High-level multiplex amplification
The present disclosure provides a “looping amplification” method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
1. A method for amplifying one or more target nucleic acids, the method comprising:
contacting sample nucleic acids with a forward primer and a reverse primer for each target nucleic acid, wherein each primer comprises a target-specific portion, a common sequence 5′ of the target-specific portion, a DNA sequencing primer binding site 5′ of the common sequence, and a first or second nucleotide tag 5′ of the DNA sequencing primer binding site, wherein the first and second nucleotide tags in the forward and reverse primers are different, and wherein the common sequence comprises a transposon end sequence; and
amplifying the target nucleic acid(s) in a first amplification to produce at least one target amplicon wherein a target nucleotide sequence is flanked by the common sequence on one end and its reverse complement on the other end, whereby a single strand of the target amplicon can form a stem loop structure, wherein the first amplification is carried out in a microfluidic device comprising a plurality of reaction chambers using a third primer, wherein the third primer comprises a portion specific for the second nucleotide tag, a barcode nucleotide sequence 5′ of the tag-specific portion, and a first flow cell attachment site 5′ of the barcode nucleotide sequence, wherein the first amplification produces first target amplicons having the structure: 5′-first nucleotide tag-first primer binding site-common sequence-target nucleotide sequence-reverse complement of common sequence-second primer binding site-second nucleotide tag-barcode nucleotide sequence-first flow cell attachment site-3′;
recovering the first target amplicons from the microfluidic device after the first amplification; and
amplifying the first target amplicons in a second amplification using a fourth primer having a portion specific for the first nucleotide tag and a second flow cell attachment site 5′ of said first nucleotide tag-specific portion and a reverse primer specific for the second flow cell attachment site, wherein the second amplification produces second target amplicons having the structure: 5′-second flow cell attachment site-first nucleotide tag-first primer binding site-common sequence-target nucleotide sequence-reverse complement of common sequence-second primer binding site-second nucleotide tag-barcode nucleotide sequence-first flow cell attachment site-3′.
2. The method of claim 1 , wherein a plurality of target nucleic acids is amplified.
3. The method of claim 1 , wherein a plurality of target nucleic acids is amplified in a single reaction mixture.
4. The method of claim 3 , wherein at least 100 target nucleic acids are amplified in a single reaction mixture.
5. The method of claim 4 , wherein at least 1000 target nucleic acids are amplified in a single reaction mixture.
6. The method of claim 1 , wherein fewer than 17,000 target nucleic acids are amplified in a single reaction mixture.
7. The method of claim 1 , wherein amplification cross-hybridization is suppressed as compared to when amplification is carried out using primers containing only target-specific sequences.
8. The method of claim 1 , wherein the average target amplicon size is greater than when amplification is carried out using primers containing only target-specific sequences.
9. The method of claim 1 , wherein the first amplification is carried out in a plurality of separate reaction mixtures, wherein each reaction mixture is in a separate reaction chamber.
10. The method of claim 1 , wherein amplification is carried out in multiplex within each of the plurality of reaction chambers.
11. The method of claim 10 , wherein more than 100 target nucleic acids are amplified in each of the plurality of reaction chambers.
12. The method of claim 1 , wherein the microfluidic device comprises a matrix-type microfluidic device.
13. The method of claim 1 , wherein the method additionally comprises sequencing the target amplicons.
14. The method of claim 11 , wherein simultaneous amplifications are carried out for at least 4800 target nucleic acids from a particular sample in the microfluidic device.
15. The method of claim 14 , wherein simultaneous amplifications are carried out for at least 4800 target nucleic acids from at least 48 samples in the microfluidic device.
16. The method of claim 1 , wherein amplification is carried out in the presence of 2-pyrrolidinone with or without trehalose.