Primers for diagnosing avellino corneal dystrophy
a technology of corneal dystrophy and primers, applied in biochemistry apparatus and processes, organic chemistry, sugar derivatives, etc., can solve the problems of corneal dystrophy, eye loss, serious troubles in both society and economics, etc., and achieve the effect of more efficiently and accurately diagnosing avellino corneal dystrophy
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example 1
Construction of Real-Time PCR Primers and MGB Probes
[0023]In order to construct primers capable of amplifying a region comprising a mutation in exon 4 of BIGH3 gene, pairs of primers of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22 and SEQ ID NOs: 23 and 24 were designed using Primer Express 3.0 software (Applied Biosystems U.S.A).
ACD Fw primer:(SEQ ID NO: 1)5′-TCC ACC ACC ACT CAG CTG TAACD Re primer:(SEQ ID NO: 2)5′-CCA TCT CAG GCC TCA GCT T(60 bp)AV Fw primer:(SEQ ID NO: 3)5′-TGC AGC CCT ACC ACT CTC AAAV Re primer:(SEQ ID NO: 4)5′-AGG CCT CGT TGC TAG G(150 bp)Real Fw primer:(SEQ ID NO: 5)5′-TAG TCT CTT ATT CTA ATA GAReal Re primer:(SEQ ID NO: 6)5′-GCT GCA GAC TCT GTG TTT AA(860 bp)ACD Fw2 primer:(SEQ ID NO: 7)5′-CCA TCC CTC CTT CTG TCT TCT GACD Re2 primer:(SEQ ID NO: 8)5′-CGG GCC CCT CCA...
example 2
Diagnosis of Avellino Corneal Dystrophy Using Real-Time PCR
[0026]Samples were taken from the blood, hair root and oral epithelial cells of test subjects, and DNA was isolated from the samples. The isolation and purification of DNA were performed using a partial modification of the phenol / chloroform extraction method (Miller, SA et al., Nucl. Acids Res. 16:1215, 1988), and the isolated DNA was dissolved in a suitable amount of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH7.4) and confirmed by electrophoresis on 1% agarose gel and used as template DNA in PCR.
[0027]The PCR reactions were performed using the primers (SEQ ID NOs: 1 to 12) for amplifying the fragment containing the mutation region, and the probes (SEQ ID NOs: 13 to 24), constructed in Example 1.
[0028]25 μg of a master mix containing 10 pmol of each primer and 5 pmol of each probe was prepared and used in the PCR reaction.
[0029]The real-time PCR reaction was performed in the following conditions: 36 cycles each consisting of 10 ...
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