Nucleic acid ladders
View Patent ↗The present invention provides nucleic acid compositions or ladders which may be used as standards for estimating the size (in base pairs) and or mass of nucleic acid molecules of unknown size and/or mass. The invention also relates to methods for producing such compositions or ladders, ladders or compositions produced by such methods, and to methods for estimating the size and/or mass of nucleic acid molecules by comparison to these nucleic acid sizing ladders.
1. A nucleic acid ladder consisting essentially of a plurality of double stranded nucleic acid fragments, each fragment having a size in base pairs of between 20 kb and 100 base pairs, a copy number, a mass, and a relative mass wherein the mass of each fragment is the size in base pairs of the fragment multiplied by the copy number of the fragment, wherein the relative mass of each fragment is the mass of the fragment divided by the sum of the masses of all of the fragments, wherein the relative mass of any one fragment of the plurality is no more than 3 time the relative mass of any other fragment of the plurality, wherein at least two of the plurality of nucleic acid fragments have a size greater than 1 kb, and wherein at least two of the plurality of nucleic acid fragments have a size less than 1 kb, wherein the nucleic acid ladder lacks a highlight fragment.
2. The nucleic acid ladder of claim 1 , wherein at least 3 of the plurality of double stranded nucleic acid fragments have a size greater than 1 kb, and wherein at least 3 of the double stranded nucleic acid fragments have a size less than 1 kb.
3. The nucleic acid ladder of claim 1 , wherein at least 4 of the plurality of double stranded nucleic acid fragments have a size greater than 1 kb, and wherein at least 4 of the plurality of double stranded nucleic acid fragments have a size less than 1 kb.
4. The nucleic acid ladder of claim 1 , wherein at least 5 of the plurality of double stranded nucleic acid fragments have a size greater than 1 kb, and wherein at least 5 of the plurality of double stranded nucleic acid fragments have a size less than 1 kb.
5. The nucleic acid ladder of claim 1 , wherein the plurality of double stranded nucleic acid fragments are stained with a detectable label.
6. The nucleic acid ladder of claim 5 , wherein the detectable label is [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+.
7. The nucleic acid ladder of claim 5 , wherein the detectable label is ethidium bromide.
8. The nucleic acid ladder of claim 1 , wherein the relative mass of any one fragment of the plurality is no more than 2.5 times the relative mass of any other fragment of the plurality.
9. The nucleic acid ladder of claim 1 , wherein the relative mass of any one fragment of the plurality is no more than 2 times the relative mass of any other fragment of the plurality.
10. The nucleic acid ladder of claim 1 , wherein the relative mass of any one fragment of the plurality is no more than 1.5 times the relative mass of any other fragment of the plurality.
11. A nucleic acid ladder comprising a plurality of double stranded nucleic acid fragments, wherein three or more of the fragments are of a size selected from the group consisting of:
(a) 100 base pairs,
(b) 200 base pairs,
(c) 300 base pairs,
(d) 400 base pairs,
(e) 500 base pairs,
(f) 650 base pairs,
(g) 850 base pairs, and
(h) 1650 base pairs; and
wherein two or more of the fragments are of a size selected from the group consisting of:
(a) 1 kilobase pairs,
(b) 2 kilobase pairs,
(c) 3 kilobase pairs,
(d) 4 kilobase pairs, and
(e) 5 kilobase pairs;
wherein each fragment has a copy number, a mass, and a relative mass wherein the mass of each fragment is the size in base pairs of the fragment multiplied by the copy number of the fragment, wherein the relative mass of each fragment is the mass of the fragment divided by the sum of the masses of all of the fragments, wherein the relative mass of any one fragment of the plurality is no more than 3 time the relative mass of any other fragment of the plurality.
12. The nucleic acid ladder of claim 11 , wherein four or more of the fragments are between 100 base pairs and 1650 base pairs.
13. The nucleic acid ladder of claim 12 , wherein five or more of the fragments are between 100 base pairs and 1650 base pairs.
14. The nucleic acid ladder of claim 12 , wherein three or more of the fragments are between 1 kilobase pairs and 5 kilobase pairs.
15. A nucleic acid ladder comprising a plurality of double stranded nucleic acid fragments, wherein three or more of the fragments are of a size selected from the group consisting of:
(a) 100 base pairs,
(b) 200 base pairs,
(c) 300 base pairs,
(d) 400 base pairs,
(e) 500 base pairs,
(f) 650 base pairs,
(g) 850 base pairs, and
(h) 1650 base pairs; and
wherein two or more of the fragments are of a size selected from the group consisting of:
(a) 1 kilobase pairs,
(b) 2 kilobase pairs,
(c) 3 kilobase pairs,
(d) 4 kilobase pairs, and
(e) 5 kilobase pairs;
wherein each fragment has a copy number, a mass, and a relative mass wherein the mass of each fragment is the size in base pairs of the fragment multiplied by the copy number of the fragment, wherein the relative mass of each fragment is the mass of the fragment divided by the sum of the masses of all of the fragments, wherein the relative mass of any one fragment of the plurality is no more than 3 time the relative mass of any other fragment of the plurality and
wherein the nucleic acid ladder further comprises at least one highlight fragment having a size in the range of 100 base pairs to 5 kilobase pairs and having a relative mass that is three times greater than the relative mass of other molecules in the composition.
16. The nucleic acid ladder of claim 15 , wherein four or more of the fragments are between 100 base pairs and 1650 base pairs.
17. The nucleic acid ladder of claim 15 , wherein five or more of the fragments are between 100 base pairs and 1650 base pairs.
18. The nucleic acid ladder of claim 15 , wherein three or more of the fragments are between 1 kilobase pairs and 5 kilobase pairs.
19. The nucleic acid ladder of claim 15 , wherein the highlight fragment has a relative mass that is at least 5 times greater than the other fragments.