DNA

Part:BBa_K3577003

Designed by: Xixi Song   Group: iGEM20_Worldshaper-Shanghai   (2020-08-27)
Revision as of 02:16, 23 October 2020 by SylviaSong (Talk | contribs)

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KLK3 mRNA

KLK3 is a kind of biomarker in prostate cancer tissue. The mRNA of KLK3 can be detected in the urine of patient with prostate cancer. KLK3 almost does not express in other cancers or just a little that can be ignored, so it has high specificity in prostate cancer. Moreover, the expression of it is significantly different between prostate cancer cells and normal cells, which shows a high sensitivity.


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]


Usage and Biology

We searched KLK3 on the UCSC Genome Browser (http://genome.ucsc.edu/), a genome browser hosted by UCSC which offers access to genome sequence data. When determining which specific transcript of the gene to use, we chose the transcript with the most amount of exons. We then copied the sequence of one particular exon shared by multiple transcripts of the gene (NCBI Reference Sequence: NM_001030047.1). The KLK3 sequence we uploaded this time is only a fragment of the whole sequence, which is the region amplified by the KLK3 primers.

Results

We obtained three pairs of primers for KLK3, all pairs of primers of KLK3 were verified by PCR ( Polymerase Chain Reaction) before we start following research, the best one pair is KLK3-2 (FIgure1).

The primer sequence was as follows:

KLK3-2F: TGGTGCTGCACCCCTCATC

KLK3-2R: GGGCAGGTCCATGACCTTC

Figure1. Primer test for both KLK3 and PCA3

We added a T7 sequence at the 5’ end of forward primer to help start the process of transcription. For the reverse primer, we designed it with a trigger at the 5’ end, so as to add the trigger sequence to the end of PCR product during the process of PCR.

The sequences of these overhang-added primers are as follow :

T-KLK3-2F：taatacgactcactatagggTGGTGCTGCACCCCTCATC

T-KLK3-2R: gtttgaatgaattgtaggcttgttatagttatgtttGGGCAGGTCCATGACCTTC

The results of PCR showed that the primers with overhang could also amplify KLK3 successfully without significantly affecting the amplification efficiency.

Figure2. PCR efficiency test of two kinds of overhang-added primers


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