Selection of Hygromycin-Resistant Arabidopsis Seedlings

tube RT-PCR and Pyrosequencing reactions to monitor the relative amounts of two alleles in a single sample helps to minimize variations due to RNA extraction and target amplification.There are several issues that may affect future applications of this technique to clinical samples.The study of a specific SNP may require additional characterization of the target by sequencing because of the inherent heterogeneity of RNA viruses.O'Meara et al. ( 8) have successfully utilized amplification oligonucleotides containing limited degeneracy at certain positions to accommodate annealing to a broad number of known HIV isolates because minor changes in the sequence can affect RT-PCR amplification of the target area, ultimately influencing the lower limits of detection for minor alleles.In addition, unexpected differences in the target can also affect results because the specific dispensation of nucleotides in the assay determines the identity of the SNP and the internal peak height controls (i.e., adjacent bases).Confirmation of the target sequence may be needed to ensure that both the SNP and the neighboring sequences are within the parameters of the analysis software.This approach should enhance both basic research on virus-host interactions and clinical analysis of viral dynamics in response to administration and/or cessation of therapies.