Mpss

1. MPSS technology enables the simultaneous sequencing of 20 base signatures from the cDNAs hybridized to every microbead. This is initiated by immobilizing one million or more microbeads in a monolayer array in a microfluidic flow cell for carrying out the sequencing biochemistry and imaging processes (see Note 7).

2. Sequencing is initiated by ligation of an adapter molecule to the GATC (DpnII) single stranded overhang. The adaptor contains a BbvI restriction site; BbvI, a type IIs restriction enzyme, cuts the DNA asymmetrically at positions 13 (5') and 9 (3') nucleotides away from the recognition site. This produces DNA strands on the bead with four-base single-stranded overhangs immediately adjacent to the DpnII site (see Fig. 2).

3. To determine which bases were revealed by the enzymatic cleavage, a set of encoded adapters are ligated to the overhang. Encoded adapters contain all possible combinations of a four-base single-stranded overhang at one end, a single-stranded decoding sequence at the other end and an internal BbvI recognition site. Four types of encoded adapters are ligated to each bead. Each of them bears a common sequence complementary to the overhang but differs by the unique decoder sequence used to identify one base of the four-base overhang (see Note 8).

4. The identity of the ligated encoded adapter is then revealed by probing the decoding region sequentially with 16 fluorescently labeled decoder probes. Knowing the identity of the encoded adapter, thus, yields the identity of the four-base overhang in the signature.

5. To collect additional sequence information, the cycle is repeated by cleavage with BbvI, which removes the first encoding adapter, and reveals the next four-base overhang for subsequent identification.

6. Samples are sequenced in two frames (called steppers) by the use of initiating adapters in which the restriction enzyme recognition site is offset by two bases (see Note 9).

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