USE OF ORGANIC CATIONIC COMPOUNDS TO ACCELERATE NUCLEIC ACID HYBRIDIZATION, SYNTHESIS, AND AMPLIFICATION
The invention provides methods for accelerated synthesis of nucleic acids, and related compositions which involve the use of organic amines in the nucleic acid synthesis reaction mixture. The invention also provides methods for reducing processing steps associated with nucleic acid synthesis. The invention further provides methods for screening compounds that have positive benefits on the synthesis of nucleic acids.
1 . A nucleic acid synthesis reaction mixture, comprising at least one organic diamine, an oligoamine, or other dication or combinations thereof.
2 . The mixture of claim 1 , wherein the at least one organic diamine is an aliphatic organic diamine.
3 . The mixture of claim 2 , wherein the at least one organic diamine is an alkyl diamine.
4 . The mixture of claim 3 , wherein the at least one organic diamine is a linear alkyl diamine.
5 . The mixture of claim 4 , wherein an amino group is disposed at each end of the linear alkyl diamine.
6 . The mixture of claim 1 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
7 . The mixture of claim 6 , wherein the at least one organic diamine is putrescine.
8 . The mixture of claim 1 , wherein the reaction mixture further comprises magnesium ion or magnesium salt.
9 . The mixture of claim 1 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
10 . The mixture of claim 1 , wherein the at least one oligoamine is an organometallic compound.
11 . A method for increasing the melting temperature of nucleic acids during nucleic acid synthesis, comprising
providing at least one organic diamine, an oligoamine, or other dication to the nucleic acid synthesis.
12 . The method of claim 11 , wherein the at least one organic diamine is an aliphatic organic diamine.
13 . The method of claim 12 , wherein the at least one organic diamine is an alkyl diamine.
14 . The method of claim 13 , wherein the at least one organic diamine is a linear alkyl diamine.
15 . The method of claim 14 , wherein an amino group is disposed at each end of the linear alkyl diamine.
16 . The method of claim 11 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
17 . The method of claim 16 , wherein the at least one organic diamine is putrescine.
18 . The method of claim 11 , further comprising the addition of magnesium ion or magnesium salt to the nucleic acid synthesis.
19 . The method of claim 11 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
20 . The method of claim 11 , wherein the at least one oligoamine is an organometallic compound.
21 . A method for increasing the specific hybridization of nucleic acids during nucleic acid synthesis, comprising
providing at least one organic diamine, an oligoamine, or other dication to the nucleic acid synthesis.
22 . The method of claim 21 , wherein the at least one organic diamine is an aliphatic organic diamine.
23 . The method of claim 22 , wherein the at least one organic diamine is an alkyl diamine.
24 . The method of claim 23 , wherein the at least one organic diamine is a linear alkyl diamine.
25 . The method of claim 24 , wherein an amino group is disposed at each end of the linear alkyl diamine.
26 . The method of claim 21 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
27 . The method of claim 26 , wherein the at least one organic diamine is putrescine.
28 . The method of claim 21 , further comprising the addition of magnesium ion or magnesium salt to the nucleic acid synthesis.
29 . The method of claim 21 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
30 . The method of claim 21 , wherein the at least one oligoamine is an organometallic compound.
31 . A method for increasing the rate of nucleic acid synthesis during a nucleic acid synthesis, comprising
providing at least one organic diamine, an oligoamine, or other dication to the nucleic acid synthesis.
32 . The method of claim 31 , wherein the at least one organic diamine is an aliphatic organic diamine.
33 . The method of claim 32 , wherein the at least one organic diamine is an alkyl diamine.
34 . The method of claim 33 , wherein the at least one organic diamine is a linear alkyl diamine.
35 . The method of claim 34 , wherein an amino group is disposed at each end of the linear alkyl diamine.
36 . The method of claim 31 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
37 . The method of claim 36 , wherein the at least one organic diamine is putrescine.
38 . The method of claim 31 , further comprising the addition of magnesium ion or magnesium salt to the nucleic acid synthesis.
39 . The method of claim 31 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
40 . The method of claim 31 , wherein the at least one oligoamine is an organometallic compound.
41 . A method for improving the amplification of nucleic acids during a nucleic acid synthesis, comprising
providing at least one organic diamine, an oligoamine, or other dication to the nucleic acid synthesis.
42 . The method of claim 41 , wherein the at least one organic diamine is an aliphatic organic diamine.
43 . The method of claim 42 , wherein the at least one organic diamine is an alkyl diamine.
44 . The method of claim 43 , wherein the at least one organic diamine is a linear alkyl diamine.
45 . The method of claim 44 , wherein an amino group is disposed at each end of the linear alkyl diamine.
46 . The method of claim 41 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
47 . The method of claim 46 , wherein the at least one organic diamine is putrescine.
48 . The method of claim 41 , further comprising the addition of magnesium ion or magnesium salt to the nucleic acid synthesis.
49 . The method of claim 41 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
50 . The method of claim 41 , wherein the at least one oligoamine is an organometallic compound.
51 . A method for reducing the background signal in a nucleic acid synthesis, comprising
providing at least one organic diamine, an oligoamine, or other dication to the nucleic acid synthesis.
52 . The method of claim 51 , wherein the at least one organic diamine is an aliphatic organic diamine.
53 . The method of claim 52 , wherein the at least one organic diamine is an alkyl diamine.
54 . The method of claim 53 , wherein the at least one organic diamine is a linear alkyl diamine.
55 . The method of claim 54 , wherein an amino group is disposed at each end of the linear alkyl diamine.
56 . The method of claim 51 , wherein the at least one organic diamine is selected from the group consisting of ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, 1,6-diaminohexane, or 2,2′-(Ethylenedixoy)bis(ethylamine) and combinations thereof.
57 . The method of claim 56 , wherein the at least one organic diamine is putrescine.
58 . The method of claim 51 , further comprising the addition of magnesium ion or magnesium salt to the nucleic acid synthesis.
59 . The method of claim 51 , wherein the at least one oligoamine is a metal ion in combination with a chelator.
60 . The method of claim 51 , wherein the at least one oligoamine is an organometallic compound.
61 . A sample processing method for nucleic acid detection that improves or eliminates at least one of sample preparation, lysing, separating, washing, or elution, comprising
heating the sample in the presence of either urine or urea, and
introducing a portion or all of said heated sample directly into a nucleic acid detection system.
62 . The method of claim 61 , wherein the detection is not inhibited.
63 . The method of claim 61 , wherein the amount of the sample used is 10% of the volume of the nucleic acid detection reaction.
64 . The method of claim 61 , wherein the amount of the sample used is 20% of the volume of the nucleic acid detection reaction.
65 . The method of claim 61 , wherein the amount of the sample used is 30% of the volume of the nucleic acid detection reaction.
66 . The method of claim 61 , wherein the amount of the sample used is 40% of the volume of the nucleic acid detection reaction.
67 . The method of claim 61 , wherein the amount of the sample used is 50% of the volume of the nucleic acid detection reaction.
68 . The method of claim 61 , wherein the concentration of urea prior to heating is from about 1 M to about 4 M.
69 . The method of claim 68 , wherein the concentration of urea prior to heating is from about 1.5 M to about 3 M.
70 . The method of claim 61 , wherein the separating is by binding to a matrix.
71 . The method of claim 61 , wherein the separating is by precipitation.
72 . A process for identifying a compound that both increases the melting temperature of nucleic acids and enhances specific hybridization during nucleic acid synthesis, comprising
contacting a compound of interest with a nucleic acid having a known melting temperature during nucleic acid synthesis and a known temperature at which hybridization occurs during nucleic acid synthesis and measuring the melting temperature of the nucleic acid after contact with the compound of interest and measuring the temperature at which hybridization occurs after contact with the compound of interest during nucleic acid synthesis, and selecting compounds that both increase the melting temperature during nucleic acid synthesis and the temperature at which hybridization occurs for said known nucleic acid.
73 . A process for identifying a compound that enhances reverse transcriptase synthesis, comprising
contacting a compound of interest with a nucleic acid and measuring whether the reverse transcriptase synthesis is more efficient than in the absence of said compound, and selecting compounds that improve the efficiency of reverse transcriptase synthesis.
It should also be understood that wherever in the present application the term comprising or including (or a term of similar scope) is recited in connection with the description of any embodiment or part thereof, a corresponding embodiment or part thereof reciting instead the term consisting essentially of or the term consisting of (or a term of similar scope) is also disclosed.
Any and all publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly exemplified in combination within.