July 1, 2006 (Vol. 26, No. 13)
Combination Makes for Easy Identification and Retrieval of Oligonucleotide Samples
GenVault’s(www.genvault.com) 384-well GenPlate features biological barcodes incorporated into Whatman(www.whatman.com) FTA paper. These barcodes, referred to as GenCodes, are a combination of nonhuman oligonucleotide sets that differ in size and sequence. This combination of oligonucleotides creates a binary number, which is directly related to the individual plate barcode number and linked to the corresponding sample identity.
In the past, we used gel electrophoresis to check the quality of incoming oligos and the GenCodes. Using traditional electrophoresis methods, this process can be cumbersome and time-consuming, depending on the number of samples that need to be processed. In a high-throughput laboratory, efficient use of time is essential to keeping down production costs.
Recently, we switched to using the HDA-GT12 system, an automated, tabletop capillary electrophoresis system designed to overcome the bottlenecks of gel electrophoresis from eGene(www.egeneinc.com). This has allowed for fast, cost-effective, and automated sample-handling analysis.
Materials and Methods – GenCode Generation
From the coding sequences of Arabidopsis thaliana genes, n sets of five oligonucleotides that consist of 50, 60, 70, 80, and 90 base pairs produce GenCodes. Each of the n sets is different in sequences, and the sets do not cross-react by PCR. The selected sequences were "BLASTed" against human genomic DNA at www. ncbi.nlm.nih.gov/BLAST, to minimize the likelihood of a positive PCR result when GenCodes are developed in the presence of human genomic DNA.
Only the oligonucleotides that were not complementary to any human genomic DNA were selected in creating the GenCode to investigate the presence of homologous sequences in the human NCI Blast-n engine with a threshold E-value of 1 against the human genome in the nonredundant database. Matching oligonucleotide sequences were not retrieved using these parameters.
Multiplex PCR Reactions
The Table lists the GenCodes Sequences and the PCR primers used in the multiplex PCR reactions (Integrated DNA Technologies; www.idtdna.com). For each oligonucleotide set, multiplex reactions were set up using the following: 1X NH4 Buffer [16-mM (NH4)2SO4, 67-mM Tris-HCl (pH 8.8 at 25 C), 0.01% Tween-20], 1.5-mM MgCl2, 20-µM of each dNTP, 0.4 units of Biolase DNA polymerase (Bioline; www.bioline.com), and 2.8-nM of each primer set. The cycling parameters for the multiplex reaction are the following: 2 minutes at 95C followed by 30 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 30 seconds, and final extension time at 72C for 45 seconds.
Multiplex PCR products were separated and visualized using the HDA-GT12 High Performance Genetic Analyzer and GCK-5000 cartridge (eGene).
Fig.1: An Electropherogram view of the GenCode 1257 separated on the HDA-GT12 High Performance Genetic Analyzer
Results and Conclusion
Each lane in the GenCode pattern shows the presence or absence of the amplified product from each of the five-oligonucleotide sets. The GenCode is then read as a binary number where the presence of a PCR product, for example the 50-base oligonucleotide in set one, would be read as a 1 and the absence as a 0. An nx5 digit binary number is read in order from bottom to top and left to right. In the case of the GenVault system, the binary number generated is the binary analog of the base-10 plate number found on the GenPlate barcode label. Accordingly, the sample itself can be linked back to the plate number and hence the sample data.
We found that using the HDA-GT12 system to verify the oligonucleotide sets and the GenCode is faster, accurate, and less expensive than gel electrophoresis. The HDA-GT12 system is automated and can characterize short nucleic acid fragments in less than ten minutes.
Figure 1 and 2 show a gel-view and electropherogram view of the GenCode (1257) separated on the HDA-GT12 High Performance Genetic Analyzer. Lane 4 is used for the check code, and the barcode is read from bottom to top and from left to right. For example, sample 1257 reads 01100 01110 10101. The binary check code is 01110 or 14, thus verifying the validity of the read.
Currently, our procedure utilizes the HDA-GT12 system three times per week in lieu of acrylimide gel electrophoresis, which requires extensive manual manipulation and often promulgates errors. The system saves time, lowers reagent and disposable cost, and enables high resolution and sensitivity. Therefore, only 30%, instead of 80%, of the experimental time is spent on performing the electrophoresis procedure.
Previously, this analytical procedure entailed testing each oligonucleotide, production, combination, spotting onto the FTA plates, running the sample on the gel, and scanning the gel. Frequently, this procedure produced additional electrophoresis bands, indicating additional PCR products that should not have appeared in the samples. If such problems occurred, researchers would need to repeat the entire procedure in order to determine which samples were problematic.
A backup system for labeling the biological samples themselves, coupled with separating the amplified oligonucleotides via an automated electrophoresis system, enables researchers to precisely identify the original DNA samples throughout the research process. The GenCodes system provides a method of always linking the sample to the plate and facilitates sampling data at any stage of the research. The HDA-GT12 Genetic Analyzer automates the workflow. The result is a biological barcoding system that enables easy tracking and retrieval of samples.
Fig.2: A gel view of the GenCode 1257 separated on the HDA-GT12 High Performance Genetic Analyzer
