Difference between revisions of "SHROB Genotyping"
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==New Method== | ==New Method== | ||
Latest revision as of 17:09, 8 June 2020
SOP
New Method
This method was a tetra-pair ARMS system designed using the tool at http://primer1.soton.ac.uk/primer1.html
Primers
- SHROB Flanking 5': GGTTGTTTCTCAATGCAGATAGTAAATT
- SHROB Inner Fwd: CCTGGACACTGTCACCTAATGATTAAA
- SHROB Inner Rev : TCCATTCAATAACCAGATATAACAGAGTA
- SHROB Flanking Rev: TAATACTTGTTAACATTCGAAGGGATTC
Expected Results
There are two outer control primers and two inner primers, one of which (the Fwd primer) is specific for the mutation as shown below.
There should always be a 248bp fragment, and one or both of the other fragments:
| Genotype | Bands | ||
|---|---|---|---|
| Wild-Type | 248 | 184 | |
| Mutant | 248 | 118 | |
| Heterozygote | 248 | 184 | 118 |
Original Method
This is the older method, suggested by Takaya et al.
Primers
- SHROB-Fwd AGT GAA TGC TGT GCA GTC
- SHROB-Rev AAG GTT CTT CCA TTC AAT
Reference
From Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ. Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat. Nat Genet 14: 130–1, 1996. [10.1038/ng1096-130 doi]
Genotyping of lean and obese Koletsky rats by PCR-RFLP analysis. Primers (5 -AGTGAATGCTGTGCAGTC-3 ; 5'-AAGGTTCTTCCATTCAAT-3') were used to amplify PCR products from 100 ng genomic DNA in a 50 uL reaction. The reaction profiles were as follows; denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s, for 30 cycles, All PCR products were digested with Tru9I, analysed by electrophoresis on a 5.0 % agarose gel (NuSieve 3:1 Agarose, Takara Shuzo Co., Ltd., Shiga, Japan), and stained with ethldium bromide. No Tru9l sites were found in the 121-bp PCR product amplified from genomic DNA derived from a wild-type lean Koletsky rat, and the PCR product was not cleaved with Tru9l (lane 1). A single Tru9l restriction site was present in the mutant allele, and Tru9I digestion of the genomic PCR products from obese Koletsky (fa/fa) rats resulted in two fragments of 82 bp and 39 bp in size (lanes 6-1 0). Tru9l digestion of the PCR products from heterozygous lean Koletsky (+/fa rats gave rise to three 121-bp,82-bp, and 39-bp fragments (lanes 2-5).
