Claims
- 1. A method for selecting primer pairs for amplifying a target sequence, comprising the steps of:
choosing a reference sequence; masking at least selected repeat regions in said reference sequence to yield a masked reference sequence; selecting primer sequences from said masked reference sequence to yield a set of primers; evaluating said set of primers for extent of coverage and overlap of said masked reference sequence; and selecting a subset of primer pairs having reduced overlap from said set of primers.
- 2. The method of claim 1, wherein said primer sequences are selected according to two or more parameters including primer length and primer melting temperature.
- 3. The method of claim 1, wherein said step of selecting a subset of primer pairs selects a subset of primer pairs with a minimal or substantially minimal number of primer pairs required to amplify said target sequence.
- 4. The method of claim 3, wherein said step of selecting a subset of primer pairs selects a subset of primer pairs with a least number of primer pairs required to amplify said target sequence.
- 5. The method of claim 3, wherein said second selecting step selects said subset of primer pairs according to at least one parameter selected from the group of overlap length, gaps between pairs of primer pairs, and necessity of adding another primer pair to the subset.
- 6. The method of claim 1, wherein said step of selecting a subset of primer pairs is performed by a computer program and said computer program executes a single-source shortest-path algorithm to select said subset of primer pairs.
- 7. The method of claim 1, wherein said step of selecting a subset of primer pairs is performed by a computer program and said computer program executes an algorithm solving a single-source shortest path problem on a weighted, directed graph G=(V,E) for the case in which all edge weights are normegative, and w(u,w)≧0 for each edge (u,v)εE.
- 8. The method of claim 1, wherein said target sequence is genomic DNA from a human species.
- 9. The method of claim 1, wherein said target sequence is genomic DNA from a non-human primate species.
- 10. The method of claim 1, wherein said reference sequence is genomic DNA from a human species.
- 11. A computer program for selecting primer pairs for amplifying a target nucleic acid sequence comprising:
computer code that receives input of a reference sequence; computer code that masks at least selected repeat regions in said reference sequence to yield a masked reference sequence; computer code that selects primer sequences from said masked reference sequence to yield a set of primers; computer code that evaluates said set of primers for extent of coverage and overlap of said masked reference sequence; and computer code that selects a subset of primer pairs having reduced overlap from said set of primers.
- 12. The computer program of claim 11, wherein said primer sequences are selected according to two or more parameters including primer length and primer melting temperature.
- 13. The computer program of claim 11, wherein said computer code executes an algorithm that in said second selecting step selects a subset of primer pairs with a minimal or substantially minimal number of primer pairs required to amplify said target sequence.
- 14. The computer program of claim 11, wherein said computer code executes an algorithm that in second selecting step selects said subset of primer pairs according to at least one parameter selected from the group of overlap length, gaps between pairs of primer pairs, and necessity of adding another primer pair to the subset.
- 15. The computer program of claim 11, wherein said computer code executes a single-source shortest-path algorithm.
- 16. A system that selects primer pairs for amplifying a target nucleic acid sequence comprising:
a processor; and a computer readable medium coupled to said processor for storing a computer program comprising:
computer code that receives input of a reference sequence; computer code that masks at least selected repeat regions in said reference sequence to yield a masked reference sequence; computer code that selects primer sequences from said masked reference sequence to yield a set of primers; computer code that evaluates said set of primers for extent of coverage and overlap of said reference sequence; and computer code that selects a subset of primer pairs having reduced overlap from said set of primers.
- 17. The system as claimed in claim 16, wherein the computer code selects primer sequences according to two or more parameters including primer length and primer melting temperature.
- 18. A method for selecting a subset of primer pairs from a set of candidate primer pairs for amplifying a target nucleic acid sequence, comprising:
providing a reference sequence; evaluating said set of candidate primer pairs by scoring the usefulness in amplifying the reference sequence of primer pairs from the candidate set of primer pairs to identify a subset of primer pairs; and selecting the subset of primer pairs from said set of candidate primer pairs.
- 19. The method of claim 18, wherein evaluating said set of candidate primer pairs includes determining the extent of any overlap at least one pair of primer pairs from said set of candidate primer pairs.
- 20. The method of claim 18, wherein evaluating said set of candidate primer pairs includes determining the extent of any gap between at least one pair of primer pairs from said set of candidate primer pairs.
- 21. The method of claim 18, wherein evaluating said set of candidate primer pairs includes considering the total number of primer pairs in the subset.
- 22. The method of claim 18, wherein evaluating the set of candidate primer pairs includes minimizing the number of primer pairs in the subset.
- 23. The method of claim 18, wherein evaluating the set of candidate primer pairs includes applying a single-source, shortest-path algorithm to the candidate set of primer pairs.
- 24. The method of claim 18, including removing similar primer pairs from the candidate set of primer pairs.
- 25. The method of claim 18, wherein said reference sequence has been masked to remove at least some repeat sequences of said target sequence.
- 26. The method of claim 18, wherein evaluating said set of candidate primer pairs includes assigning a cost to a primer pair from the set of candidate primer pairs reflecting the suitability of the primer pair for use in amplifying the target sequence.
- 27. A method for amplifying a target sequence, comprising the steps of:
mixing a reaction cocktail comprising deoxynucleotide triphosphates, target DNA, a divalent cation, DNA polymerase enzyme, a broad spectrum solvent, a zwitterionic buffer and at least one primer pair having a length of about 28 nucleotides to about 36 nucleotides and a melting temperature of about 72° C. to about 88° C.; heating said reaction cocktail at a denaturing temperature of about 90° C. to about 96° C. for about 1 second to about 30 seconds; cooling said reaction cocktail at an annealing/extension temperature of about 50° C. to about 68° C. for about 1 minute to about 28 minutes; repeating said heating and cooling steps at least 10 times; and cooling said reaction cocktail to 4° C. in a final cooling step.
- 28. The method of claim 27, wherein said reaction cocktail comprises about 50 μM to about 400 μM of each primer of said at least one primer pair, about 200 μM to about 500 μM each dNTP, about 0.02 ng/μl to about 2.5 ng/μl template (target) DNA, 0.0% to about 7.0% broad spectrum solvent, 0.0 M to about 0.75 M betaine, about 7 mM to about 35 mM NH4SO4, about 25 mM Tris to about 125 mM Tris, about 100 μM to about 500 μM MgCl2, about 0.01 units/μl to about 0.20 units/μl polymerase, and 0 mM to about 50 mM zwitterionic buffer.
- 29. The method of claim 28, wherein said reaction cocktail comprises about 100 nM to about 240 nM of each primer of said at least one primer pair, about 300 μM to about 400 μM each dNTP, about 0.05 ng/μl to about 1.5 ng/μl template (target) DNA, 1.5% to about 4.5% broad spectrum solvent, 0.2 M to about 0.6 M betaine, about 10 mM to about 20 mM NH4SO4, about 40 mM Tris to about 80 mM Tris, about 250 μM to about 400 μM MgCl2, about 0.025 units/μl to about 0.07 units/μl polymerase, and 10 mM to about 30 mM zwitterionic buffer.
- 30. The method of claim 29, wherein said reaction cocktail comprises about 192 nM of each primer of said at least one primer pair, about 385 μM each dNTP, about 1.2 ng/μl template (target) DNA, about 3.7% DMSO, about 0.24 M betaine, about 13 mM NH4SO4, about 48 mM Tris, about 385 μM MgCl2, about 0.05 units/μl polymerase, and 25 mM Tricine.
- 31. The method of claim 27, wherein a duration of each of said cooling step increases during the repeating step.
- 32. The method of claim 27, wherein said reaction cocktail further comprises about 0.005 μg/μl to about 0.10 μg/μl taq antibody.
- 33. The method of claim 27, wherein an initial heating step is performed before said heating step.
- 34. The method of claim 27, wherein an additional cooling step is performed after said repeating step and before said final cooling step.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional application U.S. Ser. No. 60/317,311 filed Sep. 5, 2001, and to U.S. Ser. No. 10/042,406, filed Jan. 9, 2002 and U.S. Ser. No. 10/042,492, filed Jan. 9, 2002, each of which is incorporated by reference in its entirety for all purposes.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60317311 |
Sep 2001 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
10042406 |
Jan 2002 |
US |
Child |
10236480 |
Sep 2002 |
US |
Parent |
10042492 |
Jan 2002 |
US |
Child |
10236480 |
Sep 2002 |
US |