WO2004034027A2

WO2004034027A2 - Method for sizing polynucleotides using electrophoresis with non-dna size standards

Info

Publication number: WO2004034027A2
Application number: PCT/US2003/032146
Authority: WO
Inventors: Zhaowei Liu; Thomas Kane
Original assignee: Spectrumedix Corp
Current assignee: Spectrumedix Corp
Priority date: 2002-10-10
Filing date: 2003-10-10
Publication date: 2004-04-22
Anticipated expiration: 2005-04-10
Also published as: JP2006507485A; EP1549927A2; WO2004034027A3; AU2003282574A8; AU2003282574A1

Abstract

The present invention relates to a method of determining a size of a sample polynucleotide. A sample polynucleotide is subjected to electrophoresis in the presence of at least: a fluorescent compound having a first fluorescence spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide, and a plurality of size standards, each of the size standards having a different mobility and wherein each of the size standards is essentially free of polynucleotides. Migration coordinates of the sample polynucleotide and plurality of size standards are determined. A size of the sample polynucleotide is determined using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

Description

METHOD FOR SIZING POLYNUCLEOTIDES USING ELECTROPHORESIS WITH NON-DNA SIZE STANDARDS

Field of the Invention

The present invention relates to a method for sizing polynucleotides by subjecting the polynucleotides to electrophoresis to obtain electrophoresis data and comparing the electrophoresis data to electrophoresis data obtained from a non- polynucleotide size standard.

Background of the Invention

Sample polynucleotides may be subjected to electrophoresis in the presence of reference polynucleotides having a known size. The size of the sample polynucleotides may be determined using migration times of the sample polynucleotides and of the reference polynucleotides. Sample polynucleotides and reference polynucleotides having the same size generally exhibit the same mobility when subjected to electrophoresis.

Detection methods, such as fluorescence resulting from intercalating dyes, are sensitive to the presence of both the sample polynucleotides and the reference polynucleotides. Thus, electrophoresis data from a separation of sample polynucleotides and reference polynucleotides may include a sample polynucleotide peak that is obscured by a peak resulting from one of the reference polynucleotides.

A polynucleotide size standard that reduces obscured sample peaks in electrophoresis data is needed.

Summary of the Invention

A first aspect of the invention relates to a method for determining a size of a sample polynucleotide. A sample polynucleotide is subjected to electrophoresis in the presence of at least (1) a fluorescent compound having a first fluorescent spectrum, wherein detection of the fluorescence of the fluorescent compound is indicative of the presence of the sample polynucleotide and (2) a plurality of size standards, each of the size standards having a different mobility. Each of the size standards is preferably at least essentially free of polynucleotides. A preferred size standard is at least essentially free of nucleotides.

Migration coordinates of the sample polynucleotide and size standards are determined. A size of the sample polynucleotide is determined using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards. The migration coordinate may be a migration time, migration distance, or a migration coordinate determined from a combination thereof, such as a mobility.

The fluorescent compound may be an intercalating compound that intercalates with the polynucleotide and exhibits enhanced fluorescence when so intercalated. An exemplary intercalating compound is ethidium bromide.

Subjecting the sample polynucleotide to electrophoresis preferably comprises irradiating the intercalating compound with light and detecting, at a first wavelength, fluorescent light indicative of the presence of the sample polynucleotides. Subjecting the size standards to electrophoresis preferably comprises irradiating the size standards with light and detecting, at a second, different wavelength, fluorescent light indicative of the presence of the size standards.

Fluorescent light associated with the intercalating compound is preferably not indicative of the presence of the size standards because the size standards are preferably essentially free of intercalated intercalating compound, e.g., the intercalating compound either does not intercalate with the size standards or does not exhibit increased fluorescence when intercalated with the size standard. The fluorescence of the intercalating compound is preferably substantially lower if associated with the size standards rather than when intercalated with the sample polynucleotides. Thus, the size standards may be said to be immune to the intercalating dye.

Detecting light at the second, different wavelength may comprise preventing at least some light at the first wavelength from reaching a detector. For example, a grating, prism, or optical filter may be placed along an optical path between the size standards and the detector to allow light having the second wavelength to reach the detector but to substantially prevent light having the first wavelength from reaching the same portion of the detector as the second wavelength. Thus, if a two dimensional detector is used, first light indicative of the presence of the sample polynucleotides may reach a first portion of the detector and second light indicative of the presence of the size standards may reach a second different portion of the detector.

The sample polynucleotides and size standards may be subjected to electrophoresis simultaneously along the same separation lane, such as within the same capillary.

The polynucleotides and size standards maybe subjected to electrophoresis in the presence of a buffer.

The size standards may have a net r.ha σe of essentially zero in the buffer. The size standards may be non-ionic.

The mass to charge ratio of size standards in accordance with the present invention may be varied to achieve different mobilities. Thus, size standards of the present invention may have similar sizes to one another but different charges that result in different mobilities. Exemplary size standards having variable mass to charge ratios include e-Tag reporters available from Aclara Incorporated.

The mobility of a given size standard may different from a mobility of a sample polynucleotide having the same mass. Determining a size of the sample polynucleotide may comprise using a predetermined relationship between the mobility of each size standard and the mobility of a reference polynucleotide having a known mass. Such a relationship may be determined using, for example, calibration data including (1) data indicative of a mobility of the size standards as a function of masses or other physical parameter thereof and (2) data indicative of a mobility of reference polynucleotides as a function of their mass or other physical parameters. Based on such calibration data one may determine the mass or other physical parameter of a sample polynucleotide based on a determination of the mobilities of the sample polynucleotide and the size standards.

The mobility of each size standard may be different from a mobility of a sample polynucleotide having the same size and determining a size of the sample polynucleotide may comprise using a predetermined relationship between the mobility of each size standard and the mobility of a polynucleotide having the same size.

The mobility of each size standard may be different from a mobility of a sample polynucleotide having the same length and determining a size of the sample polynucleotide may comprise using a predetermined relationship between the mobility of each size standard and the mobility of a polynucleotide having the same length.

Another aspect of the invention relates to a method of determining a relationship between the mobility of a plurality of size standards and a mobility of at least one reference polynucleotide, wherein the relationship between the mobilities of the size standards and the reference polynucleotide may be used in a method for electrophoretically determining a size of a sample polynucleotide. The method comprises providing a plurality of size standards, each of the size standards having a different mobility and each of the size standards preferably being at least essentially free of polynucleotides. A plurality of reference polynucleotides is provided. Each of the reference polynucleotides preferably has a different size.

The size standards and the reference nnlvnucleotides are subjected to electrophoresis. Migration coordinates of the size standards and reference polynucleotides are determined. A relationship between mobilities of the size standards and the mobilities of the reference polynucleotides is determined, whereby the size of a sample polynucleotide may be determined by (1) subjecting the sample polynucleotide to electrophoresis in the presence of size standards to determine migration coordinates of the size standards and sample polynucleotides and (2) using at least the migration coordinates of the size standards and sample polynucleotides and the determined relationship between the mobilities of the size standards and the reference polynucleotides.

A size standard that is essentially free of polynucleotides preferably lacks polynucleotide sequences longer than about 10 base pairs. Preferred size standards comprise less than about 10% by mass of nucleic acids, for example, less than about 5% by mass of nucleic acids. In one embodiment, the size standards are free of nucleic acids.

Yet another aspect of the invention relates to a computer-readable medium comprising executable software code, the code for processing electrophoresis data to determine a size of at least one sample polynucleotide, the electrophoresis data comprising (1) peaks indicative of a separation of at least one sample polynucleotide along a first separation lane and (2) peaks indicative of a separation of a plurality of size standards along the first separation lane, the size standards being essentially free of polynucleotides, the computer-readable medium comprising: code to determine a migration coordinate of at least one peak corresponding to the presence of the sample polynucleotide subjected to electrophoresis along the first separation lane; code to determine migration coordinates of peaks indicative of a presence of at least two size standards subjected to electrophoresis along the first separation lane; and code to determine the size of the sample polynucleotide based on at least mobilities of the size standards and mobilities of reference polynucleotide having known sizes.

An additional aspect of the invention relates to a method of determining a size of a sample polynucleotide. A sample polynucleotide is subjected to electrophoresis in the presence of at least (1) an intercalating dye having a first fluorescent spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide and (2) a plurality of size standards, each of the size standards having a different mobility and wherein detection of the light of the first fluorescence spectrum is essentially not indicative of a presence of the size standards. The presence of the size standards is preferably determined by detecting light having a wavelength different from the light of the first fluorescence spectrum detected to determine the presence of the sample polynucleotides.

A migration coordinate of the sample polynucleotide is determined. A migration coordinate of the each size standard is determined. A size of the sample polynucleotide is determined using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

Yet another aspect of the invention relates to a method of determining a size of a sample polynucleotide, comprising subjecting the sample polynucleotide to electrophoresis in the presence of at least (1) a fluorescent compound having a first fluorescence spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide and (2) a plurality of non-polynucleotide size standards, each of the non-polynucleotide size standards having a different mobility.

A migration coordinate of the sample polynucleotide is determined. A migration coordinate of each of the plurality of non-polynucleotide size standards is determined. A size of the sample polynucleotide is determined using the migration coordinate of the sample polynucleotide and the migration coordinates of the non- polynucleotide size standards.

A non-polynucleotide size standard is a compound which resists intercalation by etludium bromide. For example, one such compound is essentially free of double stranded polynucleotides. A preferred non-polynucleotide size standard is a linear polymer that is at least essentially free of nucleic acids or nucleotides.

Brief Description of the Drawings

The present invention is discussed below in reference to the Figures in which: Figure 1 shows a flow chart of a method of the present invention; and Figure 2 shows an electrophoresis system in accordance with the presence invention.

Detailed Description of the Present Invention

Referring to the flow chart of Figure 1, one embodiment of the present invention relates to a method of determining a size of one or more a sample polynucleotides. At least one sample polynucleotide, such as a polynucleotide comprising at least one of a single strand of DNA and a double strand of DNA, is subjected to electrophoresis along an electrophoresis lane. Exemplary electrophoresis lanes include a bore of a capillary, an electrophoresis lane of a microfabricated micro fluidic device, and a lane within a slab gel.

The electrophoresis of the sample polynucleotide preferably takes place in the presence of at least one fluorescent compound having a first fluorescent spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide. A plurality of a plurality of size standards are also subjected to electrophoresis, preferably along the same electrophoresis lane. Size standards and the sample polynucleotides are preferably subjected simultaneously to electrophoresis along the same electrophoresis lane. Each of the size standards preferably has a different mobility and is at least essentially free of polynucleotides. The size standards maybe at least essentially free of nucleic acids. The size standard may be completely free of polynucleotides and/or nucleic acids. In one embodiment, the mass of a given size standard is less than 10% nucleic acid, for example less than 5% nucleic acid, or less than 1% nucleic acid. h one embodiment the size standards may be anionic.

The size standards may comprise linear polymers. The size standards may comprise at least one of oligosaccharides and polysaccharides, such as poly-dextrans. The oligosaccharides or polysaccharides may be labeled with, for example, fluorescent dyes including 8-Amino-l,3,6-pyrenetrisulfonic acid (ATTS) (Morll et al. 1998. Electrophorsis 19, 2603-2611.)

Other suitable size standards include protein conjugates, which may be labeled with fluorescent dyes including succinimidyl ester derivatives such as the available from Molecular Probes.

Yet more suitable size standards include beads of varying size or charge to mass ratio that exhibit different mobilities when subjected to electrophoresis. The beads may be tagged with a fluorophore to facilitate their detection.

Electrophoresis data is determined from the at least one sample polynucleotide and the size standard. Exemplary electrophoresis data include fluorescence intensity versus time data. Migration coordinates, such as migration times, of the sample polynucleotide and size standards are determined. A size of the sample polynucleotide is determined using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards. It should be understood that the size standards may be subjected to electrophoresis before or after the sample polynucleotides. i one embodiment, migration coordinates of size standards are determined prior to subjecting the sample polynucleotides to electrophoresis. The size standard migration coordinate data is saved for example using a computer readable medium. Upon obtaining electrophoresis data of the sample polynucleotides, the sizes thereof are determined using the previously acquired migration coordinate data of the size standards.

Electrophoresis in accordance with the present invention may be performed using electrophoretic separation lanes known in the art. Exemplary separation lanes include the internal bores capillaries filed with a sieving matrix that causes NCC's of varying sizes to migrate with different mobilities. An exemplary instrument for performing capillary electrophoresis is disclosed in United States Patent No. 6,027,627, which patent discloses an automated parallel electrophoretic system. The patent is incorporated by reference herein.

Referring to Fig. 2, an electrophoresis system 230 in accordance with the present invention includes a plurality of capillaries 232. A first array of ends 238 of the capillaries 232 may be spaced apart in substantially the same manner as the wells 262 of a microtitre tray 264. This allows one to simultaneously perform capillary electrophoresis on volumes of material present in each of the wells 262 of the tray 264. Thus, the molecular ladder and the sample NCC internal standard mixtures may be placed in various wells of the tray. The array of capillary ends 238 are placed in contact with the volumes of material in the wells 262. Upon the brief application of a current through the capillaries, an amount of the material is drawn into the respective capillaries of the array. The current is provided by a source 272 of high- voltage (HV) electricity. The array of capillary ends 238 is placed in contact with a solution of buffer. Upon the renewed application of an electric field to the capillaries 232, compounds previously drawn into the capillaries 232 from the wells 262 migrate toward a detection zone 246.

The detection zone 246 is spaced apart along a separation axis from the array of capillary ends 238. A detection system determines the presence of compounds in the detection zone. Exemplary detection systems include a light source 252 and a detector 260. Compounds present in the detection zone are irradiated with light 256 from the light source 252. A beam steering element 254 may be used to direct light 256 toward the detection zone. The irradiated compounds or fluorophores associated with the compounds may emit fluorescence 258.

Electrophoresis data may include a subset of data generally defining a peak, which is indicative of the presence of a compound, such as a sample or a size standard subjected to electrophoreis. A migration coordinate of a peak, such as a migration time τ_p of the peak, may be determined by fitting the peak to a peak-shape model and determining the peak migration coordinate from the parameters nf the fitted peak. Alternatively, one may simply determine a peak migration time from a peak maximum of the observed migration time data.

A computer 290 includes a computer-readable medium comprising code that when executed receives the detector signal from detector 260 and process the data in accordance with the invention. The code is discussed elsewhere herein.

The electrophoresis system of the invention may include a computer or other processor configured to determine a size of one or more sample polynucleotides. The processor is typically implemented through a combination of hardware and executable software code. In the usual case, the processor includes a programmable computer, perhaps implemented as a reduced instruction set (RISC) computer, which handles only a handful of specific tasks. The computer is typically provided with at least one computer-readable medium, such as a PROM, flash, or other non- volatile memory to store firmware and executable software code, and will usually also have an associated RAM or other volatile memory to provide work space for data and additional software. Various steps that may be carried out by the computer or other processor in response to code of the computer-readable medium are discussed below.

The computer or processor may be configured to receive a detector signal. Because the computer may be either local to the electrophoresis instrument or remote therefrom, the computer may receive the detector signal through, for example, a hardwired connection, wireless connection, a network, a storage medium such as a disk, or combination thereof.

The code of the computer readable medium may, optionally, include code configured to convert the detector signal to electrophoresis data. Because the detector may output a detector signal in the form of electrophoresis data including a detector coordinate, such as an intensity, and a migration coordinate, such as a time, a conversion step may not be necessary.

In certain situations, the raw data must be subjected to initial conditioning 304, such as by data smoothing, baseline subtraction, or by using deconvolution techniques to identify overlapped peaks. Suitable data conditioning techniques, such as those discussed below, are disclosed in U.S. Application No. 09/676,526, filed October 2, 2000, titled Electrophoretic Analysis System Having in-situ Calibration, which application is hereby incorporated to the extent necessary to understand the present invention. The computer- readable medium includes code to perform such conditioning. Smoothing can be accomplished by using, for example, a Savitzky-Golay convoluting filter to improve the signal to noise ratio. Optimal properties of the filter, such as the width and order, can be determined by a user of the present invention on the basis of the signal to noise ratio of the data and the widths of peaks in the data.

Baseline subtraction can be performed to eliminate baseline drift. Typically, minima are identified in successive local sections of data, e.g., every 300 data points. Two or more minima in adjacent sections are connected, such as by a straight line or a polynomial fit to the minima. The values along the line connecting the minima are then subtracted from the intervening raw data. The new values after the baseline subtraction and smoothing are stored for further processing. The order of data smoothing and baseline subtraction can be reversed.

Overlapped peaks within the separations data can be identified and resolved using peak-fitting techniques. In most electrophoresis separations, the earlier-detected peaks are narrower than the later-detected, slower moving peaks. Within a given local section of data, however, peaks due to the presence of a single fragment have similar widths. Moreover, adjacent peaks rarely overlap exactly. Rather, the overlapped peaks a generally offset from one another. Accordingly, peaks due to the presence of multiple fragments tend to be wider than the single fragment peaks. Once a region of data containing overlapped peaks is identified, the underlying peaks can be resolved by fitting a model of the data to the observed data. Typically, the peak fitting model includes parameters that describe the amplitude, position, and width of each underlying peak.

The code may also be configured to determine (1) a migration coordinate of at least one peak corresponding to the presence of the sample polynucleotide subjected to electrophoresis along the first separation lane and (2) migration coordinates of at least two peaks respectively indicative of the presence of least two size standards subjected to electrophoresis along the first separation lane.

The code may be configured to determine the size of the sample polynucleotide based on at least a relationship between mobilities of the size standards and mobilities of a plurality of reference polynucleotides having known sizes.

While the above invention has been described with reference to certain preferred embodiments, it should be kept in mind that the scope of the present invention is not limited to these. Thus, one skilled in the art may find variations of these preferred embodiments which, nevertheless, fall within the spirit of the present invention, whose scope is defined by the claims set forth below.

Claims

CLAIMSWhat is claimed is:

1. A method of determining a size of a sample polynucleotide, comprising: subjecting the sample polynucleotide to electrophoresis in the presence of at least: a fluorescent compound having a first fluorescence spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide; and a plurality of size standards, each of the size standards having a different mobility and wherein each of the size standards is essentially free of polynucleotides; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of each of the plurality of size standards; determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

2. The method of claim 1, wherein at least two of the size standards comprise at least one of an oligosaccharide, a polysaccharide, and a protein conjugate.

3. The method of claim 2, wherein at least one of the size standards comprises a poly- dextran.

4. The method of claim 1 , wherein the migration coordinate are migration times.

5. The method of claim 1, wherein the migration coordinate are electrokinetic mobilities.

6. The method of claim 1 , wherein determining a migration coordinate of each of the plurality of size standards comprises detecting light having a wavelength of light different from a wavelength of light detected to determine the presence of the sample polynucleotide.

7. A method of determining a relationship between the mobility of a plurality of first size standards and a mobility of at least one reference polynucleotide, wherein the relationship between the mobility of the first size standards and the at least one reference polynucleotide may be used in a method for electrophoretically determining a size of a sample polynucleotide, the method comprising: providing a plurality of first size standards, each of the first size standards having a different mobility and each of the first size standards being essentially free of polynucleotides; providing a plurality of reference polynucleotides, each of the reference polynucleotides having a known, different size; subjecting the first size standards and the reference polynucleotides to electrophoresis; determining migration coordinates of the first size standards; determining migration coordinates of the reference polynucleotides; determining a relationship between the mobilities of the first size standards and the mobilities of the reference polynucleotides, wherein the relationship may be used to determine the size of a sample polynucleotide subjected to electrophoresis in the presence of second size standards having the same mobilities as the first size standards.

8. The method of claim 7, wherein at least two of the size standards comprise at least one of an oligosaccharide, a polysaccharide, and a protein conjugate.

9. The method of claim 7, wherein at least one of the size standards comprises a poly- dextran.

10. The method of claim 7, wherein the migration coordinates are migration times.

11. The method of claim 7, wherein the migration coordinate are electrokinetic mobilities.

12. The method of claim 7, wherein determining a migration coordinate of each of the plurality of size standards comprises detecting light having a wavelength of light different from a wavelength of light detected to determine the presence of the sample polynucleotide.

13. A method of determining a size of a sample polynucleotide, comprising: subjecting the sample polynucleotide to electrophoresis; subjecting a plurality of size standards to electrophoresis, each of the size standards having a different mobility and wherein each of the size standards is essentially free of polynucleotides; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of each of the plurality of size standards; determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

14. The method of claim 1, wherein at least two of the size standards comprise at least one of an oligosaccharide, a polysaccharide, and a protein conjugate.

15. The method of claim 2, wherein at least one of the size standards comprises a poly- dextran.

16. The method of claim 13, wherein the migration coordinates are migration times.

17. The method of claim 13, wherein the migration coordinates are electrokinetic mobilities.

18. The method of claim 13, wherein the step of (a) subjecting a plurality of size standards to electrophoresis is completed prior to the step of subjecting the sample polynucleotide to electrophoresis.

19. A computer-readable medium comprising executable software code, the code for processing electrophoresis data to determine a size of at least one sample polynucleotide, the electrophoresis data comprising (1) peaks indicative of a separation of at least one sample polynucleotide along a first separation lane and (2) peaks indicative of a separation of a plurality of size standards along the first separation lane, the size standards being essentially free of polynucleotides, the computer-readable medium comprising: code to determine a migration coordinate of at least one peak corresponding to the presence of the sample polynucleotide subjected to electrophoresis along the first separation lane; code to determine migration coordinates of at least two peaks respectively indicative of the presence of least two size standards subjected to electrophoresis along the first separation lane; and code to determine the size of the sample polynucleotide based on at least a relationship between mobilities of the size standards and mobilities of a plurality of reference polynucleotides having known sizes.

20. A method of determining a size of a sample polynucleotide, comprising: subjecting a sample polynucleotide to electrophoresis in the presence of at least (1) an intercalating dye having a first fluorescent spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide and (2) a plurality of size standards, each of the size standards having a different mobility and wherein detection of the light of the first fluorescence spectrum is essentially not indicative of a presence of the size standards; determining the presence of the size standards by detecting light having a wavelength different from the light of the first fluorescence spectrum used to determine the presence of the sample polynucleotides; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of the each size standards; and determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

21. A method of determining a size of a sample polynucleotide, comprising: subjecting the sample polynucleotide to electrophoresis in the presence of at least: a fluorescent compound having a first fluorescence spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide; and a plurality of non-polynucleotide size standards, each of the non- polynucleotide size standards having a different mobility; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of each of the plurality of non- polynucleotide size standards; determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the non- polynucleotide size standards.

22. A method of determining a size of a sample polynucleotide, comprising: subjecting a sample polynucleotide to electrophoresis in the presence of at least (1) a fluorescent compound having a first fluorescent spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide and (2) a plurality of size standards, each of the size standards having a different mobility and wherein detection of the light of the first fluorescence spectrum is essentially not indicative of a presence of the size standards; determining the presence of the size standards by detecting light having a wavelength different from the light of the first fluorescence spectrum used to determine the presence of the sample polynucleotides; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of the each size standards; and determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.

23. A method of determining a size of a sample polynucleotide, comprising: subjecting a sample polynucleotide to electrophoresis in the presence of at least (1) one compound having a first detection window, wherein detection of light within the first detection window is indicative of the presence of the sample polynucleotide and (2) a plurality of size standards, each of the size standards having a different mobility and wherein detection of the light within the detection window is essentially not indicative of a presence of the size standards; determining the presence of the size standards by detecting light having a wavelength different from the light of the first detection window used to determine the presence of the sample polynucleotides; determining a migration coordinate of the sample polynucleotide; determining a migration coordinate of the each size standards; and determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standards.