Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
View Patent ↗Tags and linkers specifically designed for a wide variety of nucleic acid reactions are disclosed, which are suitable for a wide variety of nucleic acid reactions wherein separation of nucleic acid molecules based upon size is required.
1. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:
T ms -L-X
wherein,
T ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a T ms -containing moiety to be cleaved from the remainder of the compound, wherein the T ms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and
X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′ end of X; and each primer pair associates with a different locus; the compound also having the formula:
wherein
G is (CH 2 ) 1-6 wherein a hydrogen on one and only one of the CH 2 groups is replaced with —(CH 2 ) c -Amide-T 4 ;
T 2 and T 4 are organic moieties of the formula C 1-25 N 0-9 O 0-9 H α F β wherein the sum of α and β is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
R 1 is hydrogen or C 1-10 alkyl;
c is an integer ranging from 0 to 4; and
n is an integer ranging from 1 to 50 such that when n is greater than 1, then G, c, Amide, R 1 and T 4 are independently selected.
2. The kit according to claim 1 wherein the plurality is at least 3.
3. A kit according to claim 1 wherein the plurality is at least 5.
4. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:
T ms -L-X
wherein,
T ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a T ms -containing moiety to be cleaved from the remainder of the compound, wherein the T ms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and
X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′ end of X; and each primer pair associates with a different locus; the compound also having the formula:
wherein
G is (CH 2 ) 1-6 wherein a hydrogen on one and only one of the CH 2 groups is replaced with —(CH 2 ) c -Amide-T 4 ;
T 2 and T 4 are organic moieties of the formula C 1-25 N 0-9 O 0-9 H α F β wherein the sum of α and β is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
R 1 is hydrogen or C 1-10 alkyl;
c is an integer ranging from 0 to 4; and
T 5 is an organic moiety of the formula C 1-25 N 0-9 O 0-9 H α F β wherein the sum of α and β is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and T 5 includes a tertiary or quaternary amine or an organic acid; and m is an integer ranging from 0–49 where when m is greater than 1, then G, c, Amide, R 1 and T 4 are independently selected.
5. The kit according to claim 4 wherein the -Amide-T 5 is selected from:
6. The kit according to claim 4 wherein -Amide-T 5 is selected from:
7. The kit according to claim 4 wherein the plurality is at least 3.
8. The kit according to claim 4 wherein the plurality is at least 5.
9. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:
T ms -L-X
wherein,
T ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;
L is an organic group which allows a T ms -containing moiety to be cleaved from the remainder of the compound, wherein the T ms -containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and
X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′ end of X; and each primer pair associates with a different locus; the compound also having the formula:
wherein
G is (CH 2 ) 1-6 wherein a hydrogen on one and only one of the CH 2 groups is replaced with —(CH 2 ) c -Amide-T 4 ;
T 2 and T 4 are organic moieties of the formula C 1-25 N 0-9 O 0-9 H α H β wherein the sum of α and β is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;
R 1 is hydrogen or C 1-10 alkyl;
c is an integer ranging from 0 to 4; and
T 5 is an organic moiety of the formula C 1-25 N 0-9 O 0-9 H α F β wherein the sum of α and β is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and T 5 includes a tertiary or quaternary amine or an organic acid; and m is an integer ranging from 0–49 where when m is greater than 1, then G, c, Amide, R 1 and T 4 are independently selected.
10. The kit according to claim 9 wherein the -Amide-T 5 is selected from:
11. The kit according to claim 9 wherein -Amide-T 5 is selected from:
12. The kit according to claim 9 wherein the plurality is at least 3.
13. The kit according to claim 9 wherein the plurality is at least 5.