Stock No: 016144
Protocol 7625: Pyrosequencing Assay - ** human TARDBP*A315T
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

T/T=Human TARDBP tg and A315T point mutation are present
 
C/C=Human TARDBP tg is present but not A315Tmutation
 
Wild type has no PCR product for pyrosequencing.
 
GAACTTTGGT(g/a)CGTTCAGCAT
 
Sequencing primer anneals to the reverse strand.

Sequence

 

ACATTTGCAGATGATCAGATTGCGCAGTCTCTTTGTGGAGAGGACTTGATCATTA
AAGGAATCAGCGTTCATATATCCAATGCCGAACCTAAGCACAATAGCAATAGAC
AGTTAGAAAGAAGTGGAAGATTTGGTGGTAATCCAGGTGGCTTTGGGAATCAG
GGTGGATTTGGTAATAGCAGAGGGGGTGGAGCTGGTTTGGGAAACAATCAAGG
TAGTAATATGGGTGGTGGGATGAACTTTGGT(g/a)CGTTCAGCATTAATCCAGCCAT
GATGGCTGCCGCCCAGGCAGCACTACAGAGCAGTTGGGGTATGATGGGCATGT
TAGCCAGCCAGCAGAACCAGTCAGGCCCATCGGGTAATAACCAAAACCAAGGC
AACATGCAGAGGGAGCCAAACCAGGCCTTCGGTTCTGGAAATAACTCTTATAGT
GGCTCTAATTCTGGTGCAGCAATTGGTTGGGGATCAGCATCCAATGCAGGGTCG
GGCAGTGGTTTTAATGGAGGCTTTGGCTCAAGCATGGATTCTAAGTCTTCTGGC

TGGGGAATGTAG

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13002 Fluorophore ATG GGT GGT GGG ATG AAC TT Forward
13003 ATA CCC CAA CTG CTC TGT AGT GCT Reverse
13004 GGC TGG ATT AAT GCT GA 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

Stock Number Strain Name
016201 B6SJL-Tg(Prnp-TARDBP)4Jlel/J
016144 STOCK Tg(Prnp-TARDBP)4Jlel/J
016143 STOCK Tg(Prnp-TARDBP*A315T)23Jlel/J
3 strains use this protocol