Integrated capture and amplification of target nucleic acid for sequencing
The invention provides efficient methods of preparing a target nucleic acid in a form suitable for sequencing. The methods are particularly amenable for preparing high quality nucleic acids for massively parallel sequencing. The methods involve capturing a target nucleic acid from a sample and PCR amplification of the target nucleic acid. The target nucleic acid is captured by binding to a capture probe, which in turn binds to an immobilized probe. The immobilized probe is typically immobilized via a magnetic bead. The captured target nucleic acid is PCR amplified by thermocycling without prior dissociation of the target nucleic acid from the beads. The efficiency of the method lies in part in that both the capture and amplification steps are performed in a single vessel. The amplified nucleic acid can then be sequenced.
1. A method of preparing a target nucleic acid, comprising
contacting a target nucleic acid with a capture probe and an immobilized probe, the capture probe comprising a first segment that binds to the target nucleic acid and a second segment that binds to the immobilized probe, wherein the target nucleic acid binds to the first segment of the capture probe, and the second segment of the capture probe binds to the immobilized probe, thereby forming a capture hybrid capturing the target nucleic acid; and
performing a PCR amplification with real time detection of the captured target nucleic acid without a step of dissociating the capture hybrid before initiating thermocycling in the PCR amplification; wherein the PCR amplification is performed in the same vessel as the contacting step.
2. The method of claim 1 , wherein the target nucleic acid is an RNA molecule and the PCR amplification is an RT-PCR amplification.
3. The method of claim 2 , wherein the target nucleic acid is a viral RNA population.
4. The method of claim 1 , wherein the immobilized probe is immobilized via attachment to a magnetic bead.
5. The method of claim 4 , wherein the concentration of immobilized probe linked to magnetic beads is 10-30 pg/ml.
6. The method of claim 1 , wherein the PCR involves thermocycling between temperature ranges of 90-99° C., and 55-65° C.
7. The method of claim 1 , wherein the concentration of the capture probe is 0.2-0.8 pmol/ml.
8. The method of claim 1 , wherein the target nucleic acid is present in a serum or plasma sample.
9. The method of claim 8 , wherein the serum or plasma sample is treated with detergent to release viral RNA.
10. The method of claim 1 , wherein the first segment includes a nucleic acid of 10-30 bases complementary to the target nucleic acid.
11. The method of claim 3 , wherein the contacting is performed with a plurality of capture probes, the capture probes having the same second segment and different first segments, the different first segments being complementary to different conserved regions of a viral RNA target.
12. The method of claim 1 , wherein the first segment includes a random sequence of nucleotides that binds nonspecifically to the target nucleic acid.
13. The method of claim 12 , wherein the second segment includes a nucleic acid of 10-30 bases complementary to a nucleic acid of 10-30 contiguous bases in the immobilized probe.
14. The method of claim 13 , wherein the nucleic acid of the second segment is a homopolymer and the nucleic acid of the immobilized probe constitute a complementary homopolymer.
15. The method of claim 14 , wherein the homopolymer of the second segment is poly-A and the homopolymer of the immobilized probed is poly-T or vice versa.
16. The method of claim 12 , wherein the second segment of the capture probe and the complementary segment of the immobilized probe are L-nucleic acids.
17. The method of claim 1 , wherein the target nucleic acid is contacted with the capture probe and immobilized probe simultaneously.
18. The method of claim 1 , wherein the binding of the target nucleic acid to the capture probe occurs under first hybridization conditions and the binding of the capture probe to the immobilized probe occurs under second hybridization conditions and the first conditions are more stringent than the second conditions.
19. The method of claim 18 , wherein the first conditions are at a higher temperature than the second conditions.
20. The method of claim 18 , wherein the first conditions include a temperature of 50-70° C. and the second conditions include room temperature.