Methods for separation of polymeric compounds
View Patent ↗Recently two techniques using free solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: End Labeled Free Solution Electrophoresis (ELFSE) for DNA sequencing, and Free Solution Conjugate Electrophoresis (FSCE) for molar mass profiling of uncharged polymers. Previous attempts have been made to analyze experimental data generated by these new techniques for the electrophoresis of molecules with varying charge distributions. However, the importance of the ends of the polymers in determining the polymer's overall mobility was neglected in previous work. Through a careful investigation and a reanalysis of the experimental data, it is determined here that this “end effect” critically impacts the behavior of polymers and charged-uncharged polymer conjugates during electrophoresis. In this way, the invention provides for methods that exploit this “end effect” for the separation of polymeric molecules on the basis of size, including for example DNA separation and sequencing techniques.
1. A method for separating single-stranded polymeric compounds according to their relative molecular lengths, the method comprising:
attaching a chemical moiety at or near each end of each of said single-stranded polymeric compounds to generate doubly end-labeled polymeric compounds; and
subjecting the doubly end-labeled polymeric compounds to free-solution electrophoresis, each chemical moiety suitable to impart increased hydrodynamic friction to each end of each doubly end-labeled polymeric compound thereby to facilitate separation of the doubly end-labeled polymeric compounds according to their electrophoretic mobilities during said free-solution electrophoresis.
2. The method of claim 1 , wherein the single-stranded polymeric compounds to be separated are linear polymeric compounds.
3. The method of claim 1 , wherein the single-stranded polymeric compounds to be separated are charged polymeric compounds.
4. The method of claim 1 , wherein the chemical moieties attached as end-labels are uncharged or substantially uncharged chemical moieties.
5. The method of claim 1 , wherein the single-stranded polymeric compounds to be separated are selected from among polypeptides or polynucleotides.
6. The method of claim 5 , wherein the single-stranded polymeric compounds to be separated are selected from among the polynucleotides, including single-stranded DNA, and RNA.
7. The method of claim 1 , wherein the chemical moieties attached as end-labels are selected from polypeptides, polypeptoids, and polypeptide-polypeptoid conjugates.
8. The method according to claim 1 , wherein the chemical moieties are selected from the group consisting of Streptavidin, or a derivative thereof, N-methoxyethylglycine (NMEG)-based polymers of length up to 3100 monomer units, and a molecule consisting of a poly(NMEG) backbone optionally grafted with oligo (NMEG) branches.
9. A method for sequencing a section of a DNA molecule the method comprising:
(a) synthesizing a first plurality of ssDNA molecules each comprising a sequence identical to at least a portion at or near the 5′ end of slid section of DNA, said ssDNA molecules having substantially identical 5′ ends but having variable lengths, the length of each ssDNA molecule corresponding to a specific adenine base in said section of DNA;
(b) synthesizing a second plurality of ssDNA molecules each comprising a sequence identical to at least a portion at or near the 5′ end of said section of DNA, said ssDNA molecules having substantially identical 5′ ends but having variable lengths, the length of each ssDNA molecule corresponding to a specific cytosine base in said section of DNA;
(c) synthesizing a third plurality of ssDNA molecules each comprising a sequence identical to at least a portion at or near the 5′ end of said section of DNA, said ssDNA molecules having substantially identical 5′ ends but having variable lengths, the length of each ssDNA molecule corresponding to a specific guanine base in said section of DNA;
(d) synthesizing a fourth plurality of ssDNA molecules each comprising a sequence identical to at least a portion at or near the 5′ end of aid section of DNA, said ssDNA molecules having substantially identical 5′ ends but having variable lengths, the length of each ssDNA molecule corresponding to a specific thymine base in said section of DNA;
(e) attaching a chemical moiety to end nucleotides at or near each end of said ssDNA molecules to generate doubly end-labeled polymeric compounds; and
(f) subjecting each plurality of ssDNA molecules to free-solution electrophoresis; and
(g) identifying the nucleotide sequence of the section of DNA in accordance with the relative electrophoretic mobilities of the ssDNAs in each plurality of ssDNAs;
wherein any of steps (a), (b), (c), and (d) may be performed in any order or simultaneously; and
whereby each chemical moiety imparts increased hydrodynamic friction to each end of each doubly end-labeled polymeric compound thereby to facilitate separation of the doubly end-labeled polymeric compounds according to their electrophoretic mobility.
10. The method of claim 9 , wherein the chemical moieties are uncharged chemical moieties.
11. The method of claim 9 , wherein the chemical moieties are selected from among polypeptides and polypeptoids.
12. The method of claim 9 , wherein the chemical moieties are selected from the group consisting of Streptavidin, or a derivative thereof, N-methoxyethylglycine (NMEG) based polymers comprising up to 300 monomer units, and a molecule consisting of a poly(NMEG) backbone optionally grafted with oligo (NMEG) branches.
13. The method according to claim 9 , wherein the section of DNA comprises less than 2000 nucleotides.
14. The method according to claim 13 , wherein the section of DNA comprises less than 1000 nucleotides.
15. The method according to claim 14 , wherein the section of DNA comprises less than 500 nucleotides.
16. The method according to claim 15 , wherein the section of DNA comprises less than 300 nucleotides.
17. The method according to claim 16 , wherein the section of DNA comprises less than 100 nucleotides.
18. A method for separating single-stranded polymeric compounds differentiated in size by at least one polymer unit, the method comprising:
attaching a chemical moiety at or near each end of the single-stranded polymeric compounds; and
subjecting the single-stranded polymeric compounds with the attached chemical moieties to free-solution electrophoresis, each chemical moiety imparting increased hydrodynamic friction to each end of said polymeric compounds to thereby modify the electrophoretic mobility of said single-stranded polymeric compounds.
19. The method of claim 18 , wherein the difference in relative size of the single-stranded polymeric compounds is a single polymer unit.
20. The method of claim 19 , wherein the single-stranded polymeric compounds comprise single-stranded DNA molecules, and each polymer unit is a nucleotide.