2.4 Screening of samples for MD by PCR
The samples were screened for MD using genomic DNA extracted by PCR. Meq
gene specific PCR targeting BamH1-H 132 bp tandom repeats by using M1 &
M2 primers described previously by Davidson and Borenstein (1999) was
employed (Supplementary Table 2). Briefly, the PCR amplification was
carried out using 2 µl of template DNA in a total volume of 25 µl
reaction volume containing 2.5 µl of 10x Taq buffer with
MgCl2, 1 µl of each primer (20 pmol), 0.5 µl (10 mMol)
of dNTPs and 0.25 µl (5 U/ml) of Taq DNA polymerase and nuclease free
water. The PCR conditions were as follows: 94˚C for 4 min, 35 cycles of
94˚C for 1 min, 55 ˚C for 1 min, 72˚C for 1 min and final elongation at
72˚C for 10 min. The presence of 434 bp product after amplification in
the samples were considered MD positive and these samples were used for
further amplification of MD oncogenes.