The assessment of genetically modified (GM) crops for regulatory approval currently requires a detailed molecular characterization of the DNA sequence and integrity of the transgene locus. In addition, molecular characterization is a critical component of event selection and advancement during product development. Typically, molecular characterization has relied on Southern blot analysis to establish locus and copy number along with targeted sequencing of polymerase chain reaction products spanning any inserted DNA to complete the characterization process. Here we describe the use of next generation (NexGen) sequencing and junction sequence analysis bioinformatics in a new method for achieving full molecular characterization of a GM event without the need for Southern blot analysis. In this study, we examine a typical GM soybean [Glycine max (L.) Merr.] line and demonstrate that this new method provides molecular characterization equivalent to the current Southern blot‐based method. We also examine an event containing in vivo DNA rearrangement of multiple transfer DNA inserts to demonstrate that the new method is effective at identifying complex cases. Next generation sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, efficiency, and consistency of the method, and provides a viable alternative for efficiently and robustly achieving molecular characterization of GM crops.

We have fabricated a low-cost disposable polymerase chain reaction thermal chamber that uses buoyancy forces to move the sample solution between the different temperatures necessary for amplification. Three-dimensional, unsteady finite element modeling and a simpler 1-D steady-state model are used together with digital particle image velocimetry data to characterize the flow within the device. Biological samples have been amplified using this novel thermal chamber. Time for amplification is less than 30 min. More importantly, an analysis of the energy consumption shows significant improvements over current technology.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays. R olling circle amplification (RCA) is a molecular cytogenetic technique used with a padlock oligonucleotide probe to detect single base changes in isolated nucleic acids (1-5). Padlock probes are composed of Ϸ100 nucleotides that hybridize to targets of Ϸ30 bases. The 30-base target-binding region of the probe is split into two 15-base segments placed in opposite orientation at each end of the linear probe so that a circle must be formed for hybridization to occur (6, 7). At 10 bases per helical turn, the hybridized probe wraps around its target three times, and the remaining 70 bases form an unhybridized singlestranded loop. Posthybridization DNA ligation connects the two ends of the probe in the middle of the 30-base binding region. The unbound 70-base loop facilitates probe circularization and permits Ϸ20 bases to serve as a primer recognition site for DNA polymerase to replicate the circle. RCA is an isothermal process in which the polymerase progresses continuously around the loop until the 100 bases have been replicated hundreds or thousands of times. Incorporating a labeled nucleotide during the RCA reaction produces sufficient signal for easy visualization of the target.Application of RCA to in situ targets in fixed or permeabilized cells has not been uniformly successful to date. Whereas recent work has demonstrated that the concept is viable (8), DNA detection efficiencies of 20-30% lessen the utility of RCA as an assay. Lack of success has been attributed to possible blocking of the polymerase by the target strand, and it was suggested that this problem might be overcome by cutting the target DNA strand near the RCA probe's hybridization site (5). Under these conditions, DNA polymerase could free the probe from the target, in effect spinning the probe away from the target, keeping the polymerase from being blocked during the amplification process. Here, we report that in addition to restriction enzyme digestion of DNA, additional steps were required to achieve consistent and satisfactory results for RCA in situ. Whereas heat denaturation is typically used to render the target DNA single stranded, we found that complete removal of the nontargeted DNA strand by digestion with exonuclease III significantly increased the efficiency of the process.We also demonstrate the use of RCA to detect mRNA in cytological preparations. Using appropriate image analysis techniques, the RCA assay is sufficiently quantitative to enable transcriptionally mediated dose-response curves to be generated. Inc...

The inference of an individual's geographic ancestry or origin can be critical in narrowing the field of potential suspects in a criminal investigation. Most current technologies rely on single nucleotide polymorphism (SNP) genotypes to accomplish this task. However, SNPs can introduce homoplasy into an analysis since they can be identical-by-state. We introduce the use of insertion polymorphisms based on short interspersed elements (SINEs) as a potential alternative to SNPs. SINE polymorphisms are identical-by-descent, essentially homoplasy-free, and inexpensive to genotype using a variety of approaches. Herein, we present results of a blind study using 100 Alu insertion polymorphisms to infer the geographic ancestry of 18 unknown individuals from a variety of geographic locations. Using a Structure analysis of the Alu insertion polymorphism-based genotypes, we were able to correctly infer the geographic affiliation of all 18 unknown human individuals with high levels of confidence. This technique to infer the geographic affiliation of unknown human DNA samples will be a useful tool in forensic genomics.