Protocol 7642: Pyrosequencing Assay - ** human MAPT*E10+14
Version 1.0

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 = T/T
Wild type has no PCR product for pyrosequencing.
 
TGAGTACCTTCA(c/t)ACGTCCCATGC
Sequencing primer anneals to the forward strand.

Sequence

TCCCCTGGGATGTGACTCAACCTCCCGTCACTCCCCAGACTGCCTCTGCCAAGTC
CGAAAGTGGAGGCATCCTTGCGAGCAAGTAGGCGGGTCCAGGGTGGCGCATGTC
ACTCATCGAAAGTGGAGGCGTCCTTGCGAGCAAGCAGGCGGGTCCAGGGTGGCG
TGTCACTCATCCTTTTTTCTGGCTACCAAAGGTGCAGATAATTAATAAGAAGCTGG
ATCTTAGCAACGTCCAGTCCAAGTGTGGCTCAAAGGATAATATCAAACACGTCCC
GGGAGGCGGCAGTGTGAGTACCTTCA(c/t)ACGTCCCATGCGCCGTGCTGTGGCTTG
AATTATTAGGAAGTGGTGTGAGTGCGTACACTTGCGAGACACTGCATAGAATAAA
TCCTTCTTGGGCTCTCAGGATCTGGCTGCGACCTCTGGGTGAATGTAGCCCGGCT
CCCCACATTCCCCCACACGGTCCACTGTTCCCAGAAGCCCCTTCCTCATATTCTAG
GAGGGGGTGTCCCAGCATTTCTGGGTCCCCCAGCCTGCGCAGGCTGTGTGGACAG
AATAGGGCAGATGACGGACCCTCTCTCCGGACCCTGCCTGGGAAGCTGAGAATAC
CCATCAAAGTCTCCTTCCACTCATGCCCAGCCCTGTCCCCAGGAGCCCCATAGCCC
ATT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13068 GGC TAC CAA AGG TGC AGA TAA Forward
13069 Fluorophore ATG CAG TGT CTC GCA AGT GTA Reverse
13070 CGG CAG TGT GAG TAC CT SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
Primer 1 0.25
Primer 2 0.25
Kapa 2G HS taq polym 0.01
DNA 0.00

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- Go to step 2-4, 10 times
6 94.0 --
7 50.0 --
8 72.0 --
9 -- Go to step 6-8, 28 times
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.