Hi,
Im doing a HUMARA assay to investigate X-chromosome inactivation. Right now the shorter allele has a great advantage in my PCR resulting in very different allele frequncies for my microsatellites in control patients that "should" have a 1:1 ratio between the two alleles. How can I make sure that the longer allele is amplified as good as the shorter one?
In other words, how can one optimize a PCR so that a longer allele is not less amplified then a shorter one.
These are my conditions at the moment in a 20ul reaction:
Buffer 2ul
dNTP 1μl of 2mM (0.1mM)
F and R primers 0.5μl of 1μM (25nM)
MgCl2 1μl of 25mM (1.25mM)
DNA 50ng/2ul
Taq 0.2ml of 5U/ml (0.05U/ml)
95° 5’, for 20 cycles (95° 20’’, 60-50° 30'’, 72° 30'’) and 25 cycles (95° 20’’, 55° 30'’, 72°).
Thanks!
PCR optimization was performed using DNA extracted from the maize cultivar LH10HtHt and the inbred lines L30HtHt, L30Htht, and L30htht, kindly provided by Sementes Agroceres S/A (Uberlândia/MG). The latter three lines, nearly isogenic for a gene that confers resistance against Exserohilum turcicum, were developed by backcrossing using the cultivar as the donor parent. DNA was extracted according to Hoisington et al. (1994) and quantified by fluorometry.
Amplification reactions were done in PTC-100 (MJ Research, USA) thermocyclers in a final reaction volume of 30ml containing 10 ng of DNA template, 0.1 mM of each dNTP, 1 U of Taq DNA polymerase (Promega, USA), 1X reaction buffer (50 mM KCl, 1.5 mM MgCl2, 100 mM Tris-HCl, pH 9, and 0.1% Triton X-100), additional 0.5 mM MgCl2, and varying concentrations of primers (0.165, 0.33, 0.5, 0.67 or 1.0 mM). Amplification reactions were optimized for 125 maize MapPairsä primers (Research Genetics, USA), that amplify microsatellite loci from all maize chromosomes. Primer sequences were obtained from the Maize Database.
A two-stage touchdown amplification program was designed for each locus based on the Tm of both primers, calculated according to Tm = [2oC (A + T) + 4oC (C + G) - 5oC] (Newton and Graham, 1997) so that, when possible, both Tm were included in the program. An initial denaturation step of 3 min at 94oC was used in all programs, followed by a first stage in which the Tm was gradually decreased by 1oC following every 2 cycles. This treatment was followed by a second stage consisting of either 19 or 29 amplification cycles, in which the lowest Tm remained constant. Denaturation and elongation steps were held constant for 1 min at 94oC and 2 min at 72oC, respectively, during both stages. An elongation step of 6 min at 72oC was performed in all programs after stage 2.
I think you must increase more cycle of PCR condition to 35 cycles. Also, re-edit your program of PCR conditions.
Honestly, if both templates are an exact match to your primers you will always have an amplification bias that favors the smaller product. Can you adjust your primers so that you include a different forward or reverse?
Hi all, Thank you for your answers.
Katie, what do you mean with including a different forward or reversed? That I should include an extra primer in my reaction or change on of the two? Why would this help? Thank you!
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