Amplicons of calpastatin gene were analyzed with RFLP method by using both MspI
and NcoI enzymes.
We digested genome DNA with HpaII (sensitive to methylation of CpG) or MspI
(digests methylated CpG), and then amplified the exon 1 regions by PCR with specific primers.
Aliquots of four il of the amplicons were subjected to enzymatic digestion with the restriction enzymes Dral, HaeIII and MspI
(Invitrogen), according to the instructions.
The fragment containing the Asp449Asp (T/C) or MspI
polymorphism region was amplified using the forward and reverse primers of 5,-CAGTGAAGAGGTGTAGCCGCT-3' and 5'-TAGGAGTCTTGTCTCATGCCT-3', respectively.
The 290 bp fragment from VKORC1-1639 G allele created a MspI
restriction site and, on MspI
digestion resulted in 123 bp and 167 bp fragments.
demonstrated linkage disequilibrium in DRD3 polymorphism loci BalI and MspI
, which was not associated with TS in 16 pedigrees.
xMELP is performed as previously described (17) with the following alterations: digestion and ligation reactions are combined using 500 ng of DNA along with 7.5 [micro]L of annealed oligonucleotides [3 A/mL JHpaII 12XXXX (A is absorbance; XXXX indicates xMELP primers) and 6 A/mL JHpa24XXXX], 0.5 [micro]L BSA [10 g/L, New England Biolabs (NEB)], 0.5 [micro]L ATP (100 mmol/L, pH 7.0, NEB), 5 [micro]L digestion buffer (NEB), 4 U MspI
or 2 U HpaII (NEB), 2 U[T.sub.4] DNA ligase (Life Technologies).
In one study investigation of MSPI
polymorphism showed no significant association with T2DM .
Genomic DNA (1 pg) was extracted with a FlexiGene DNA kit (Qiagen, Germany) and digested by 1 unit of MspI
or HpaII (FastDigest, Fermentas, Germany) at 37[degrees]C overnight and resolved on 1% agarose gel.