WO1989001620A1

WO1989001620A1 - Optoelectric electrophoresis analysis systems

Info

Publication number: WO1989001620A1
Application number: PCT/US1988/002679
Authority: WO
Inventors: Michael D. Morris; Reed A. Shick; Konan Peck; Stephen J. Parus
Original assignee: The Regents Of The University Of Michigan
Priority date: 1987-08-13
Filing date: 1988-08-11
Publication date: 1989-02-23

Abstract

A system for evaluating electrophoresis samples using optical means for sensing orientation of macromolecules suspended within the gel or fluid. An electrophoresis sample (16) is positioned such that a probe beam passes through the sample. Optical elements (14, 16) and detectors (22) are employed to detect changes in the probe beam caused by orientation of the molecules within the sample. The optical systems can be based on induced birefringence within the sample, electric dichroism, fluorescence, light scattering, etc. Systems in accordance with this invention can be used to locate areas within an electrophoresis gel sample where separation has occurred to locate the presence of sample molecules. Alternately, unseparated samples can be analyzed by timing the characteristic relaxation or orientation time of various molecules as they go from an orientated condition within the sample gel or fluid to a random orientation after the external field is relieved or reversed in polarity, or from a random orientation to an oriented condition after the external field is applied or reversed in polarity.

Description

OPTOE ECTRIC ELECTROPHORESIS ANALYSIS SYSTEMS

BACKGROUND AND SUMMARY OF THE INVENTION

This invention relates to systems for analyzing electrophoresis samples and particularly to such systems which detect the presence of molecules by inducing orientation of the molecules by an external electrical or magnetic field and detecting the oriented molecules by optical means.

Nucleic acids, proteins and other biological macromolecules are commonly separated by electrophoresis in which the molecules are caused to migrate within a media such as agarose or polyacrylamide gels. For certain electrophoresis methods the gel is not needed and migration takes place in a narrow tube through a fluid. Molecule characteristics determine their mobility and thus molecules become separated by type during migration. Depending on the complexity of the sample, the separation process may take as little as forty-five minutes or as long as twelve hours. Nucleic acid electrophoresis must be performed in a low DC field and is therefore slow. The reason is that the molecules become elongated in high electric fields. When this happens, molecules of different sizes tend to migrate at similar rates, causing a loss in resolution. The components separated by electrophoresis are visualized by staining them. The most coππton stain is ethidium bromide. Color stains and silver staining are occasionally employed, but are less convenient and sensitive than ethidium. Ethidium staining is a fairly rapid procedure, requiring only about twenty minutes of operator time if carried out manually. Ethidium bromide is, however, a known potent carcinogen, and must be used carefully.

In some procedures, proteins or nucleic acids are reacted with fluorescent molecules or molecules containing radioisotopes before separation, thus making the separated macromolecules fluorescent or radioactive. Gene sequencing procedures involve such fluorescent or radioactivity labelling of nucleic acids, with molecules which are specific for the base which terminates one end of the chain.

Protein electrophoresis itself is generally faster than nucleic acid separation. Times of thirty minutes to several hours are coπmon. The staining procedures are, however, time consuming. An automated procedure for staining with Coo assie stain takes about an hour. An automated silver staining procedure takes about three hours. Accordingly, present electrophoresis evaluation techniques are both time consuming in that a significant time period must elapse to enable adequate separation of molecules to occur, and also since staining procedures are often complex and time consuming. A technique to examine molecules separated by electrophoresis without staining would be welcome, even if some sensitivity were sacrificed. In addition, procedures for detecting molecule mobility in various media without separation would also be desirable.

In accordance with a first set of systems according to the present invention, characterization of electrophoresis samples after molecule separation is carried out without staining using optical systems for detecting orientation of the molecules caused by exposure to an external electromagnetic or other field. For example, the birefringence characteristics of the sample can be used to detect orientation. When molecules become oriented within an electrophoresis sample, they cause the sample to become birefringent in the area in which they are present. Accordingly, a linearly polarized light ray passing through the sample in the area of the oriented molecules will become circularly or elliptically polarized as it exits the sample. Linear and/or circular polarizers are used to detect polarization changes in the sample caused by birefringence. This birefringence characteristic can be used to detect the presence of molecules at particular regions of the sample after molecule separation without staining, which is ordinarily necessary for evaluation by visual inspection or by conventional densitometers.

In accordance with another aspect of this invention, optical systems can be used to detect molecular orientation for unseparated samples. For such systems the molecules become oriented in the presence of an electric or magnetic field which is periodically relaxed or reversed. Molecule length, configuration, charge and other characteristics determine their mobility within the medium. This mobility is related to the time necessary for the molecules to move from an oriented state within the medium to random orientation (or vice versa) . The change from orientation to randomness is detected by an optical system. For example, birefringence change in the sample with respect to time can be evaluated. Highly mobile molecules will return to randomness or become oriented in a very short time, whereas longer, less mobile molecules will have a longer characteristic change 5^" of state time period. By using a birefringence detector and cyclically applying an electric field to the sample, a distribution of change of state times can be generated which is characteristic of the constituents of the sample. Change of state time can be measured by observing birefringence changes after the external field is removed, 10 or as a function of light transmission through a range of applied AC signal frequencies.

Other optical properties or oriented molecule can be used to detect molecular orientation. For example, if the wavelength of the light source is absorbed by the sample molecules, then orientation in 15 an electromagnetic field causes a difference in the absorption of light polarized at different angles. The time or frequency dependence of circular dichroism may also be used to characterize the orientation of the molecules. The techniques are similar to those used to monitor birefringence changes, except for the wavelength of the light source. 20 If wavelengths characteristic of different classes of molecules can be found, then electric dichroism at several wavelengths may be monitored at the same time, thus increasing the resolving power of the technique.

Another optical technique for evaluating molecular 25o orientation employs fluorescence. If a molecule is itself fluorescent or has been labelled with a fluorescent reagent, then the fluorescence of the oriented molecule will be polarized. As the molecule returns to random orientation or becomes oriented, the change of this fluorescence polarization is monitored by techniques similar to those 30. used to monitor birefringence decay.

Additional benefits and advantages of the present invention will become apparent to those skilled in the art to which this invention relates from the subsequent description of the preferred embodiments and the appended claims taken in conjunction with the

35. accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagrammatic view showing an electrophoresis analysis system in accordance with a first embodiment of this invention adapted for characterizing separated gel samples using a pair of linear polarizers for detecting birefringence changes in the sample.

Figure 2 is a diagrammatic view showing an electrophoresis analysis system in accordance with a second embodiment of this invention adapted for characterizing separated gel samples using a pair of linear polarizers and a quarter-wave plate which detect birefringence changes in the sample.

Figure 3 is a diagraππiatic view showing an electrophoresis analysis system in accordance with a third embodment of this invention for detecting birefringence changes having a translating stage for scanning a gel sample particularly suited for use with samples previously subjected to electrophoresis migration.

Figure 4 is a diagraππiatic view showing an electrophoresis analysis system in accordance with a fourth embodiment of this invention for detecting birefringence changes particularly adapted for column electrophoresis.

Figure 5 is a diagrammatic view of an electrophoresis analysis system for detecting birefringence changes in accordance with a fifth embodiment of this invention adapted for relaxation time measurements of unseparated samples. Figure 6 is a diagraππatic view of an electrophoresis analysis system according to a sixth embodiment of this invention adapted for measuring orientation time or relaxation time, or frequency of samples based on changes in non-linear optical properties of the sample. Figure 7 is a diagrammatic view of an electrophoresis analysis system in accordance with a seventh embodiment of this invention which detects molecular orientation by changes in electric dichroism.

Figure 8 is a diagrairmatic view of an electrophoresis analysis system in accordance with an eighth eπbodiment of this invention which detects molecular orientation by fluorescence polarization. Figure 9 is a diagrai iatic view of an electrophoresis analysis system in accordance with a ninth embodiment of this invention vfaich detects molecular orientation by light scattering.

Figure 10 is a diagrammatic view of an electrophoresis

55 analysis system in accordance with a tenth embodiment of this invention which detects molecular orientation by forming an image of the birefringence or dichroism of fluorescence polarization or light scattering by means of a photographic camera or video camera.

W.2 DETAILED DESCRIPTION OF THE INVENTION

An electrophoresis analysis system in accordance with a first embodiment of this invention is shown in Figure 1 and is generally designated there by reference number 10. System 10 includes low power laser 12 as a light source which may be a helium-neon lazer

15..: with approximately a one milliwatt output. The light output from laser 12 needs to be linearly polarized for this system since it is based on measuring birefringence. This is achieved in accordance with the embodiment of Figure 1 by using a laser 12 of a^' type which inherently provides a polarized output and further increasing the

20 level of polarization using linear polarizer 14 which may be a sheet or prism polarizer. Sample 16 would typically be an agarose or polyacrylamide gel in which macromolecules are present and have been separated by electrophoresis migration. Power supply 18 applies an electric field to sample 16 in a manner which orients molecules within

25 the sample.

The direction of the electric field within sample 16 is oriented 45 degrees frcm the plane of polarization of the light from laser 12. This orientation maximizes the sensitivity to birefringence change. Analyzing polarizer 20 is interposed within the ray emitted

30 from sample 16. Detector 22 senses the intensity of light transmitted through analyzing polarizer 20.

In operation, the light from laser 12 is made well polarized after passing through polarizer 14. Induced birefringence within sample 16 causes linearly polarized light passing through the sample

35. to become circularly or elliptically polarized. This occurs since a portion of the ray passing through the sample is delayed with respect to another portion of the ray due to a variation in index of refraction. If sample 16 is not birefringent, either because an electromagnetic field is not applied to the sample or because macromolecules are not present in the probed area, the light will remain principally linearly polarized. Analyzing polarizer 20 may be oriented to normally extinguish the light where there is no birefringence such that induced circular or elliptical polarization will cause some light to be transmitted through polarizer 20 and sensed by detector 22 since birefringence causes the plane of polarization of some of the light to change. Alternately, polarizer 20 may be oriented to normally permit light to pass which would be reduced in intensity upon birefringence through sample 16. In use, system 10 can be used to detect the presence of molecules in selected areas of sample 16 which where separated by electrophoresis, or may be used to characterize unseparated samples, as explained in greater detail below.

An electrophoresis analysis system in accordance with the second embodiment of this invention is shown in Figure 2 and is generally designated by reference number 30. Elements of this embodiment and others described below which are identical to those described in connection with the first embodiment are identified by like reference numbers. System 30 varies from the embodiment shown in Figure 1 in that quarter-wave plate 32 is added. Quarter-wave plate 32 causes linearly polarized light passing through it to become circularly polarized. Therefore, absent birefringence being induced within sample 16, the light will be circularly polarized as it passes through quarter-wave plate 32. In the event of induced birefringence, quarter-wave plate 32 partly reverses the elliptical polarization to make it more closely approach linear polarization. The degree of linear polarization is evaluated by analyzing polarizer 20 and detector 22.

Figure 3 illustrates an electrophoresis analysis system in accordance with a third embodiment of this invention which functions as a scanning densitometer to detect birefringence changes and is designated by reference number 40. Light transmitted through polarizer 14 is directed through sample 16 by mirror 42. Sample 16 is immersed in a bath 44 of electrophoresis buffer. Bath 44 contains two electrodes 46 which are conventionally platinum wire and run along the sides of the gel sample. The bottom of bath 44 is transparent glass

48 and a glass cover over the gel (not shown) serves to keep it in a fixed position. Sample 16 is scanned by translating it through the fixed optical path (as shown in phantom lines) , most conveniently with a motor driven X-Y translation stage. In use, molecules are deposited on a gel sample and subjected to an electromagnetic field to cause molecular migration and separation. Light from laser 12 passes through selected areas of sample 16 to detect the presence of the molecules of interest. Signal generator 50 produces an AC signal with a frequency range, for example, of 4 to 16 Hz. The AC signal is used to prevent net movement of molecules which occurs When they are exposed to DC fields. For maximum sensitivity, the signal from detector 22 is processed through lock-in amplifier 52 which provides synchronous demodulation.

An electrophoresis analysis system according to a fourth embodiment of this invention is shown in Figure 4 and is generally designated by reference number 60. This embodiment is similar in configuration to that shown in Figure 3 except that it is adapted for use with column electrophoresis procedures. Power supply 18 and signal generator 50 are operated so that the sample receives a voltage with a net DC component, which causes migration, and an AC component, which can be detected by synchronous demodulation or other techniques. For this embodiment, macromolecules within column 62 are detected as they cross the optical path of the analyzer. Column 62 is typically a length of glass tubing filled with a gel media or with a fluid buffer only with buffer reservoirs 64 and 66 at both ends. Power connections are made to each of reservoirs 64 and 66. Column 62 is preferably made of tubing having a rectangular cross section to minimize distortion of the probing laser beam as it passes through the column.

Figure 5 illustrates an electrophoresis analysis system in accordance with a fifth embodiment designated by reference number 80.

This embodiment, and that shown in Figure 6, differ from those shown in Figure 3 and 4 in that they are particularly adapted for characterizing unseparated samples of molecules. For this embodiment, a sample of molecules is provided which is mixed with a gel such as agarose or polyacrylamide. In some embodiments the mixture is poured - 8 - into sample chamber 82 which preferably has a light path length on the order of one to ten millimeters. Chamber 82 is constructed from Lucite (Trademark) or other material which is both electrically insulating and waterproof and fitted with glass windows 86. A field 3 is presented to the sample by platinum foil electrodes 84. In operation, voltage is applied, and when it is turned off, molecular mobility is measured as a change in birefringence as the oriented molecules returning to random orientation or change frcm randomness to orientation. It is convenient to repeat the measurement many times,

10 reversing the field direction between each measurement to prevent net movement of the sample molecules. Detector 22 generates data which are collected by an analog-to-digital converter and stored.

Using system 80, a characteristic exponential relaxation time is observed for each molecule such as nucleic acid fragments.

1 Relaxation times will be about one microsecond for the smallest nuclear acid fragments and about one minute for the largest. Repeated measurements allow signal averaging and also allow measurements on several different time scales to encompass the range of expected molecules. Data are analyzed by fitting the measurements to a sum of

20 exponentials or by examining the logarithm of the measurement data for straight line sections.

As another means for operating system 80, the frequency of applied voltage to sample chamber 82 can be varied, for example, between about 1 Hz to 1 MHz. The relaxation times are observed as the

25, frequency at which the observed birefringence signal is 45 degrees out of phase with the second harmonic of the driving signal. The harmonic is required because birefringence is observed as a change in transmitted intensity, which depends on the magnitude of the external field, not its polarity.

30 The above described embodiments detect birefringence changes as a means of detecting molecule orientation in separated or unseparated samples. Numerous other optical systems may be used to detect orientation. Some of the alternate optical systems are described in connection with the embodiments of this invention

35- described below. The techniques described below may be employed in systems for separated samples operating as a scanning densito eter as in Figure 3, or for systems adapted for unseparated samples such as that shown in Figure 3.

The embodiment illustrated in Figure 6 designated by reference number 90 utilizes the phenomenon of electric birefringence in which a second harmonic of the fundamental frequency of light from laser 12 is generated when the sample molecules are oriented. For this embodiment, laser 12 is pulsed. The light at the optical second harmonic frequency is separated from the unconverted laser light (at the fundamental frequency) by interference filter 92. The second harmonic light is then sensed by detector 22 and the signal is processed through waveform analyzer 54. This means for detecting molecular orientation can be used in systems for use with separated or unseparated samples.

Figure 7 shows a system for detection of molecular orientation by electric dichroism which is designated by reference number 110. The dichroism is measured as the change in absorption of laser light, which need not be laser light, as the electromagnetic field applied to the sample is varied. If an incoherent source is used, then filters such as 112 or a monochro atαr may be used to isolate several characteristic wavelengths.

Figure 8 shows a system for measurement of fluorescence polarization which is designated by reference number 120. The sample is illuminated with a light source and the fluorescence is measured through lens 122 and polarizer 124. As the molecules change orientation in response to a pulsed varying electric field, the fluorescence component which is passed by polarizer 124 will change. A laser 12 is the preferred light source, because the fluorescence intensity is proportional to the source light intensity. Several filters 12 and detectors, or a monochrcanator, may be used to monitor fluorescence at several wavelengths simultaneously.

Figure 9 shows a system according to this invention for measurement of molecule orientation by detecting changes in light scattering. Sample chamber 82 is illuminated with a light source 12 designated by reference number 130 and light scattering is measured through analyzing polarizer 20 by detector 22. The measurement is made at an angle to the illuminating beam, preferably 90 degrees, to discriminate against transmitted light. As the molecules change - 10 - orientation in response to a varying electromagnetic field, the scattering component which is passed by polarizer 20 will change. Laser 12 is the preferred light source, because the scattering intensity is proportional to the source light intensity. 5 Figure 10 shows a system designated by reference number 140 for measurement of birefringence, dichroism, fluorescence polarization or scattering by formation of the image of an extended region of a gel or of an entire gel. Sample 82 is illuminated with a light source 12 through lenses 142 and 144, and polarizer 14 and the birefringence or

Iff dichroism is measured through analyzing polarizer 20 by_. means of focusing the beam by lens 148 onto a two dimensional imagery device such as a photographic camera or a video camera 146. Two measurements are required. The dichroism or birefringence is measured with the external field applied. A background measurement is made in the

15 absence of external field. The induced birefringence or dichroism is then calculated as the difference between these measurements. By placing the camera 146 at an angle to the light beam, the fluorescence polarization or light scattering can be measured.

While the above description constitutes the preferred

20 embodiments of the present invention, it will be appreciated that the invention is susceptible to modification, variation and change without departing from the proper scope and fair meaning of the accompanying claims.

Claims

CLAIMS:

1. A system for detecting the presence of molecules suspended in electrophoresis samples comprising: means for orienting said molecules, and a light source generating a beam of light passing into the sample, optical means receiving said beam of light after passing into the sample for detecting that orientation of said molecules has occurred, thereby enabling the presence of said molecules to be detected.

2. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said means for orienting comprises exposing said sample to an electromagnetic field.

3. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises detecting changes in birefringence in said sample caused by orientation of said molecules.

4. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises detecting ' changes in electric dichroism in said sample caused by orientation of said molecules.

5. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises detecting changes in fluorescence in said sample caused by orientation of said molecules.

6. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises detecting changes in light scattering in said sample caused by orientation of said molecules.

7. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises measuring the transmission of a harmonic of the fundamental frequency of said light source.

5

8. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 further comprising means for causing said light beams to pass through selected areas of said sample such that the location of said molecules may be Q determined.

9. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 2 further comprising said electromagnetic field is varied cyclically such that a 5 relationship between the frequency of said field and the detection by said optical means can be determined.

10. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 further 0 comprising means for timing the change of orientation of said molecules in response to said electromagnetic field thereby providing a characteristic time for the change in orientation of said molecules which is a characteristic of the type of said molecules.

5 11. A system br detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said optical means comprises a two dimensional imagery device which received said light beam after passing into the sample.

0 12. A system for detecting the presence of molecules suspended in electrophoresis samples defined in Claim 1 wherein said light source generates a beam of polarized light which passes through said samples, said sample being oriented with respect to said source such that birefringence induced within said sample upon exposure to 5 said means for orienting causes the polarization of said light to change, said optical means including analyzer means for transmitting light in accordance with its polarization such that the intensity of light passing through said analyzer means is different when said birefringence is induced frcm when birefringence is not induced, and a detector for sensing the intensity of light transmitted through said analyzer means.

13. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 wherein said polarized light source includes a lazer.

14. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 wherein said polarized light source includes a linear polarizer.

15. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 wherein said analyzer means comprises a linear polarizer oriented such that light passing through said analyser means when there is no induced birefringence is minimized and wherein light passing through said analyzer means when birefringence is induced is maximized.

16. A system for detecting the presence of molecules in electrophroesis samples as defined in Claim 12 wherein said analyzer means comprises a linear polarizer oriented such that light passing through said analyzer means when there is no induced birefringence is maximized and wherein light passing through said analyzer means when no birefringence is induced is minimized.

17. A system for detecting the presence of molecules -in electrophoresis samples as defined in Claim 12 wherein said anlayzer means includes a quarter-wave plate for decreasing the extent of elliptical polarization of the light passing through said sample caused by induced birefringence.

18. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 further comprising a translation stage for moving a gel sample with respect to said beam whereby said gel sample may be translated and various areas of said gel may be characterized.

19. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 wherein said sample comprises a column having an upper -and a lower reservoir with electrodes in each of said reservoirs and wherein light from said light source passes through said column.

20. A system for detecting the presence of molecules in electrophroesis samples as defined in Claim 12 further comprising means for providing an alternating field of variable frequency to said sample whereby the light transmitted through said sample may be measured as a function of the frequency of said alternating field.

21. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 20 wherein said analyzer means comprises an interference filter whereby the second harmonic of said light beam is sensed by said detector.

22. A system for detecting the presence of molecules in electrophoresis samples as defined in Claim 12 wherein said detector cαnprises a two dimensional imagery device which receives said light beam after passing into the sample.