Difference between revisions of "Part:BBa K4613209"
 
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The locked hairpin is composed of the sequence of Ochratoxin A (OTA) aptamer, the sequence of RCA primer and the locked sequence. In the absence of OTA, the locked hairpin should be stable as "close status", eliminating nonspecific recognition. In the presence of OTA, the hairpin structure would transform into “open” conformation due to the recognition event between the aptamer and OTA.
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The locked hairpin is composed of the sequence of Ochratoxin A (OTA) aptamer, the sequence of rolling circle amplification(RCA) primer and the locked sequence. In the absence of OTA, the locked hairpin should be stable as "close status", eliminating nonspecific recognition. In the presence of OTA, the hairpin structure would transform into “open” conformation due to the recognition event between the aptamer and OTA.
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We predicted the structure of LHP.
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<center><img src="https://static.igem.wiki/teams/4613/wiki/parts/parts/structure-lhp.png"with="1000" height="" width="750" height=""/></center>
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<p style="text-align: center!important;"><b>Fig. 1 Tertiary structure prediction of LHP.</b></p>
 
As shown in Fig. 2a (Lane 1 and Lane 2), RCA products was formed after adding the RCA primers. Then we explored the effect of different primers' concentrations on the amount of RCA products. Fig. 2b shows that within the same reaction time, the efficiency of RCA amplification increases along with the rising of concentration.
 
As shown in Fig. 2a (Lane 1 and Lane 2), RCA products was formed after adding the RCA primers. Then we explored the effect of different primers' concentrations on the amount of RCA products. Fig. 2b shows that within the same reaction time, the efficiency of RCA amplification increases along with the rising of concentration.
  
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<p style="text-align: center!important;"><b>Fig. 1 The principle of locked hairpin structure-switching.</b></p>
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<p style="text-align: center!important;"><b>Fig. 2 The principle of locked hairpin structure-switching.</b></p>
  
  
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<p style="text-align: center!important;"><b>Fig. 2 a. Verification of RCA Reation using Dumbbell Template. M: Marker. Lane2: negative control. Lane3: 100 μM RCA primer. b. Effect of different RCA primers' concentrations on the amount of RCA products. M: Marker. Lane2-7, different concentrations of RCA primers (0 to 100 μM).</b></p>
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<p style="text-align: center!important;"><b>Fig. 3 (a) Verification of RCA Reation using Dumbbell Template. M: Marker. Lane2: negative control. Lane3: 100 μM RCA primer. (b) Effect of different RCA primers' concentrations on the amount of RCA products. M: Marker. Lane2-7, different concentrations of RCA primers (0 to 100 μM).</b></p>
  
  
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<p style="text-align: center!important;"><b>Fig. 3 The response of the method to OTA at varied concentrations (0 pM to 20000 pM). The fluorescence values were detected by quantitative PCR instrument.
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<p style="text-align: center!important;"><b>Fig. 4 The response of the method to OTA at varied concentrations (0 pM to 20000 pM). The fluorescence values were detected by quantitative PCR instrument.
 
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==== Reference ====
 
==== Reference ====

Latest revision as of 14:36, 12 October 2023

Locked Hairpin (LHP)

The locked hairpin is composed of the sequence of Ochratoxin A (OTA) aptamer, the sequence of rolling circle amplification(RCA) primer and the locked sequence. In the absence of OTA, the locked hairpin should be stable as "close status", eliminating nonspecific recognition. In the presence of OTA, the hairpin structure would transform into “open” conformation due to the recognition event between the aptamer and OTA.

We predicted the structure of LHP.


Fig. 1 Tertiary structure prediction of LHP.

As shown in Fig. 2a (Lane 1 and Lane 2), RCA products was formed after adding the RCA primers. Then we explored the effect of different primers' concentrations on the amount of RCA products. Fig. 2b shows that within the same reaction time, the efficiency of RCA amplification increases along with the rising of concentration.


Fig. 2 The principle of locked hairpin structure-switching.


Fig. 3 (a) Verification of RCA Reation using Dumbbell Template. M: Marker. Lane2: negative control. Lane3: 100 μM RCA primer. (b) Effect of different RCA primers' concentrations on the amount of RCA products. M: Marker. Lane2-7, different concentrations of RCA primers (0 to 100 μM).


Results of the system detected by quantitative PCR instrument were shown below.


Fig. 4 The response of the method to OTA at varied concentrations (0 pM to 20000 pM). The fluorescence values were detected by quantitative PCR instrument.

Reference

  1. Zhang J, Lu Y, Gao W, et al. Structure-switching locked hairpin triggered rolling circle amplification for ochratoxin A (OTA) detection by ICP-MS[J]. Microchemical Journal, 2023, 186: 108365.

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]