ARMS-PCR - (Jun/25/2013 )

I'm having some trouble when using single cell ARMS-PCR for Beta thalassemia HbS cd6 A>T.

After the PCR, mutant band was observed in normal sample. any suggestion to improve the specificity of primers?

PCR Master mix-final volume 20ul
5x Bioline MyTaq Buffer 4ul
primer final conc. 0.2uM
Taq 2.5U

Annealing temperature at 65C

below is the primer sequence.

HbS-Wildtype 5'- AAC AGA CAC CAT GGT GCA TCT GAC TCC TGA -3'
HbS-Mutant 5'- CCC CAC AGG GCA GTA ACG GCA GAC TTC TCC A -3'
A primer 5'- CCC CTT CCT ATG ACA TGA ACT TAA -3'
B primer 5'- ACC TCA CCC TGT GGA GCC AC -3'
A-out primer 5'- GAG ACT TCC ACA CTG ATG CAA TC-3'
B-out primer 5'- GAA GTC CAA CTC CTA AGC CAG T-3'

Below is the reference I used.
http://www.ithanet.eu/ithapedia/index.php/Protocol:Amplification_refractory_mutation_system_(ARMS)
1st PCR was run using HbS-Wildtype,HbS-Mutant, A-out and B-out primer.
2nd PCR was run using HbS-Wildtype,HbS-Mutant, A and B-primer.

I also tried touchdown pcr, which is Ta-70C for first cycle then minus 0.5C for 10cycles, final Ta-65C for 20 cycles. The mutant band still can be observed after the PCR.

Specificity you can increase with a higher Mg²⁺ concentration and some additives such as TMAC.