Reagents for DNA isolation

1. TKM1: 10 mM Tris-HCl, pH 7.6. 10 mM KCl, 10 mMMgCl2, 2 mM EDTA, and 2.5% Nonidet P-40.

Fig. 1. PCR-ELISA detection of GALT gene mutations. Three regions of the GALT gene are amplified by multiplex PCR. The PCR product is labeled with Digoxigenin (DIG) by the incorporation of DIG-labeled nucleotides in the PCR reaction. The PCR product is denatured and hybridized to biotinylated sequence-specific oligonucleotide probe. A separate hybridization reaction is used to detect the normal and mutant sequence at each of the loci. The specificity of the hybridization is optimized by the design of oligonucleotides and the temperature of hybridization. Further, nonbiotinylated blocking oligonucleotides are used to reduce background hybridization. The hybrids with biotinylated oligonucleotides are then immobilized on streptavidin-coated microtiter plates. Unbound hybrids and PCR products are washed away. The plates are then treated with anti-DIG antibodies conjugated with peroxidase. Unbound antibodies are washed away. The presence of allele specific hybridization is detected by activity of peroxidase enzyme on a colorimetric substrate.

Fig. 1. PCR-ELISA detection of GALT gene mutations. Three regions of the GALT gene are amplified by multiplex PCR. The PCR product is labeled with Digoxigenin (DIG) by the incorporation of DIG-labeled nucleotides in the PCR reaction. The PCR product is denatured and hybridized to biotinylated sequence-specific oligonucleotide probe. A separate hybridization reaction is used to detect the normal and mutant sequence at each of the loci. The specificity of the hybridization is optimized by the design of oligonucleotides and the temperature of hybridization. Further, nonbiotinylated blocking oligonucleotides are used to reduce background hybridization. The hybrids with biotinylated oligonucleotides are then immobilized on streptavidin-coated microtiter plates. Unbound hybrids and PCR products are washed away. The plates are then treated with anti-DIG antibodies conjugated with peroxidase. Unbound antibodies are washed away. The presence of allele specific hybridization is detected by activity of peroxidase enzyme on a colorimetric substrate.

2. TKM2: 10 mM Tris-HCl, pH 7.6. 10 mM KCl, 10 mMMgCl2, 2 mM EDTA, 0.4 M NaCl, 0.05% sodium dodecyl sulfate (SDS).

4. Absolute Ethanol.

Table 1 PCR Primers

Size of

Primer Sequence (5'—>3') Mutation PCR product

GALT 9: GGT CAG CAT CTG GAC CCC AGG K285N, 668

INJR: GGG GTC GAC GCC TGC ACA TAC TGC ATG TGA Y209C, N314D

GALT6: AGG AGG GAG TTG ACT TGG AGT Q188R, L195P 428 IN7R: GGG GAC ACA GGG CTT GGC TCT CTC CCA

S135LF: GAG TGA TAC TCC TTT ACC TCA GGA CCC AGT S135L 252 S135LR: GGA CCG ACA TGA GTG GCA GCG TTA CAT TC

0 0

Post a comment