CN1607257A

CN1607257A - Capillary electrophoresis molecular beacon gene detecting technology

Info

Publication number: CN1607257A
Application number: CN 200310101118
Authority: CN
Inventors: 姜晓峰
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-10-16
Filing date: 2003-10-16
Publication date: 2005-04-20

Abstract

A sensitive gene detection technology-capillary electrophoresis molecule beacon gene detection technology which improves the current technology using advantages of capillary electrophoresis technology that quick, high sensitivity, low cost and capable of automation, and combined with strong specificity and high sensitivity of beacon detection technology. Said invention can be used in the gene diagnosis of clinic disease.

Description

Capillary electrophoresis molecular beacon technique of gene detection

One, technical field:

The present invention relates to a kind of novel quick, sensitive technique of gene detection, especially can detect transgenation rapidly and sensitively, be applicable to the gene diagnosis of clinical disease.

Two, background technology:

The gene diagnosis of disease mainly is the diagnosis to DNA variation, and the mutant dna sequence that causes disease comprises the variation of various sudden changes and polymorphism.The short battle array sequence of for example one or more nucleotide transversions, disappearance or insertion, different quantities repeats etc.The method that transgenation/polymorphism commonly used at present detects has single-strand conformation polymorphism analysis (SSCP), heteroduple analysis (HA), capillary electrophoresis technique (CE) and molecular beacons technology (MB) etc.

1, single-strand conformation polymorphism analysis (SSCP) equal length, not homotactic dna fragmentation are because of the different electrophoretic mobilities of its secondary structure also difference, the medium and small sequence variation to a base of dna molecular can both change its original secondary structure, thereby changes its electrophoretic mobility in gel.SSCP utilizes the wild-type electrophoretic mobility different with the mutant DNA fragment that mutant gene is screened.SSCP does not need special equipment, be detect in the traditional method of transgenation the quickest and easy.But also there are some shortcomings in SSCP: (1) can only point out that there is sudden change in a certain zone of DNA, can not make an explanation or accurate location to sudden change; (2) SSCP sudden change recall rate is 80% under the single deposition condition; (3) the suitable pcr amplified fragment of length between 100～300bp that detect of SSCP, fragment length will reduce detection efficiency greater than 300bp; (4) the SSCP influence factor is a lot, needs the analyst that rich experience is arranged; (5) SSCP length consuming time, complete sscp analysis process needs more than 6 hours usually.

2, heteroduple analysis (HA) with the sex change of PCR product after again annealing, can form four kinds of double-stranded DNAs in the heterozygote sample, two homoduplexs and two heteroduplexs, heteroduplex form the bubble structure distortion at the base mismatch place, the rate of migration in gel is slower than homoduplex.The sudden change recall rate of HA is 80%, and analytic plate segment length scope and SSCP are close, or slightly longer.Traditional HA also has the general defective of plate gel electrophoresis simultaneously, is not suitable as the routine inspection project.

Except that SSCP and HA, plate gel electrophoresis DNA analysis of variance method commonly used also has denatured gradient gel electrophoresis (DGGE), base mismatch chemical cracking method (CCM) etc., because of its shortcoming such as loaded down with trivial details, time-consuming, and limited their application aspect routine analysis, can only bring into play its advantage at scientific research field.

3, capillary electrophoresis screening-gene sudden change (CE-SSCP) capillary electrophoresis is to be motivating force with the high-voltage electric field, is split tunnel with the kapillary, according to the difference in mobility and the distribution behavior and realize an isolating liquid-like phase isolation technique between each component in the sample.The sudden change of capillary electrophoresis screening-gene has not only shown the susceptibility (90%) of height, and quick, easy, low cost and other advantages.It is wide to detect the dna fragmentation length range simultaneously, little of tens bases, arrives thousands of bases greatly.But capillary electrophoresis screens transgenation and still exists and can not make an explanation or pinpoint shortcoming to sudden change.

4, (Molecular Beacons, MB) MB is 3 ' and 5 ' terminal mark fluorescent molecule and a quencher molecule respectively at same probe to molecular beacon.This probe 5 ' and hairpin structure of 3 ' terminal self formation, this moment, fluorescence molecule and quencher molecule were contiguous, therefore can not produce fluorescence.When in the solution special template being arranged, this probe and template are hybridized, thereby have destroyed the hairpin structure of probe, so solution just produces fluorescence, intensity of fluorescence is directly proportional with the amount of template in the solution, therefore can be used for the PCR quantitative analysis.A fundamental characteristics of molecular beacon is, because the rigidity of dna double spiral, probe-target sequence hybridization product can not coexist with the stem complementary state.The complementary probe-target sequence hybridization is more stable than loop-stem structure on energy fully, and complementary its energy of incomplete crossbred is stable not as good as loop-stem structure.This characteristic also is the basis of molecular beacons detection single nucleotide polymorphism high degree of specificity.The molecular beacons detection technology is an important breakthrough on the nucleic acid detection method.But the molecular beacon technique of gene detection still has its shortcoming, detects when can't carry out a plurality of transgenation point as it.Simultaneously because molecular beacon background problem causes false positive probably.

At present, gene diagnosis was in by the stage of scientific research field to clinical universal transition, did not still have a kind of sophisticated DNA variation screening method as routine clinical checkup item.This mainly is because traditional detection method all has its limitation.Therefore it is extremely urgent to set up easy, responsive, special and rapid automatized gene test means.

Three, summary of the invention:

In order to overcome the limitation of traditional gene tester, but the present invention improves traditional technique of gene detection---utilize advantages such as capillary electrophoresis technique is quick, highly sensitive, the low automatization of cost, advantages such as binding molecule beacon detection technique high specificity, sensitivity height, found new super-sensitive technique of gene detection---capillary electrophoresis molecular beacon technique of gene detection.It has not only overcome the capillary electrophoresis detection technique and can not make an explanation or pinpoint shortcoming to transgenation, has also overcome to detect when molecular beacons technology can't be carried out a plurality of transgenation point and because molecular beacon background problem causes false-positive shortcoming.This technology can realize automatization simultaneously, is applicable to the gene diagnosis of clinical disease.

Four, description of drawings:

Describe below in conjunction with the advantage of accompanying drawing this technological invention:

Fig. 1 is the electrophorogram of molecular beacon after the sex change, and we occur about 11 seconds at visible its electrophoresis peak;

Fig. 2 is the electrophorogram after dna fragmentation and the molecular beacon hybridization, and as seen we the molecular beacon peak occurs about 11 seconds, and the segmental peak of purpose after hybridization occurring about 15 seconds.

From these two figure we as can be seen, even there is the background fluorescence problem in molecular beacon probe, can utilize capillary electrophoresis technique to molecular beacon probe with hybridization after the purpose fragment separate, can not influence detected result, thereby avoid causing false positive.

Five, embodiment:

We are example to detect tumour cancer suppressor gene p53, p16 and p73 simultaneously, and the embodiment of this detection technique is described.

1, according to the distinguished sequence of a certain gene to be checked or the known mutations site design molecular beacon of gene:

When detecting tumour cancer suppressor gene p53, p16 and p73 simultaneously, we can utilize the molecular beacon probe design software according to the common mutations site of each gene, design molecular beacon respectively.

2, the gene to the known site sudden change carries out pcr amplification:

According to the purpose fragment design primer of above-mentioned cancer suppressor gene, and utilize multiplex PCR to increase.But be noted that designed DNA purpose fragment length is not close, avoid separation difficulty.

3, pcr amplification product and molecular beacon are hybridized:

Under liquid-phase condition, hybridize (method is with the hybridizing method of common probe).Can't hybridizing with molecular beacon probe of sudden change or genetically deficient arranged, and do not have can hybridizing of sudden change.

4, the PCR product after the hybridization is carried out capillary electrophoresis separation, and the online laser induced fluorescence detector that utilizes detects.

Earlier fluorescent DNA Marker is carried out capillary electrophoresis separation, determine the electrophoresis peak position of various fragment lengths; Then the PCR product after the hybridization is separated, can send fluorescence, can be detected, determine it is which kind of gene fragment by the position of Marker by laser induced fluorescence detector with molecular beacon probe bonded dna fragmentation.This detection technique is not only carried out separation detection to the dna fragmentation of different fragments, also can separate unconjugated molecular beacon probe with other dna fragmentations simultaneously, reaches the purpose that detects transgenation.

Claims

A kind of technique of gene detection, it overcomes the limitation of traditional gene tester, it is characterized in that: but advantages such as capillary electrophoresis technique is quick, highly sensitive, the low automatization of cost utilized, advantages such as binding molecule beacon detection technique high specificity, sensitivity height, can be easy, responsive, special and rapid automatized gene and transgenation are detected.