This invention relates to methods for improving the discrimination of hybridization of target molecules to probes on substrate-bound oligonucleotide, peptide, or protein arrays. Therefore, it relates to the fields of molecular biology and biophysics.
An efficient method of sequencing DNA is by means of hybridization to known nucleotide sequences arranged in microarrays. See e.g., PCT WO 89/10977. In this method a solution of single strands of unknown DNA is mixed with an array of oligonucleotides which are fixed to a substrate. The oligonucleotides vary in sequence, and each unique sequence occupies a small region on the substrate whose position is known. If the sequence of a given oligonucleotide region is complementary to the unknown DNA sample, then the DNA strands will hydrogen bond or hybridize to the oligonucleotides at that site. Since the oligonucleotide sequence is known, that part of the sample which bound to the oligonucleotide is then determined as well. If the DNA sample is fragmented into lengths comparable to the lengths of the oligonucleotides, then the entire DNA sample can, in principle, be sequenced.
The sites in the microarray at which the DNA binds can be identified by attaching tags to the sample fragments before hybridization. These tags can be radioactive, fluorescent, or luminescent, for example. By scanning the hybridized microarray for radioactivity or fluorescence, the hybridized sites can be identified.
The power of this technology lies in the discriminatory ability of the hybridization process. For DNA fragments on the order of 20 nucleotides in length or less, a single mismatch in nucleotide base pairs can significantly affect the hybridization process, and more than one adjacent base pair mismatch can effectively prevent hybridization. The degree of discrimination is controlled by the conditions in the solution: the types and concentrations of buffers and the temperature. The degree to which nucleic acids hybridize is referred to as “stringency”. In a state of high stringency conditions, hybridization rate is reduced and the probability of base pair mismatches is reduced even more. In a condition of low stringency, hybridization becomes more likely and the probability of base pair mismatches increases. In general, high stringency conditions and high discrimination against base pair mismatches are characterized by higher temperature, lower ionic strength, low reactant concentration, and short reaction times. In addition, many washings of the microarray with hybridization buffers are done to remove sample DNA strands which have not hybridized to probe oligonucleotides.
The initial DNA sample is often very limited in size, and to increase the probability of detecting a successful hybridization in the microarray, the DNA is amplified using polymerase chain reaction (PCR) or other means. Despite the amplification process, the DNA concentration is often still very limited, so hybridization of a substantial fraction of the sample may be needed for reliable detection. Thus conditions of high stringency may also limit the detectability of hybridized samples.
Hybridization rates in the microarray are ultimately limited by diffusion of the DNA samples in their buffer to the substrate. More specifically, the microarray is mounted within a structure (i.e., a cell) which serves as a reservoir for the DNA sample. Various techniques are used to circulate the samples within the cell to expedite hybridization, such as circulation of the sample from the cell to an external reservoir and back, or by agitation of the cell, but hybridization times can still be many hours. Furthermore, washing the microarray in buffer to remove DNA samples which did not hybridize to oligonucleotide sites, thereby increasing the stringency conditions, can take a comparable amount of time. See, for example, U.S. Pat. No. 6,114,122.
One technique to speed things up is to use eletrophoresis to attract the negatively charged DNA samples to the oligonucleotides. This requires adding electrodes and an electrical grid to the microarray, so that an electric field with the right polarity can be established to attract the DNA to the oligonucleotides. The electrical mobility of the DNA can be much greater than the intrinsic diffusion rate in solution. After hybridization has taken place, the polarity of the field can be reversed, thereby driving the non-hybridized DNA samples away from the microarray, and making the washing steps more effective. This can greatly increase the stringency of the process while reducing the overall hybridization time. See, e.g., U.S. Pat. No. 5,849,486.
However, these improvements are purchased at the expense of added complexity. The microarray must be provided with an electrical grid. Moreover, the grid must be covered by a permeation layer which isolates and protects the DNA from the metallic grid, excludes electrolysis products from the DNA buffer, and provides support for the oligonucleotide probes. High density microarrays are typically scanned for fluorescence through their transparent substrates. This is not possible if an electrical grid is present. The buffer properties must be adjusted to accommodate the electrophoresis. In particular, the buffer electrical conductivity must not be low. These constraints may not permit using buffers which are optimal for hybridization.
The concept of a microarray to identify unknown samples can be extended to other molecules such as proteins. In the technique known as ELISA (enzyme linked immunosorbent assay), an array of known antibodies is created on a substrate. The array is then exposed to a solution of unknown proteins (i.e., antigens). After washing, proteins which remain bound to their corresponding antibodies can be fluorescently tagged, and identified from their locations in the array. While electrophoresis can be used here, the variety of charge states of different proteins complicates the experiments.
Temperature gradient gel electrophoresis, as described in German Application DE-OS 36 22 591, is a method for detecting slight structural differences or peculiarities of biological macro-molecules such as nucleic acids or proteins. This technique relies upon the use of temperature gradients in combination with electrophoresis for the separation of biological macro-molecules. This technique, however, is restricted to the operation of flat-bed gel electrophoresis.
Thermophoresis refers to a process in which particles, residing in a gas supporting a temperature gradient, are driven away from warm surfaces toward cooler surfaces. The thermophoretic drift velocity is found to be directly proportional to the temperature gradient in the gas. Although the phenomenon has been well studied with aerosols, there has been little reported on the use of thermophoresis with particles in liquid. McNab et al. (1973) J. Colloid and Interface Science 44:339 have presented an equation describing thermophoretic drift velocity based on a study of small latex spheres in water and hexane. No dependence on particle size was detected within the limited particle size range studied. The thermophoretic drift velocity was found to be directly proportional to the temperature gradient in the fluid.
Thus, there is still a need for methods for improving the stringency of and/or decreasing the time required for hybridization experiments. The present invention addresses this and other needs.
USPTO Backgrounds
USPTO Backgrounds