Part:BBa_K3060005

RCA&MCA Primer3 for DNA hydrogel

This part is a ssDNA, cooperating with BBa_K3060002-BBa_K3060007 to form the DNA hydrogel. This part can not only help the linear template to form the circular template, but also complimentary with circular template. The conjugate can be recognized by phi29 DNA polymerase and triggers the rolling circle amplification (RCA, or R). After the RCA completed, adding this part and primer2 (BBa_K3060004) 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 part can not only help the linear template to form the circular template, but also complimentary with circular template. The conjugate can be recognized by phi29 DNA polymerase and triggers the rolling circle amplification (RCA, or R). After the RCA completed, adding this part and primer2 (BBa_K3060004) to start the multi-primed chain amplification (MCA, or M).

Characterization

The formation of circular template

In order to examine the successful preparation of the circular template, different samples were characterized by polyacrylamide gel electrophoresis (PAGE) As shown in Fig. 1, ligation products of ssDNA (Lane 2) exhibited a series of dispersive bands with slower migration than that of ssDNA (lane 1), indicating the formation of circular templates and other byproducts. After the ligation products were treated with Exo I and Exo III, only one bright and well-defined band still existed, proving the complete digestion of ligation byproducts and successful preparation of the circular template (lane 3).

Figure 1. PAGE image analysis of ssDNA (Lane 1), ligation products of ssDNA by reaction with Primer 3 and T4 DNA ligase (Lane 2), purified circular template by digesting above-mentioned ligation products with Exo I, and Exo III (Lane 3).

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.

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]