Cyc

Fig. 2 FCCM analysis of the common British COL7A1 mutation 7786delG. FCCM of fragment 19 (exons 103-108) for two patients heterozygous for the mutation 7786delG (lanes 1-2) and a negative control (lane 3). Lane 4 contains the size standards. Cleavage peaks (458 + 1082 bp) in lanes 1 and 2 are absent for the negative control. The uncleaved peak is 1540 bp.

Fig. 2 FCCM analysis of the common British COL7A1 mutation 7786delG. FCCM of fragment 19 (exons 103-108) for two patients heterozygous for the mutation 7786delG (lanes 1-2) and a negative control (lane 3). Lane 4 contains the size standards. Cleavage peaks (458 + 1082 bp) in lanes 1 and 2 are absent for the negative control. The uncleaved peak is 1540 bp.

because of the toxic nature of osmium tetroxide, work has been undertaken to validate a safer replacement. Potassium permanganate/tetraethylamine chloride[16,19,20] is now preferred because in addition to resolving the safety concerns of osmium tetroxide it can also overcome the lack of reactivity of some thymine-guanine mismatches.1-17-1 Recently, a combination single-tube protocol has been developed, termed Single-Tube Chemical Cleavage of Mismatch Method, that permits the chemical modification of heteroduplexes by potassium permanga-nate/tetraethylamine chloride and hydroxylamine without the need for intermediate purification steps.[19]

Solid-phase technology

The original CCM protocol[4] required many lengthy ethanol precipitation stages that limited the quantity of work performed each day. However, the development of an ultrarapid protocol that made use of biotinylated PCR primers, in conjunction with streptavidin-coated paramagnetic beads, yielded a solid-phase CCM protocol. Exploitation of the affinity of biotin for streptavidin along with microtitre plate technology permits easy and effective purification of the cleavage products, thereby allowing high throughput screening to be performed.[13] Very recently, the solid-phase CCM protocol has seen further development whereby the DNA is immobilized on solid silica supports. This version of solid-phase

CCM is fast and sensitive for detection of sequence changes.[21]

Detection protocols

Depending on the labeling protocol used, cleavage products can be detected using standard denaturing polyacrylamide gel electrophoresis (PAGE) apparatus and detected using standard X-ray film, or on a fluorescent DNA fragment analyzer utilizing PAGE or, more recently, capillary electrophoresis.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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