Efficient arrays of amplified polynucleotides
The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are arrays of polynucleotides in which the polynucleotides have undergone multiple rounds of amplification in order to increase the strength of signals associated with single polynucleotide molecules.
1. A method of making a random array of amplicons for determining sequences of a plurality of different target polynucleotides presented by the array, said method comprising:
a) providing a flow cell comprising a surface that contains discrete regions arranged in a regular array, and inter-regional areas surrounding the discrete regions,
wherein substantially every discrete region is occupied by a single branched macromolecular structure with a target polynucleotide, wherein different discrete regions comprise different target polynucleotides;
b) amplifying the target polynucleotides in situ to produce multiple copies of the respective target polynucleotide at each discrete region such that the copies are confined to the area of the discrete region; and
c) carrying out sequencing reactions with the target polynucleotide copies in situ to determine sequences of a plurality of said target polynucleotides.
2. The method of claim 1 , wherein the target polynucleotides in step (a) are products of a first amplification reaction and said first amplification reaction is carried out in solution.
3. The method of claim 2 , wherein the first amplification reaction comprises amplification of genomic DNA sequences of an organism.
4. The method of claim 2 , wherein the regular array is in a rectilinear pattern.
5. The method of claim 2 , wherein the regular array is in a hexagonal pattern.