Genotyping

Applications for genotyping are extensive, ranging from linkage mapping for research or diagnostic purposes to forensic or paternity testing, microbiological typing, and veterinary or agricultural trait mapping.[5-7] Electropho-retic conditions with high precision are essential in many of these applications. For 2-bp microsatellites, standard deviations of ±0.3 nucleotide are required to ensure with 99.7% probability that the tested alleles are identical or different.[8] This level of precision is achievable with CGE.

Capillary gel electrophoresis can be problematic when it is used to determine the absolute size of a DNA fragment. This is illustrated by DNA triplet repeat expansions, such as those in Huntington disease.[9]

Fig. 2 (a) Detecting eight SNPs in the 5'UTR and intron 2 of the MT3 gene. The various alleles are numbered 1-8 and demonstrate the utility of CGE in multiplexing and distinguishing various fragments on the basis of size and/or color. SNPs #3 and #6 illustrate how comigrating alleles for each SNP are distinguishable by primers labelled with two different fluorochromes. SNPs #4 and #6 are distinguishable by their migratory distance. (b) QA graph for normal and mutant (AF508) fragments for cystic fibrosis. It can be seen that over a period of 10 months, there has been little drift outside the acceptable limit of 1 bp for the mutant (91 bp) and normal (94 bp) fragments. (View this art in color at www.dekker.com.)

Fig. 2 (a) Detecting eight SNPs in the 5'UTR and intron 2 of the MT3 gene. The various alleles are numbered 1-8 and demonstrate the utility of CGE in multiplexing and distinguishing various fragments on the basis of size and/or color. SNPs #3 and #6 illustrate how comigrating alleles for each SNP are distinguishable by primers labelled with two different fluorochromes. SNPs #4 and #6 are distinguishable by their migratory distance. (b) QA graph for normal and mutant (AF508) fragments for cystic fibrosis. It can be seen that over a period of 10 months, there has been little drift outside the acceptable limit of 1 bp for the mutant (91 bp) and normal (94 bp) fragments. (View this art in color at www.dekker.com.)

Although the resolving power of CGE can distinguish variations in DNA fragments as small as 1 bp, the sizing of a DNA fragment can be a potential source of error. With the Huntington disease example, we observed that the sizes of triplet repeats determined through CGE were always one to two repeats (3-6 bp) less than the gold standard obtained by the 32P slab gel technique.[10] This discrepancy is unacceptable in the above triplet repeat scenario for which an accurate measure of the repeat number is essential because the cutoff for predicting the development of disease occurs exactly at 40 repeats.

Sizing in CGE depends on the algorithm that converts mobility data for an unknown peak into size information based on size vs. mobility results from the size standards.1-11-1 Incorrect size calls can come from poor resolution of two or more peaks, and of particular relevance to CGE, the aberrant mobility of unknown peaks. Incomplete denaturation leading to sequence-specific secondary structures is a recognized cause of aberrant mobility and can be avoided by optimizing the denaturing conditions in CGE.[11] Another way around this potential problem is to normalize the automated size measurements to known fragment sizes. We take the latter approach in Huntington disease by incorporating a CAG allelic ladder with known CAG fragment sizes as an additional sizing standard, thereby allowing exact sizing of the triplet repeat.[12]

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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