Nucleic acid sequencing

A method for sequencing a heterogeneous population of single stranded DNA molecules simultaneously and without spatial separation, wherein each DNA molecule is present in a unique amount and each DNA molecule bears a primer that provides a double stranded portion, which method comprises the following steps:

• a) contacting the plurality of single stranded DNA molecules with hybridization probes, each probe comprising a label cleavably attached to a known base sequence of predetermined length, the array containing all possible base sequences being incapable of ligation to each other, wherein the contacting is carried out in the presence of ligase under conditions to ligate the double stranded portion of each DNA molecule, the probe bearing the base sequence complementary to the single stranded DNA molecule adjacent to the double stranded portion thereby to form an extended double stranded portion which is incapable of ligation to further probes;
• and
• b) removing all unligated probes;
• followed by the steps of:
• c) cleaving the ligated probes to release each label;
• d) recording the quantity of each label;
• and
• e) activating the extended double stranded portion to enable ligation thereto;
• wherein
• f) steps (a) to (e) are repeated in a cycle for a sufficient number of times to determine the sequence of each single stranded DNA molecule by determined the sequence of release of each label;
• wherein the sequence of each DNA molecule is identified on the basis of the relative quantities of each mass label recorded in sted (d) and the unique amount of each DNA molecule.