Genotyping STR Alleles on an Electronically Active DNA Chip
Ron Sosnowski, David Canter, Melanie Duhon, Lana Feng , M. Muralihar, Ray Radtkey,
James O’Connell, Michael Heller, and Michael Nerenberg
Nanogen Inc., San Diego, CA
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We are combining the technologies of microelectronics and molecular biology for use in DNA analysis. Other efforts in this area have emphasized array or microfabrication techniques, however the kinetics of the molecular biology reactions remain controlled by conventional means. Manipulation of the kinetics of highly specific DNA hybridization has been achieved here by the application of precisely regulated electric fields, establishing the use of an electric field as a parameter which is equivalent or superior to temperature and chemical denaturants.
A DC electric field applied to specific 80
mm diameter test sites directly controls denaturation of DNA. Electric fields contained in a microelectronic device also regulate DNA transport and concentration thereby facilitating DNA hybridization. Dehybridization discrimination among different DNA hybrids is provided by varying the strength of the electric field. Additionally, electric field denaturation control allows single base pair mismatch discrimination of HLA-DQa to be carried out rapidly (<30 seconds) and with high resolution (1). Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and may constitute a novel method of DNA denaturation.The use of STR loci is becoming more widespread overseas and in the United States. Therefore, we have used this approach to achieve single repeat unit discrimination of STR alleles. This has been accomplished on a microscopic array format in less than one minute.
We are currently in the process of developing this assay for use in a product. In addition to assay development, we are developing a portable instrument capable of point of use genetic fingerprinting.
Ultimately, the miniaturization and increased speed of genetic analysis permitted by this technology should broaden the use of DNA fingerprinting in forensic applications.
RESULTS AND DISCUSSION
We present here a report on our progress with STR analysis on an electronically active DNA chip. Details of the chip, including a description of the permeation layer are shown in Figure 1. It is important to note that the electronic profile of each electrode, or test site, can be individually controlled. Therefore, we are able to develop assays which have 25 different hybridization tests, each with a unique electronic stringency level, in a 1 mm2 array. This feature is important from the perspective of not only miniaturization, but also in the area of automation, since it is much easier to move reagents short distances by electronics than to move fluidic sample robotically (1).
Figures 2, 3 and 4 show experiments on the TH01, TPOX and CSF1PO loci respectively. These results demonstrate that our assay is capable of identifying the STR alleles present in the DNA sample. Allele identification is accomplished by analyzing the relative amount of fluorescence remaining on test sites after treatment. High levels of signal indicate stable hybrids, which are present only at test sites that have cognitive capture oligo.
Simultaneous analysis of several STR loci is necessary to produce a useful genotyping assay. The data in Figure 5 demonstrate that this type of analysis can be accomplished for TH01 and TPOX. It should be noted that these loci share the STR nucleotide sequence AATG. Therefore, this experiment represents resolution of a particularly challenging locus-allele combination. We are currently developing assays which would include simultaneous analysis of several loci.
Conformation with existing practices in genotyping dictates that our assay be compatible with analysis of PCR products, and further, that this be done with maximum efficiency. The data shown in Figure 6 indicates that PCR product from amplification of the TH01 locus can be directly analyzed on the chip, with minimal sample preparation. The ability to provide accurate identifications of PCR generated targets, in both control and unknown situations, establishes the feasibility of the Nanogen STR assay. Experiments are currently underway to make incorporate multiplex PCR into the assay.
These results demonstrate that we have make significant progress toward developing an assay which can be applied to genotyping. We anticipate that this technology will broaden this field by making DNA identity testing easier to perform and much faster than current methods (1,2).
ACKNOWLEDGMENTS
This work was supported by a contract with The Bode Technology Group and a grant from the National Institutes of Justice.
REFERENCES
1. Sosnowski R.G., Tu E., Butler W.F., O’Connell J.P. and Heller M.J. Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci. USA 1997; 94:1119-1123.
2. Edman C.F., Raymond D.E., Wu D.J., Tu E., Sosnowski R.G., Butler W.F., Nerenberg M. and Heller M.J. Electric field directed nucleic acid hybridization on microchips. Nucleic Acid Research, in press.
Figure 1- Nanogen Silicon Device with a 25 Electrode Microarray.
(A) The processed chip is 1 cm square. Contact electrodes, indicated by dark squares around the perimeter of the chip, are connected to a power supply thereby conducting current through insulated platinum leads, indicated by dark lines, to the exposed platinum electrode. (B) The 1mm square array is located in the center of the chip and consists of 25 test electrodes (80
mm) and 4 larger corner electrodes (160mm) which are used as counter electrodes in establishing an electric field. The chip has been made by microfabrication techniques commonly employed by the microelectronics industry. (C) In a cross section of a test electrode as it appears when the permeation layer of agarose and streptavidin is laid down, the DNA is shown attached to the agarose above the electrode. Streptavidin in the permeation layer reacts with the biotinylated DNA probe to immobilize the DNA capture probe at the test site. This single-stranded oligo is then used to selectively interrogate sample DNA.
Figure 2 - Identification of TH01 Target DNA Alleles
TH01 contains tetranucleotide repeat (AATG) present in various copy numbers. Figure 2 depicts data from an experiment designed to determine the identity of the alleles present in an unknown target DNA sample. The chip was prepared by electronically addressing capture DNA representing all TH01 alleles to individual sites on the chip, so that each test site is capable of detecting a different TH01 allele. A mix of complementary target DNA, composed of TH01 alleles 5 and 9, was then electronically hybridized to each of the sites containing addressed capture DNA. The Nanogen assay was then applied to determine allelic identity.
These results show that a heterozygous mix of TH01 DNA can be resolved into match and mismatch hybrids, with the match hybrids representing the identity of the alleles present in the DNA sample. All possible homozygote and heterozygote TH01 STR allelic combinations have been analyzed by our chip format, with similar excellent levels of discrimination among alleles.
Figure 3 - Identification of TPOX Target DNA Alleles
TPOX, or the human thyroid peroxidase gene, contains within its noncoding region the tetranucleotide repeat (AATG). To demonstrate that chip arrays are a viable medium on which to discriminate variable numbers of tandem repeat alleles, numerous homozygote and heterozygote TPOX combinations have been analyzed.
Figure 3 depicts data from an experiment where target DNA containing the TPOX 8 and 11 alleles was analyzed. Oligo capture DNA containing all allelic possibilities was electrically addressed to individual sites on the chip. A mix of complementary target DNA, composed of TPOX alleles with 8 and 11 STRs, was then electronically hybridized to each of the pads containing addressed capture DNA. Our assay was then used to determine allelic identity.
The results show that a mix of TPOX 8 STR/11 STR DNA can be unequivocally discriminated from all mismatches. Further, all other homozygous and heterologous TPOX combinations analyzed yield comparable discrimination.
Figure 4 - Identification of CSF1PO Target DNA Alleles
Capture oligos containing CSF1PO alleles 7 through 15 inclusive were electronically addressed to representative sites. Target DNA containing CSF1PO 9 and 14 alleles was then electronically hybridized to each of the sites. The Nanogen assay was applied to determine discrimination of correct capture-target complexes. Figure 4 shows the fluorescent signals at various capture sites, demonstrating the ability of the assay to correctly discriminate the alleles present in the target sample.
Figure 5 - TH01/TPOX Multiplex Analysis
Locus-allele specific capture oligos were individually addressed to different sites on a single chip. The DNA chip containing capture oligos was then hybridized with a mixture of TH01 and TPOX target DNA containing heterozygote alleles. The chip was then washed and analyzed by the Nanogen hybridization assay, in which relative fluorescent levels are used to determine whether sites contain capture-target match or mismatch DNA hybrids.
The results showed that under our assay conditions, 7 and 9 STR alleles of TH01 hybridized very well with their cognitive capture sites. Hybridization to other capture alleles was not detectable (6x, 8x, 10x and 11x), indicating an excellent discrimination of TH01 7/9 heterozygote. For the TPOX locus, we also obtained a good matched capture/target interaction (10x and 12x). Further, hybridization of 10 and 12 STR targets to mismatched captures was either undetectable (8xc and 13xc), or low enough to yield a discrimination ratio of 15 fold or higher (11xc and 9xc respectively), resulting in easy discrimination of TPOX 10/12 heterozygote.
Figure 6 - Identification of TPOX Alleles in Double Stranded PCR-amplified DNA
This experiment was done to determine the utility of the Nanogen STR assay when using double-stranded DNA. Figure 6 provides an example of the ability of our system to accurately identify PCR generated targets.
The TPOX 1 locus was PCR amplified using control template K562 following standard conditions outlined in the Promega STR Users manual (3). The genotype of K562 is heterozygous for the 8 and 9 repeat alleles. Following amplification the amplicon was denatured at 95
°C and hybridized to a Nanogen APEX chip. As previously discussed, the chip had capture probes unique for PCR products containing each number of repeat length...The figure shows the relative amount of signal present on the positive (8C, 9C match) and negative (7C, 10C mismatch). As seen in the previous experiments the level of discrimination attainable ranges from 20-fold to infinite. Similar results have been obtained using CSF1 and TH01 from both K562 control DNA and genomic DNA isolated from anonymous donors.
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