Nucleic acid marker ladder for estimating mass
View Patent ↗The invention relates to a nucleic acid marker ladder which is a restriction endonuclease digest, wherein a nucleic acid restriction endonuclease digest is a collection of nucleic acid fragments resulting from complete digestion of one or more nucleic acids by one or more restriction endonucleases; the restriction endonuclease digest contains at least 3 fragments; and the size of the fragments in base pairs is a multiple of an integer, wherein the integer is 10 or more.
1. A nucleic acid marker ladder comprising 3 or more nucleic acid fragments, wherein the size of each said fragment in base pairs is a multiple of an integer between 10 and 100, and wherein the mass of each said fragment is equal in its size in base pairs divided by the sum of the sizes in base pairs of all said fragments times the total mass of all said fragments.
2. The nucleic acid marker ladder of claim 1 , wherein said fragments are derived from one or more nucleic acid molecules by digestion with one or more restriction endonucleases.
3. The nucleic acid marker ladder of claim 2 , wherein said fragments are derived from a single nucleic acid molecule.
4. The nucleic acid marker ladder according to claim 1 , wherein the integer is 10.
5. The nucleic acid marker ladder according to claim 1 , wherein the integer is 25.
6. The nucleic acid marker ladder according to claim 1 , wherein the integer is 50.
7. A method of using a nucleic acid marker ladder to estimate the mass of a nucleic acid comprising:
(a) electrophoresing a known amount of the marker ladder of any of claims 1 - 6 and an unknown amount of said nucleic acid on an agarose gel,
(b) comparing the mass of said marker ladder with the mass of said nucleic acid; and,
(c) estimating the mass of said nucleic acid.
8. A method of preparing a nucleic acid marker ladder comprising:
(a) generating two or more polymerase chain reaction (PCR) products wherein each product is generated from a template comprising a first restriction endonuclease site and a primer comprising a second restriction endonuclease site;
(b) joining the PCR products to produce a nucleic acid molecule; and
(c) digesting one or more nucleic acid molecules with one or more restriction endonucleases wherein a nucleic acid marker ladder is produced wherein the ladder contains three or more fragments and the size of the fragments in base pairs is a multiple of an integer, wherein the integer is 10 or more.
9. The method of claim 8 wherein the restriction endonuclease site in the template is located internally.
10. The method of claim 8 wherein the restriction endonuclease sites are located at one and or both ends of the PCR product.
11. The method of claim 8 wherein the template is selected from the group consisting ofplasmid DNA, phage DNA, plant DNA, animal DNA and bacterial DNA.
12. The method of claim 11 wherein the template is plasmid DNA.
13. The method of claim 8 wherein one of the PCR products further comprises an origin of replication.
14. The method of claim 8 wherein one of the PCR products further comprises a selectable marker.
15. The method of claim 8 wherein the first and second restriction endonuclease sites are the same.