Difference between revisions of "Part:BBa K4683003:Experience"

Revision as of 02:27, 12 October 2023

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4683003

Lambert_GA 2022
Specificity Testing While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock BBa_K4245200 in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence,BBa_K4245006, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV, BBa_K4683003, and one with three SNVs, No part name specified with partinfo tag.. hsa-miR-133a-3p, No part name specified with partinfo tag. UNIQf244323f4e7724ee-partinfo-00000005-QINU

@@ Line 8: / Line 8: @@
 <br>
 Specificity Testing
-	While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock  <partinfo>BBa_K4245200 </partinfo> in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence  ,<partinfo>BBa_K4245006 </partinfo>, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV,  <partinfo>(BBa_K4683003) </partinfo>, and one with three SNVs,  <partinfo>BBa_K4683004 </partinfo>.  hsa-miR-133a-3p, <partinfo>BBa_K4683004<partinfo/>, was also included to ensure the padlock would not ligate to any miRNA.
+While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock  <partinfo>BBa_K4245200 </partinfo> in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence,<partinfo>BBa_K4245006 </partinfo>, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV,  <partinfo>BBa_K4683003 </partinfo>, and one with three SNVs,  <partinfo> BBa_K4683004 </partinfo>.  hsa-miR-133a-3p, <partinfo> BBa_K4245103<partinfo/>, was also included to ensure the padlock would not ligate to any miRNA.
 <html><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/screenshot-2023-10-11-at-4-21-01-pm.png
 "
@@ Line 15: / Line 15: @@
 <br>
-Figure 1. Comparison of sequences used to test specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p
+Figure 1. Comparison of sequences used to test the specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p
 <br>
 <br>