DEGENERATE OLIGONUCLEOTIDES AND THEIR USES
The present invention provides a plurality of oligonucleotides comprising a semi-random sequence, wherein the semi-random sequence comprises degenerate nucleotides that are substantially non-complementary. Also provided are methods for using the plurality of oligonucleotides to amplify a population of target nucleic acids.
1 . A method for amplifying a population of target nucleic acids, the method comprising:
(a) contacting the population of target nucleic acids with a plurality of oligonucleotide primers to form a plurality of nucleic acid-primer duplexes, each of the oligonucleotide primers comprising the formula N m X p Z q , wherein:
N is a 4-fold degenerate nucleotide selected from the group consisting of adenosine (A), cytidine (C), guanosine (G), and thymidine/uridine (T/U);
X is a 3-fold degenerate nucleotide selected from the group consisting of B, D, H, and V, wherein B is selected from the group consisting of C, G, and T/U; D is selected from the group consisting of A, G, and T/U; H is selected from the group consisting of A, C, and T/U; and V is selected from the group consisting of A, C, and G;
Z is a 2-fold degenerate nucleotide selected from the group consisting of K, M, R, and Y, wherein K is selected from the group consisting of G and T/U; M is selected from the group consisting of A and C; R is selected from the group consisting of A and G; and Y is selected from the group consisting of C and T/U;
m, p, and q are integers, m either is 0 or is from 2 to 20, p and q are from 0 to 20; provided, however, that no two integers are 0, and further provided that oligonucleotides comprising N, which have at least two N residues, have at least one X or Z residue separating the two N residues.
(b) replicating the plurality of nucleic acid-primer duplexes to create a library of replicated strands, wherein the amount of replicated strands exceeds the amount of target nucleic acids used in step (a), indicating amplification of the population of target nucleic acids.
2 . The method of claim 5 , wherein the formula of the plurality of oligonucleotide primers is selected from the group consisting of N m X p , N m Z q , and X p Z q , m is from 2 to 8, p and q are each from 1 to 8, and the sum total of the two integers is 9.
3 . The method of claim 6 , wherein the oligonucleotide primers comprising N have no more than three consecutive N residues.
4 . The method of claim 7 , wherein each of the oligonucleotide primers has a sequence selected from the group consisting of KNNNKNKNK, NKNNKNNKK, and NNNKNKKNK.
5 . The method of claim 5 , wherein each oligonucleotide primer further comprises a sequence of non-degenerate nucleotides at the 5′ end, the non-degenerate sequence being constant among the plurality of oligonucleotides, and the constant non-degenerate sequence being about 14 nucleotides to about 24 nucleotides in length.
6 . The method of claim 5 , wherein replication of the target nucleic acid is catalyzed by an enzyme selected from the group consisting of Exo-Minus Klenow DNA polymerase, Exo-Minus T7 DNA polymerase, Phi29 DNA polymerase, Bst DNA polymerase, Bca polymerase, Vent DNA polymerase, 9°Nm DNA polymerase, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, a variant thereof, and a mixture thereof.
7 . The method of claim 5 , further comprising amplifying the library of replicated strands using a polymerase chain reaction.
8 . The method of claim 11 , wherein amplification utilizes at least one primer selected from the group consisting of a primer having substantial complementary to a constant region at the ends of the replicated strands and a pair of primers.
9 . The method of claim 11 , wherein the amplified library is labeled by incorporation of at least one modified nucleotide during the polymerase chain reaction, the modified nucleotide selected from the group consisting of a fluorescently-labeled nucleotide, aminoallyl-dUTP, bromo-dUTP, and a digoxigenin-labeled nucleotide.
10 . The method of claim 5 , wherein the target nucleic acid is fragmented by a method selected from the group consisting of mechanical, chemical, thermal, and enzymatic means.
11 . The method of claim 11 , wherein the target nucleic acid is DNA, the replication is catalyzed by Exo-Minus Klenow DNA polymerase, and the amplification is catalyzed by Taq DNA polymerase.
12 . The method of claim 11 , wherein the target nucleic acid is RNA, the plurality of oligonucleotide primers further comprises an oligo dT primer, the replication is catalyzed by MMLV reverse transcriptase and/or Exo-Minus Klenow DNA polymerase, and the amplification is catalyzed by Taq DNA polymerase.
13 . The method of claim 16 , wherein the replication comprises a first reaction utilizing the oligo dT primer and MMLV reverse transcriptase and a second reaction utilizing the plurality of oligonucleotide primers and Taq DNA polymerase.