Part:BBa_K4118006

hsa_circ_0005962 H2 probe

Modifications:

Fluorescent Dye used: ATTO 610

Quencher: BHQ2

The H2 probe is a hairpin-shaped structure, enhanced with a fluorophore and a quencher used for signal production. It hybridizes to the loop and stem 2 domains of the hsa_circ_0005962 H1 probe. When the H1-H2 complex is created the fluorophore is distanced from the quencher leading to signal production. It contains a twelve-nucleotide long dangling end for hybridization to the RCA backbone and a 30 nucleotide long domain for H1 probe hybridization.

Based on the H1 sequence, the H2 probe consists of the following domains: A domain complementary to the RCA product (5), A domain complementary to sequences (2) and (3) that is responsible for the hybridization of the two probes (6), A domain complementary to 20 bases of sequence (6) to create a 20-base hairpin stem (7) The loop domain (8)

When designing the above structure special care must be taken, so that no artifacts are present it the secondary structure. Unexpected secondary structures can affect the probe's ability to hybridize to the corresponding H1 and as result little to no signal will be produced.

Bellow the excitation and emission spectra for ATTO 610 are presented.

ATTO 610 has an Excitation maximum of 615 nm and Emission maximum of 634 nm.

Below the absorption spectrum of BHQ2 is presented.

BHQ2 has a Quenching range of 550-650nm.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]