SHROB Genotyping: Difference between revisions
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==Primers== | ==New Method== | ||
This method was a tetra-pair ARMS system designed using the tool at http://primer1.soton.ac.uk/primer1.html | |||
* SHROB Flanking 5': GGTTGTTTCTCAATGCAGATAGTAAATT | |||
* SHROB Inner Fwd: CCTGGACACTGTCACCTAATGATTAAA | |||
* SHROB Inner Rev : TCCATTCAATAACCAGATATAACAGAGTA | |||
* SHROB Flanking Rev: TAATACTTGTTAACATTCGAAGGGATTC | |||
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: | |||
{| class="wikitable" | |||
! style="font-weight: bold;" | Genotype | |||
! colspan="3" style="text-align: center; font-weight: bold;" | 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-Fwd AGT GAA TGC TGT GCA GTC | ||
* SHROB-Rev AAG GTT CTT CCA TTC AAT | * SHROB-Rev AAG GTT CTT CCA TTC AAT | ||
==Reference== | ===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] | 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] | ||