Sequence amplification with linear primers
The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. The use of these primers, as described herein, allows for the reduction in the amplification of nonspecific hybridization events (such as primer dimerization) while allowing for the amplification of the target nucleic acid sequences.
1. A method of amplifying a nucleic acid sequence, comprising:
a) generating a plurality of cDNA target sequences by a performing reverse transcription reaction on a plurality of RNA;
b) hybridizing the plurality of cDNA target sequences with a plurality of first linear primers which contain (i) a terminal 3′ region having a first unconstrained random sequence and (ii) a 5′ region having a first universal sequence,
wherein the first unconstrained random sequences of the terminal 3′ region include inosine, adenosine, guanosine, and cytidine, and the first random sequences exclude thymidine,
wherein the plurality of the first linear primers include different random sequences,
wherein the random sequences hybridize to a sequence of the plurality of cDNA target sequences, and
wherein the plurality of the first linear primers include the same universal sequence which does not hybridize to the plurality of cDNA target sequences; and
c) forming a plurality of extended linear primer products by extending the first linear primers that are hybridized to the plurality of cDNA target sequences;
d) hybridizing the plurality of the extended linear primer products to a plurality of second linear primers which contain (i) a terminal 3′ region having a second unconstrained random sequence and (ii) a 5′ region having a second universal sequence,
wherein the second random unconstrained sequences of the terminal 3′ region include inosine, adenosine, guanosine, and cytidine, and the second random sequences exclude thymidine,
wherein the plurality of the second linear primers include different random sequences, and
wherein the random sequences hybridize to a portion of the extended linear primer products,
wherein the plurality of the second linear primers include the same universal sequence as the universal sequence in the first linear primers; and
e) forming a plurality of double-extended linear primer products by extending the plurality of second linear primers that are hybridized to the plurality of the extended linear primer products.
2. The method of claim 1 , further comprising: forming a plurality of hairpin structures by intramolecular hybridizing the plurality of double-extended linear primer products in step (e).
3. The method of claim 1 , further comprising: sequencing the double-extended linear primer products in step (e).
4. The method of claim 1 , further comprising: forming amplification products by amplifying the plurality of double-extended linear primer products of step (e) in a single primer amplification reaction with a plurality of amplification primers that hybridize to the first or the second universal sequence regions of the plurality of double-extended linear primer products.
5. The method of claim 4 , further comprising: forming a plurality of hairpin structures by intramolecular hybridizing the amplification products.
6. The method of claim 4 , further comprising: sequencing the amplification products.
7. The method of claim 1 , further comprising: forming amplification products by amplifying the plurality of double-extended linear primer products of step (e) in an exponential amplification reaction with (i) a plurality of amplification primers that hybridize to the first universal sequence regions of the plurality of double-extended linear primer products, and (ii) a plurality of amplification primers that hybridize to the second universal sequence regions of the plurality of double-extended linear primer products.
8. The method of claim 7 , further comprising: forming a plurality of hairpin structures by intramolecular hybridizing the amplification products.
9. The method of claim 7 , further comprising: sequencing the amplification products.
10. The method of claim 1 , wherein the first and second random sequences in the terminal 3′ regions of the first and second linear primers is 4-12 nucleotides in length.
11. The method of claim 1 , wherein the plurality of RNA is isolated from a single cell.
12. The method of claim 1 , wherein the plurality of RNA is isolated from fewer than 10 cells.
13. The method of claim 1 , wherein the plurality of RNA is isolated from fewer than 100 cells.
14. The method of claim 1 , wherein the plurality of RNA is isolated from a tumor.