Protocol 15033: Pyrosequencing Assay - ** Hfe*C294Y
Version 2.0

Notes

This assay will NOT distinguish hemizygous from homozygous trangenic animals.  This assay only confirms the point mutation.

This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = A/A

Wild type has no PCR product for pyrosequencing.
 
AAGGTTCACCT(g/a)TCAAGTGGAG
 
Sequencing primer anneals to the forward strand.
 
C294Y mutation.  This is a change from TGT to TAT.
 
JR16519 is a truncated form of Hfe cDNA and will not have an amplification product with these primers

Sequence

 

AGCTGGGGAGAAAGTGCCTACTTTGGTGAAAGTGACTCGCCACTGGGCCTC
TACGGGGACCTCTCTAAGGTGTCAGGCTCTGGACTTCTTCCCCCAGAACATC
ACTATGAGGTGGTTGAAGGACAACCAACCACTGGATGCCAAAGATGTCAAC
CCCGAGAAGGTGCTACCTAACGGGGATGAGACCTATCAAGGCTGGCTGACA
TTGGCCGTGGCCCCTGGGGACGAGACAAGGTTCACCT(g/a)TCAAGTGGAG
CACCCAGGCCTGGACCAGCCTCTCACTGCCTCTTGGGAGCCCTTGCAATCC
CAGGCCATGATTATCGGAATCATCAGTGGAGTCACCGTCTGTGCCATCTTCTT
GGTTGGAATTCTGTTCCTAATCTTAAGGAAAAGGAAGGCTTCAGGAGGAACC
ATGGGTGGCTATGTCTTAACAGACTGTGAGTGATCTGCAGCCTGCTGAACCA

CGGAAGAGAGAAAACTCAGCCAAAGACTTGGAGGGGGCACACTTGCTCCAC

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12312 TAC CTA ACG GGG ATG AGA CCT A Forward
12313 Fluorophore GGC TGC AGA TCA CTC ACA GTC Reverse
12314 GGA CGA GAC AAG GTT CA SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
12312 0.50
12313 0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.