Method for sizing polynucleotides using electrophoresis with non-DNA size standards
View Patent ↗According to a method of determining a size of a sample polynucleotide, a sample polynucleotide is subjected to electrophoresis in the presence of a fluorescent compound having a first fluorescence spectrum. Detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide. One or more size standards are also subjected to electrophoresis, optionally in the presence of the sample polynucleotide. If more than one size standard is used, the different size standards typically have different mobilities. The size standards are generally essentially or completely free of polynucleotides. Migration coordinates, e.g., migration times, of the sample polynucleotide and size standard(s) are determined. A size of the sample polynucleotide can be determined using the migration coordinate of the sample polynucleotide and the migration coordinate(s) of the size standard(s).
1. A method of determining a size of a sample polynucleotide, comprising:
subjecting a sample polynucleotide to electrophoresis in the presence of at least:
a fluorescent intercalating compound having a first fluorescence spectrum, wherein detection of light of the first fluorescence spectrum is indicative of the presence of the sample polynucleotide, a size standard having a net charge in solution, the size standard being essentially free of polynucleotides;
determining a migration coordinate of the sample polynucleotide;
determining a migration coordinate of the size standard; and
determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinate of the size standard.
2. The method of claim 1 , wherein:
the step of subjecting comprises subjecting the sample polynucleotide to electrophoresis in the presence of 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; and
the method comprises determining the migration coordinates of each of the size standards.
3. The method of claim 2 , wherein said plurality of size standards comprises at least two size standards, wherein said at least two size standards comprise at least one of an oligosaccharide, a polysaccharide, and a protein conjugate.
4. The method of claim 2 , wherein a first size standard of said plurality of size standards is a polypeptide including a number of peptides and a second size standard is a different polypeptide having a different electrophoretic mobility.
5. The method of claim 4 , wherein the first and second size standards have different numbers of peptides.
6. The method of claim 4 , wherein the polypetides comprises a fluorescent tagging agent.
7. The method of claim 2 , wherein the migration coordinates are migration times.
8. The method of claim 2 , wherein the migration coordinates are electrokinetic mobilities.
9. The method of claim 2 , 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.
10. The method of claim 1 , wherein the fluorescent compound having a first fluorescence spectrum is selected from intercalating dyes, groove binding dyes, other dyes that non-covalently associate with the polynucleotide, or any combination thereof.
11. A method of determining a size of a sample polynucleotide, comprising:
subjecting the sample polynucleotide to electrophoresis in the presence of an intercalating compound;
subjecting a size standard, having a net charge in solution, to electrophoresis, the size standard being essentially free of polynucleotides;
detecting the presence of the intercalating compound to determine a migration coordinate of the sample polynucleotide;
determining a migration coordinate of each of the plurality of size standard;
determining a size of the sample polynucleotide using the migration coordinate of the sample polynucleotide and the migration coordinates of the size standard.
12. The method of claim 11 , wherein:
the step of subjecting the subjecting a size standard to electrophoresis comprises subjecting a plurality of size standards to electrophoresis, each of the size standards having a different mobility and each of the size standards being essentially free of polynucleotides; and
the method comprises determining the migration coordinates of each of the size standards.
13. The method of claim 12 , wherein said plurality of size standards comprises at least two size standards, wherein said at least two size standards comprise at least one of an oligosaccharide, a polysaccharide, and a protein conjugate.
14. The method of claim 12 , wherein a first size standard of said plurality of size standards is a polypeptide including a number of peptides and a second size standard is a different polypeptide having a different electrophoretic mobility.
15. The method of claim 14 , wherein the first and second size standards have different numbers of peptides.
16. The method of claim 14 , wherein the polypetides comprises a fluorescent tagging agent.
17. The method of claim 14 , wherein the steps of subjecting comprise separating the sample polynucleotide to electrophoresis in the presence of the size standards along the same separation lane.
18. The method of claim 11 , wherein the migration coordinates are migration times.