Part:BBa_K3060004

RCA&MCA Primer2 for DNA hydrogel

This part is a ssDNA, cooperating with BBa_K3060002-BBa_K3060007 to form the DNA hydrogel. This conjugate can carry out the rolling circle amplification (RCA, or R). After the RCA completed, adding this part and primer2 (BBa_K3060005) to start the multi-primed chain amplification (MCA, or M).

Usage

This part is a ssDNA, cooperating with BBa_K3060002-BBa_K3060007 to form the DNA hydrogel. This conjugate can carry out the rolling circle amplification (RCA, or R). After the RCA completed, adding this part and primer2 (BBa_K3060005) to start the multi-primed chain amplification (MCA, or M).

Characterization

The formation of DNA Hydrogel.

The circular template we preparation is used for the RCA and MCA processes to form the DNA hydrogel. The method can be found in 2019 DUT_China_A. （https://2019.igem.org/Team:DUT_China_A/Protocols） We use a variety of methods to characterize the formation of DNA hydrogel.

a.Agarose Gel Electrophoresis

Agarose gel electrophoresis was used to evaluate the formation of DNA hydrogel. DNA hydrogel is difficult to migrate through the agarose gel and remain the retention in home position.

Figure 2. AGE image analysis of phi29(+) (Lane 1), phi29(-) (Lane 2).M1, DL2000 DNA size marker. M2, DL500 DNA size marker.

b.SEM

Scanning electron microscopy (SEM) was used to obtain the morphology of theDNA hydrogel. As shown in Fig. 3, there are a large number of hydrogel particles with a diameter of about 2 μm in the buffer.

Figure 3. SEM images showing structures of DNA hydrogel particles.

c.DLS

We dynamic light scattering (DLS) analysis to get the size of the DNA hydrogel. As shown in Fig. 4, the size of hydrogel particles is between 100 nm and 3000 nm.

Figure 4. DLS size distribution of DNA hydrogel particle.

References

[1] Song P, Ye D, Zuo X, et al. DNA hydrogel with aptamer-toehold-based recognition, cloaking, and decloaking of circulating tumor cells for live cell analysis[J]. Nano letters, 2017, 17(9): 5193-5198.
[2] Lee J B, Peng S, Yang D, et al. A mechanical metamaterial made from a DNA hydrogel[J]. Nature Nanotechnology, 2012, 7(12): 816.
[3] Joosse S A, Gorges T M, Pantel K. Biology, detection, and clinical implications of circulating tumor cells[J]. EMBO molecular medicine, 2015, 7(1): 1-11.
[4] Dean F B, Nelson J R, Giesler T L, et al. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification[J]. Genome research, 2001, 11(6): 1095-1099.

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]