HET = C/T
MUT = T/T
WT = C/C
>chr7:142678732-142678788 57bp GCAGAAGCGTGGCATTGTAG AGCTGGTAGAGGGAGCAGAT
GCTCTTCTCTATCCAGCTAGAAGCATCAGGGGTGGGGGTCATGATCCTCCCGGACAACT
CAGGGTGGGGGTTATGGGCTACCTGACGGTATTGGTTATCTTGGGGAAAGGAACTGTCT
ATGTTCCAGGTTTGGTCTAACAGGTACCTGTGGGAGGTGCCCAGGGAGCCAGGAGAAA
ACTGGGGATGCAGAGGGAACCAAAGAAGTCTTGGGTAGATCTTTTAGGGGCTATTGTGG
CTAGGCAGATAGATAGATTAAGCTGTGGCTACCCTACCAGCCCCACTCATGTCTCCTGGT
CCCACATATGCACATGCACATACACACACAACTTGTGCTCATTCAAAGGTTTTATTCATT
GCAGAGGGGTAGGCTGGGTAGTGGTGGGTCTAGTTGCAGTAGTTCTCCAGCTGGTAGA
GGGAGCAGATGCTGGTG(C/T)AGCACTGATCTACAATGCCACGCTTCTGCTGGGCCACC
TCCAGTGCCAAGGTCTGAAGGTCACCTGCTCCCGGGCCTCCACCCAGCTCCAGTTGTG
CCACTGCCAGGGAGGTTGTGTCAGAGCAGGCCAGGGCATCAGCAGCACAGAAGCAAA
GACACATAGGAAAATCCAGTCAGGAGTAGCTGAGCAACCTCCTACTACCCCAGGTTTTC
TCGCCCCTTAACCCCTGCAGGTGGCAGGGGTTGCATGCCTGTGGGACAAGTCACTGGTC
ATGGATGGACACATGGGCCTCTAAAGCCTACTCATCTTCAGTCAGTGCAGTGCTCTATGA
GGGCCCTAAATAGGTACCTATCCTGGGATCTAGTCACATGTCCCAAGTTACTACCACCAA
GGACCCCTTAAGGCAGGATTCTTACTCATTCCTGGCAGCTGCCATGTTTACATATGTCTAG
CTTCCCCCAGAAACGTGTCCCCACTCCCTGGTTATCCCGAGACCCTCCACACCTAGGACA
CAATTTACAATGTCCCAAGTGTGAAGAAAACCAGGGTAGTTAGCACTGGGGACAG
Single Point Mutation: In the mutant allele a transition from G to A at nucleotide 1907 disrupted an Fnu4HI site in exon 3. This mutation changed the seventh amino acid in the A chain of mature insulin, Cys96 (TGC), to Tyr (TAC). The authors predict that the transition would disrupt a disulfide bond between the A and the B chains and would likely induce a major conformational change in insulin 2 molecules. RT-PCR studies suggest that both normal and mutant Ins2 alleles are transcribed similarly in pancreatic islets of heterozygous mice, although immunofluorescence and immunoblot analyses of heterozygous islets detected reduced levels of insulin and proinsulin.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13606 | Fluorophore-1 | GCA GAA GCG TGG CAT TGT AG | Quencher-1 | Forward | A | |
13607 | Fluorophore-2 | AGC TGG TAG AGG GAG CAG AT | Quencher-2 | Reverse | A | |
13608 | Fluorophore-3 | TCA GTG CTG CAC CAG C | Quencher-3 | WT Probe | ||
13609 | Fluorophore-4 | ATC AGT GCT ACA CCA GC | Quencher-4 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
13606 | 0.40 uM |
13607 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |
Stock Number | Strain Name |
---|---|
007688 | 129S6.B6-Ins2Akita/CofJ |
012307 | B6.129-Apoetm3(APOE*4)Mae Ldlrtm1(LDLR)Mae/J |
006860 | B6.Cg-Ins2Akita Bdkrb2tm1Jfh/SmiJ |
006580 | B6.Cg-Ins2Akita Ldlrtm1Her/J |
004369 | B6.Cg-Rag1tm1Mom Ins2Akita/J |
003548 | C57BL/6-Ins2Akita/J |
007562 | D2.B6-Ins2Akita/MatbJ |
006867 | FVB.B6-Ins2Akita/MlnJ |
008659 | NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ |
014568 | NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ |
10 strains use this protocol |