Electrophoretic techniques applied to the detection and analysis of the human microsatellite DG10s478.
Short nucleotide repetitions (STRs) are commonly used as genetic markers; thus their detection and analysis constitutes a very important tool for the mapping of genetic diseases, as well as for gathering information about genetic polymorphisms at the population level. STRs can be detected with agarose- or acrylamide-based electrophoretic techniques, followed by visualization of the DNA sample with ethidium bromide, silver nitrate, or fluorophore labeling. In this work, we analyzed genomic DNA from five individuals affected with type II diabetes mellitus (T2DM) and five controls (unaffected individuals) in order to know the most precise and reproducible technique for the analysis of the existing polymorphism in the STR DG10S478 of the TCF7L2 gene. The combination of PCR with labeling of the products with the CY5 fluorophore, followed by detection on an ALFexpress sequencer, offered the required resolution to detect the variability in this STR, based solely on size analysis. Our methodology offers similar accuracy and reproducibility at lower costs than existing methods based on the sequencing of PCR products, and is a faster alternative when applied to genotyping studies.
Taylor
Clara Y
CY
Genomics and Diagnostic Division, Center for Genetic Engineering and Biotechnology, PO Box 6162, 10600, Havana, Cuba.
Guillén
Isabel A
IA
Nazabal
Marcelo
M
Fernández
Julio R
JR
Silva
José A
JA
D000483:Alleles; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics; D005240:Feasibility Studies; D005819:Genetic Markers; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D011110:Polymorphism, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics; D051820:TCF Transcription Factors / Q000737:chemistry / Q000235:genetics*; D058118:Transcription Factor 7-Like 2 Protein
D005819:Genetic Markers; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; D058118:Transcription Factor 7-Like 2 Protein