Protocol 34971 - Human APP Indiana (V617F)

Notes

This assay cannot distinguish hemi from hom

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

Tg= 96 bp

IPC = 74 bp

>chr16:84962983-84963078 96bp CGCCATCATCGGACTCAT TGGATGGATGTGTACTGTTTCTTC

These "transgene" primers amplify mouse.  The probe is what makes this assay specific.

Sequence

Tg Sequence:

Indiana (V617F) 642 of 695aa

Hu WT V TTGTCATAGCGACAGTGATCGTCATCACCTTGGTGATGCTGAAG

Hu V->I TTGTCATAGCGACAGTGGTCATCATCACCTTGGTGATGCTGAAG I716V(FL),V717I(Lon)

Hu V->F TTGTCATAGCAACCGTGATTTTCATCACCCTGGTGATGTTGAAG Indiana allele

Mouse B6J TTGTCATAGCAACCGTGATTGTCATCACCCTGGTGATGTTGAAG

A T V I V I T L V M L K

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
45231	Fluorophore-1	AAC CGT GAT TTT CAT CAC C	Quencher-1	MUT Probe
45232		CGC CAT CAT CGG ACT CAT		Transgene Forward	A
45233		TGG ATG GAT GTG TAC TGT TTC TTC		Transgene Reverse	A
oIMR1544		CAC GTG GGC TCC AGC ATT		Internal Positive Control Forward	A
oIMR3580		TCA CCA GTC ATT TCT GCC TTT G		Internal Positive Control Reverse	A
TmoIMR0105	Fluorophore-2	CCA ATG GTC GGG CAC TGC TCA A	Quencher-2	IC Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
45232	0.40 uM
45233	0.40 uM
oIMR1544	0.40 uM
oIMR3580	0.40 uM
Wt Probe	0.15 uM
Mutant Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--
4		--	repeat steps 2-3 for 40 cycles
5	40.0	--	Forever

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.