chip is exactly silicon chip, slide, the plastic sheet that is fixed with a large amount of gene probes (oligonucleotide probe or cDNA probe) by specific arrangement mode.Biochip technology is to obtain the main means of associated biomolecule information efficiently on a large scale.At present, this technical applications mainly contains gene expression spectrum analysis, new gene discovery, transgenation and polymorphism analysis, genomic library mapping, medical diagnosis on disease and prediction, drug screening, gene sequencing etc.The early 1990s is the main development that begins the various biochips that carry out with the U.S., and less than the time in 10 years, chip technology was developed rapidly, and presented the development peak.The preparation of gene chip at present can be divided into point sample preparation and the synthetic preparation method of original position.Aspect the use of gene chip, no matter be to detect target nucleic acid molecules by the hybridization between the nucleic acid of fixed DNA nucleic acid probe and mark, still earlier by and fixed DNA nucleic acid probe and nucleic acid between hybridize, utilize the double-stranded DNA that forms to detect protein molecule at last.The most important thing is after hybridization between fixed DNA nucleic acid probe and the nucleic acid, the double-spiral structure of fully just joining that the nucleic acid intermolecular hybrid forms can provide maximum hybridization signal, and the double-spiral structure of the mispairing that the nucleic acid intermolecular hybrid forms and the remaining labels targets sequence of hybridization are wished and can be removed fully, provide minimum hybridization signal, make things convenient for accurately obtaining of useful information like this.At present, the removing of non-special bonding probe adopts two kinds of means to realize usually: for hybridizing the labels targets sequence that constitutes base mispairing with stationary probe, then adopt and optimize hybridization temperature, make it have the form of minimum formation duplex structure; For the remaining labels targets sequence of hybridization, adopt the method for damping fluid washing chip.Clearly, the hybridization of gene chip detects can only adopt a hybridization temperature, owing to have thousands of fixed nucleic acid probe molecules on the chip, therefore the hybridization temperature that satisfies all probes by optimization is very difficult, sometimes or even impossible, like this, just provide stronger hybridization signal probably with the labels targets sequence probe of base mispairing sequence hybridization, distinguish the single base mismatch sequence and become difficult, especially all the more so for the not so good situation of hybridization signal intensity homogeneity.And the method that adopts damping fluid washing chip is for hard carriers such as glass, quartz, because the carrier adsorptive power is poor, washing can be removed the remaining labels targets sequence of hybridization effectively.But,, cause the mistake of analytical results owing to the target sequence of its vesicular structure strong adsorption mark makes the background noise of detection very high for porous supports such as gels.The appeal situation shows, adopts present washing methods can not remove the target sequence of non-special bonding effectively, causes the data of chip detection unreliable.

Summary of the invention

The invention provides the electrophoresis removing method of the non-special bonding probe in the wide biochip hybridization of a kind of recognition capability that can improve detection signal and suitability.

The present invention adopts following technical scheme:

A kind of electrophoresis removing method of non-special bonding probe of the biochip hybridization that is used for cleaning biochip, biochip after the hybridization is dipped in the damping fluid, and this damping fluid placed electric field, the electric field nucleic acid molecule that free is electronegative is shifted to the electric field positive pole, makes non-special bonding probe leave chip and enters in the electrophoretic buffer and be eliminated.

Compared with prior art, the present invention has following advantage:

The present invention can effectively remove non-special bonding probe behind the ground chip hybridization, improve the recognition capability of signal to noise ratio and single base mismatch, make the information obtained accurately and reliably.

The present invention replaces traditional washing process with electrophoresis behind microarray hybridization.In the electrophoresis process, hybridize remaining labels targets sequence and at first be removed efficiently.Owing to have running balance between the target of participation formation duplex structure and the free target, constantly to remove at electrophoresis under the situation of target, the target that participates in hybridization can be removed by electrophoresis with opening also of two strands.Because the duplex structure of fully just joining is more stable than the duplex structure of mispairing, by electrophoresis, has only complete matching sequence just to have tangible hybridization signal like this, the hybridization fluorescent signal of mismatch is then identical with background and have nothing to do in what of base mismatch number.Can distinguish fully the sequence of coupling and the sequence of single base mismatch by electrophoresis.In the treating processes, the non-special bonding probe that the traditional washing methods of employing electrophoresis replacement is removed behind the gene chip hybridization is more effective.Because nucleic acid molecule has negative charge, in the electrophoresis process, the label probe that participates in hybridization is not at first removed, proceed along with electrophoretic, the duplex structure that label probe and immobilized nucleic acid molecule hybridization form is removed under electrophoresis owing to the free label probe, and weighing apparatus is broken, the duplex structure that makes hybridization form begins to unwind, owing to fully just join to such an extent that duplex structure is more stable than the duplex structure that contains base mismatch, have only complete matching sequence just to have tangible hybridization signal, what of the then identical and irrelevant base mismatch number of the hybridization fluorescent signal of mismatch with background.The sequence of can more effective differentiation mating fully by electrophoresis and the sequence of single base mismatch can be used for single base mutation identification and SNP somatotype.Because this method and present widely used DNA chip compatibility can obtain to detect biomolecules information such as nucleic acid, protein more accurate and reliably.

Because arrangement mode is fixed with a large amount of gene probes on the chip, these probes are hybridized the Tm value that can't keep identical under a lot of situations with target sequence, therefore select one " optimization hybridization temperature " and be not suitable for the hybridization temperature of gene probes all on the chip, thereby the traditional method of the mode of " optimization hybridization temperature " and the damping fluid washing of hybridization back can not effectively have been distinguished

Just joining entirely under sequence and the uneven situation of single base mismatch sequence, especially hybridization signal, this differentiation difficulty is bigger.And the method for employing electrophoresis aftertreatment, no matter whether the hybridization temperature of gene probe is consistent, adopt the pattern that normal temperature hybridization might be hybridized institute to finish hybridization, and then then can will fully just join sequence and single base mismatch sequence well by the control deposition condition and realize effective differentiation.

Description of drawings

Fig. 1 is a kind of hybridization fluorescent image of oligonucleotide microarray.

Fig. 2 is a kind of hybridization fluorescent image of oligonucleotide micro-array chip.

Fig. 3 is a kind of hybridization fluorescent image of cDNA micro-array chip.

Embodiment

A kind of electrophoresis removing method of non-special bonding probe of the biochip hybridization that is used for cleaning biochip, biochip after the hybridization is dipped in the damping fluid, and this damping fluid placed electric field, the electric field nucleic acid molecule that free is electronegative is shifted to the electric field positive pole, making non-special bonding probe leave chip enters in the electrophoretic buffer and is eliminated, in the present embodiment, voltage of electric field is 1-600V, strength of current is 1-100mA, for example: voltage of electric field is 520V, 260V or 90V, strength of current is 15mA, 50mA or 90mA, best voltage of electric field is 50-100V, strength of current is 10-20mA, and above-mentioned direction of an electric field is parallel or vertical with the chip direction, and probe is fluorescently-labeled probe, the probe of chemiluminescent labeling, a kind of in the probe of enzyme labelling or the probe of colloid gold label.

The assorted of (with reference to accompanying drawing 1) a kind of oligonucleotide microarray improves signal to noise ratio by the effective labels targets sequence that participates in hybridization of removing of electrophoresis method.With the acrylamide modification of nucleic acids by nucleic acid being fixed on a kind of microarray that contains oligonucleotide probe and polyacrylamide gel (blank sample that does not contain nucleic acid) for preparing on the sheet glass with acrylamide monomer polymerization shape method.Fig. 1 is the comparative result that conventional washing and electrophoresis method are adopted in the hybridization aftertreatment.The 3rd, 4 row are 3% polyacrylamide gels that do not have few nucleic acid among the figure, and 1,2 row are 3% polyacrylamide gels that contain the 1uM oligonucleotide.Figure 1A be with complementary fluorescent mark sequence hybridization after adopt common washing methods to obtain the fluorescent scanning image.Can not effectively remove and cause high background from adopting strong mode of washing to obtain this as can be seen method of scintigram.3rd, the average fluorescent strength of 4 row is 48431 and 47105.1st, the average fluorescent strength of 2 row is 55110 and 54349, and this intensity is the intensity of background (polyacrylamide gel) no better than, and the ratio of its fluorescence intensity only is 1.14.So the intensive fluorescence background is can not be received, and therefore blank sample does not contain few nucleic acid can should not have fluorescent signal with target hybridization naturally, causes so strong fluorescent signal to be summed up as the strong adsorption effect of polyacrylamide gel.Therefore hybridizing aftertreatment, to adopt traditional mode of washing be the target indicia thing that can not remove non-special bonding effectively.Figure 1B is the scintigram that the hybridization aftertreatment adopts the electrophoresis mode to obtain.Target by the non-special bonding of electrophoresis is effectively removed, the relative intensity of fluorescence of blank sample also only is 981, the relative intensity of fluorescence of few nucleic acid then is 24772, though the relative intensity of fluorescence of few nucleic acid has also reduced, has then risen to 25.3 from 1.14 with the ratio of blank sample.By electrophoresis can increase greatly letter (number) so just very clearly a result shows electrophoresis (sound) ratio of can making an uproar, and knows sample and blank with removing.

Adopt the electrophoretic process method that nucleic acid oligomer is fully just being joined differentiation with base mispairing behind (with reference to accompanying drawing 2) a kind of oligonucleotide microarray hybridization.Specific as follows: (for example with four kinds of not homotactic oligonucleotides and the blank sample that do not contain nucleic acid, fully just join with Cy3 pigment flag sequence Cy3-TGC TGT GAG TGA ACC TGC TGT GTT GA-3 ' respectively, middle position mispairing 1, the sequence 5 of 2,3 bases '-TCA ACA CAG CAG GTT CAC TCA CAG CA-3 ' is (P0); 5 '-TCA ACA CAG CAG CTT CAC TCA CAG CA-3 ' is (P1); 5 '-TCA ACA CAG CACGAT CAC TCA CAG CA-3 ' is (P2); 5 '-TCA ACA CAG CAC CAT CAC TCA CAG CA-3 ' (P3) is prepared into nucleic acid array (the letter representation base mismatch of following watchband underscore.Represent P0 respectively, P1, P2, P3 sequence and each 10 the few nucleic acid lattice point that does not contain the blank sample P of nucleic acid constitute few nucleic acid microarray).The dna microarray chip at normal temperatures with Cy3-TGC TGT GAG TGA ACC TGC TGT GTT GA-3 ' sequence hybridization of 10uM 2 hours, chip placed under 1 * tbe buffer liquid 5V/cm condition of normal temperature (24 ℃) or 50 ℃ electrophoresis 30 minutes after hybridization was finished.Obtain two kinds of hybridization fluorescent images (Fig. 2 A and Fig. 2 B) under the electrophoresis temperature respectively with scanner scanning.The label probe that electrophoresis not only will not participate in hybridizing in Figure 1B has been disposed, and also removed fully with the label probe that contains the base mismatch probe hybridization, therefore have only complete matching sequence (P0) to have tangible hybridization signal, the hybridization fluorescent signal of mismatch is then identical with background; And the label probe that electrophoresis only will not participate in hybridizing in Figure 1A has been disposed, and do not removed fully with the label probe that contains the base mismatch probe hybridization, therefore have only complete matching sequence (P0) and mismatch all to have corresponding hybridization signal, its mispairing 1,2, the fluorescence intensity of 3 bases be respectively fully just joining 51.9%, 2.48% and 0.899%.Clearly strength of signal descends very fast along with the increase of base mispairing number, just joining and the single base mismatch sequence though also can distinguish under this condition, but obviously there is not among Fig. 2 B the deposition condition inferior segment divide easily, especially under the bad situation of probe lattice point strength of signal sequence homogeneity, the easier differentiation of the deposition condition of Figure 1B is just being joined and the single base mismatch sequence.

Its hybridization back of (with reference to accompanying drawing 3) a kind of cDNA micro-array chip adopts the electrophoretic process method that the single nucleotide polymorphism (SNPs) in low density lipoprotein receptor gene (Ox-LDL receptor 1 gene) 14417 sites of oxidation is analyzed.Three known 14417 loci polymorphism coronary heart disease patients' fresh blood sample fixedly obtains corresponding cDNA microarray by the PCR product behind PCR.The cDNA micro-array chip at normal temperatures with the flag sequence 5 of 10uM '-Cy3-GGA AAA CAGCCA A-3 ', 5 '-Cy5-GGA AAA GAG CCA A-3 ' sequence hybridization 2 hours, chip placed under normal temperature (24) 1 * tbe buffer liquid 5V/cm conditions electrophoresis 8 minutes after hybridization was finished.Scanning obtains hybridization fluorescent image (Fig. 3).Genotype by three samples of hybridization image is easy to be determined.Homozygote 14417C/C provides strong CY3 fluorescent signal (4,5 row among Fig. 3 A, green fluorescence).Homozygote 14417G/G provides strong Cy5 fluorescent signal (7,8 row among Fig. 2 B, red fluorescence).The fluorescent signal that heterozygous 14417C/G promptly provides Cy3 provides the fluorescent signal (1,2 row among Fig. 3 A and Fig. 3 B) of Cy5 again simultaneously, after two kinds of dyestuffs stacks, provides strong yellow fluorescence signal (1,2 row among Fig. 3 C).This somatotype result and traditional result consistent (Fig. 3 D) who checks order and be listed as.

Claims (5)

1, a kind of electrophoresis removing method of non-special bonding probe of the biochip hybridization that is used for cleaning biochip, it is characterized in that the biochip after the hybridization is dipped in the damping fluid, and this damping fluid placed electric field, the electric field nucleic acid molecule that free is electronegative is shifted to the electric field positive pole, makes non-special bonding probe leave chip and enters in the electrophoretic buffer and be eliminated.

2, the electrophoresis removing method of the non-special bonding probe in the biochip hybridization according to claim 1 is characterized in that voltage of electric field is 1-600V, and strength of current is 1-100mA.

3, the electrophoresis removing method of the non-special bonding probe in the biochip hybridization according to claim 2 is characterized in that best voltage of electric field is 50-100V, and strength of current is 10-20mA.

4, the electrophoresis removing method of the non-special bonding probe in the biochip hybridization according to claim 1 is characterized in that direction of an electric field is parallel or vertical with the chip direction.

5, the electrophoresis removing method of the non-special bonding probe in the biochip hybridization according to claim 1 is characterized in that probe is a kind of in the probe of the probe of probe, enzyme labelling of fluorescently-labeled probe, chemiluminescent labeling or colloid gold label.

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