Development of an internal restriction control in the PCR detection of the methylenetetrahydrofolate reductase (MTHFR) C677T mutation.
BACKGROUND
Detection of the presence of 677C/T allele in the human MTHFR gene is easily achieved by amplification using primers designed to span this region. Hinf I digestion, occurring only in the 677T allele, subsequently discriminates between the two alleles. Existing methods, however, do not control for failed restriction endonuclease digestion.


RESULTS
A new forward, modified primer was designed and placed further upstream to create a Hinf I site, which, because it is present in both alleles, would serve as an internal restriction control.


CONCLUSIONS
By allowing an additional restriction to take place in both the normal and mutated alleles, the use of the new primer provided for an internal restriction control.
Lastrucci
R M
RM
Department of Haematology, School of Pathology, University of the Witwatersrand and The South African Institute for Medical Research, Johannesburg, South Africa.
Dawson
D A
DA
Münster
M
M
D000483:Alleles; D017931:DNA Primers; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*
D017931:DNA Primers; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)