CN201313902Y - Single wavelength DNA sequencing device - Google Patents

Abstract

The utility model discloses a single wavelength DNA sequencing device, comprising a working platform which is provided with a fluorescence excitation system, an array capillary system, a fluorescence detecting system and a data processing system, wherein the fluorescence excitation system consists of a laser with wavelength ranging from 760-960nm, a reflecting mirror arranged at the laser beam position, a low-power objective arranged on the position with 45 DEG to the central axis of the reflecting mirror and an eyepiece arranged on the vertical line of the low-power objective beam; the utility model uses a laser to replace a plurality of lasers in prior sequencing device and excites multi-photon fluorescence of four different fluorescent dyes simultaneously, so as to realize capillary electrophoresis separation and multi-photon fluorescence detection of DNA sequence, thus greatly simplifying the traditional detecting system. The single wavelength DNA sequencing device has the advantages of high sensibility, low instrument cost, low noise and small volume.

Description

The dna sequencing device of single wavelength

(1) technical field:

The utility model relates to a kind of dna sequencing device, specifically is the dna sequencing device of single wavelength.

(2) background technology:

Dna sequence analysis is one of most important technology of genomics and modern molecular biology field, is the basis of understanding gene structure and function.At present the dna sequence analysis device that uses mainly design based on the electrophoretic separation theory, has simple to operate, economical and practical, detection sensitivity, separation efficiency advantages of higher.People such as Smith adopt 4 kinds of fluorescent reagent marks, 4 spectrum channel detection systems, carried out the dna sequence analysis that the capillary electrophoresis laser fluorescence detects first, Mathies and Kambara etc. propose another kind of sequencing device, promptly adopt 20 kapillary electrophoresis arranged side by side, laser fluorescence electric charge lotus root is closed array detector (CCD) and detects, and has further improved the sensitivity that detects.Yeung etc. have proposed 100 capillary array electrophoresis devices again, adopt the CCD detector to detect 100 DNA sample separation capillaceous simultaneously, all obtain promising result.But above-mentioned detection device has a common ground, promptly be all to adopt a plurality of LASER Light Source, correspondence excites the multiphoton fluorescence of a plurality of different fluorescence dyes, survey with capillary electrophoresis separation and the multiphoton fluorescence of realizing dna sequence dna, therefore, price is very expensive, and volume is bigger, is unfavorable for the miniaturization and the immobilization of instrument; In addition, when indigo plant, green glow irradiation, the fluorescence background of gel coat, glass etc. is stronger, influences the sensitivity of instrument.

(3) summary of the invention:

The utility model just provides and a kind ofly can effectively reduce system cost, and volume is less and be easy to fix the dna sequencing device of highly sensitive single wavelength.

The dna sequencing device of single wavelength, comprise worktable, be respectively arranged with the fluorescence excitation system on the worktable successively, the array capillary system, fluorescence detection system and data handling system, it is characterized in that: described fluorescence excitation system is by a laser apparatus of 760-960nm wavelength, and the speculum that is provided with on the laser beam position, is with the speculum axis that 45 ° position is provided with a low power objective and the observation eyepiece that is provided with on the vertical line position of low power objective light beam constitutes.

The difference of the utility model and prior art is exactly to be to be that the laser apparatus of 760-960nm replaces a plurality of laser apparatus of the prior art with wavelength region, realizes multiphoton excitation(MPE).

So-called Excited Fluorescence Combined, generally, molecule or atom can only absorb a photon at every turn, from the ground state transition to excited state, when light intensity is enough high, will produce multiphoton transition, promptly once can absorb a plurality of photons.

The multiphoton excitation(MPE) spectrum of many dye fluorescent probes is broader than one-photon excitation spectrum, like this, can use the multiple dyestuff of exciting light simultaneous excitation of single wavelength, thereby obtain the different information in the same biological phenomena, is convenient to mutual contrast.So, can replace a plurality of laser apparatus with a laser apparatus, the multiphoton fluorescence of four kinds of different fluorescence dyes of simultaneous excitation is realized the capillary electrophoresis separation and the multiphoton fluorescence detection of dna sequence dna, has greatly simplified traditional detection system.Adopt the long-wavelength excitation light source, can avoid Raman scattering and Rayleigh scattering, and, help reducing the noise of background, improve the sensitivity of instrument system away from emmission spectrum.Also have the low and low characteristics of noise of cost simultaneously.

The utility model comprises the fluorescence excitation system, array capillary system, fluorescence detection system and data handling system four major parts.The fluorescence excitation system provides high-power exciting light, and focuses on the kapillary, realizes the multiphoton excitation(MPE) of different fluorescent reagents simultaneously; The array capillary system is made of side by side square kapillary, under the driving of high-voltage power supply, realizes the separation of 4 kinds of fluorescently-labeled DNA base-pair sequences of difference; The fluorescence detection system surveys the fluorescence of the multiphoton excitation(MPE) of four kinds of different fluorescent reagents by 4 passages; Data handling system is collected fluorescence and processing by automatic control, finally obtains dna sequence dna.

(4) description of drawings:

Fig. 1 is a structural representation of the present utility model;

Fig. 2 utilizes the separation graph spectrogram of the utility model to 6 kinds of luminescent dye molecules;

Fig. 3 is with the separation graph spectrogram of the utility model to DNA plasmid sample.

Among the figure: the 1-worktable, the 2-laser apparatus, the 3-speculum, the 4-low power objective, 5-observes eyepiece, the 6-array capillary, 7-phosphor collection object lens, 8-four-way fluorescence filter wheel, 9-fiber array detector, the 10-photorectifier, 11-data collecting card, 12-computer, 13-controller.

(5) embodiment:

Shown in Fig. 1 is to constitute in fluorescence excitation of the present utility model system, array capillary system, fluorescence detection system and the data handling system between each instrument by tactic relative position of light path and formation situation.

The utility model is included in the fluorescence laser system that is provided with on the worktable 1, and it is that wavelength region is the laser apparatus 2 of 760-960nm by the long-wavelength excitation light source, speculum 3, and numerical aperture is the low power objective 4 of 0.22-0.45, observation eyepiece 5 constitutes.Laser apparatus 2 is realized multiphoton excitation(MPE)s, and speculum is arranged on the light-beam position of laser apparatus, and low power objective is arranged on and is 45 ° position with the speculum axis, the observation eyepiece then be arranged on low power objective on the vertical line of luminous bundle; Described capillary system is an array capillary 6, and it can adopt single square kapillary or many square kapillaries to form side by side; Described fluorescence detection system is by phosphor collection object lens 7, four-way fluorescence filter wheel 8, fiber array detector 9 and photorectifier 10 constitute, phosphor collection object lens and above-mentioned observation eyepiece are separately positioned on the array capillary both sides, four-way fluorescence filter wheel is arranged on after the phosphor collection object lens, and fiber array detector and photorectifier all are arranged on the rear portion of four-way fluorescence filter wheel.Described data handling system comprises computer data acquiring processing hardware and process software, wherein hardware comprises data collecting card 11, computer 12 and controller 13, this controller connects computer and four-way fluorescence filter wheel respectively, and four-way fluorescence filter wheel is controlled.

Working process of the present utility model is: laser apparatus provides the long-wavelength excitation light source, realize multiphoton excitation(MPE), speculum reflects laser beam through the low power objective vertical irradiation on array capillary, realizes high-throughput, high-level efficiency separation, and realizes focusing on the positive center of kapillary by the observation eyepiece; The phosphor collection object lens are collected behind the fluorescence of array capillary, through four-way fluorescence filter wheel, filter out the diffusing veiling glare of environment, and fluorescent signal is surveyed by the fiber array detector, and realizes conversion in photorectifier, and optical signal becomes current signal; After data collecting card in the computer data processing system is collected the photodiode current signal, further amplify and filtering, the signal that data collecting card is collected, in conjunction with the control signal of controller to four-way fluorescence filter wheel, the utilization corresponding software calculates and handles out dna sequence dna.

Concrete experimental implementation requires:

1, kapillary is handled:

1. multiple fluorescence dye separates: respectively with 0.1mol/L HCl, 0.1mol/L sodium hydroxide, water and 25mmol/L borax pH9.50 solution, high pressure washing is coating quartz capillary (57cm * 75 μ m) 10min not before each sample introduction;

2. DNA separates: respectively with 0.1mol/L HCl, 0.1mol/L sodium hydroxide, water and 2%HEC/1 * TBE/20mmol/L high pressure washing coating quartz capillary (57cm * 75 μ m) 10min not, with 1%PVA the kapillary after handling is carried out surface modification again before each sample introduction;

3. separate high pressure: 20KV.Before the experiment, kapillary needs advanced horizontal high voltage balance 10 minutes.

2, input mode: 1. multiple fluorescence dye sample introduction: adopt the siphon sample introduction, 15cm * 60s;

2. DNA sample introduction: electrokinetic injection ,-6kV * 10s.

3, dna sample derivative reaction: P1 and P2 are that 5 ' end FITC modifies.

4, experimental procedure:

1) fluorescence detecting system regulates, and fluorescence focal is aimed at square kapillary central authorities.

2) high pressure balance kapillary is 10 minutes;

3) regulate computer, controller and four-way fluorescence filter wheel are in running order, make detection system simultaneously, and capillary system etc. are in running order.

4) sample detection.

In order to prove the utility model, carried out following experiment:

1, single wavelength is realized the simultaneous excitation of the multiple fluorescent reagent that excitation wavelength is different.

The excitation wavelength of 6 kinds of luminescent dye molecules and emission wavelength such as table 1 if adopt one-photon excitation then need multi-wavelength's laser apparatus, are also just said the simultaneous excitation that will realize 6 kinds of luminescent dye molecules simultaneously, the laser apparatus of a plurality of wave bands of needs.The utility model adopts single wavelength can realize simultaneously exciting and collecting.Show that Success in Experiment the utility model realizes exciting of multiple dye molecule simultaneously, and realize capillary electrophoresis separation and fluorescence detection.Experimental result such as Fig. 2.

The excitation wavelength and the emission wavelength of table 16 kind of luminescent dye molecule

Luminescent dye molecule	Excitation wavelength (nm)	Emission wavelength (nm)
			Rhodamine 6g	525	555
Rhodamine B	540	625
			The sulfonic acid rhodamine B	520	595
Tonka bean camphor	445	525
			Calcium flavin	494	517
The amino pyrene-1,3 of APTS8-, 6-trisulfonic acid sodium salt	424	505

2, adopt the utility model dna sample to be carried out the multiphoton fluorescence electrophoretic separation, experimental result such as Fig. 3.Experiment shows and can check order to DNA.

Claims (1)

1. the dna sequencing device of single wavelength, comprise worktable, be respectively arranged with the fluorescence excitation system on the worktable successively, the array capillary system, fluorescence detection system and data handling system, it is characterized in that: described fluorescence excitation system is by a laser apparatus of 760-960nm wavelength, and the speculum that is provided with on the laser beam position, is the low power objective that 45 ° position is provided with the speculum axis and an observation eyepiece that is provided with on the vertical line position of low power objective light beam constitutes.

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