BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to a method for nucleic acid hybridization, and specifically relates to a method and apparatus with temperature control for nucleic acid hybridization and hydrogen bond denaturation to accelerate nucleic acid hybridization.
1. Related Art
Based on rapidly developed techniques, such as PCR (polymerase chain reaction) and nucleic acid hybridization, molecular biotechnology has been gradually integrated with different areas, including material science, bioinformatics, and electronic technology, to bring about a new area of Biochips. The emergence of bio chips could significantly decrease needed detection time for some diseases. For example, the initial detection time of 3-5 days by cell culture techniques can be decreased to be less than 6 hours by utilizing biochip technology. However, such 6-hour detection time still cannot satisfy the demands of certain diseases. For instance, some diseases take less than 2 days from diagnosis to cause death. Therefore, current bio chip research has been focused on shortening the needed detection time of the biochip.
To shorten the detection time, it is necessary to start from the most time-consuming technique during the test. PCR takes approximately 1.5 hours and nucleic acid hybridization takes about 4 hours Together, these two techniques account for 92% of the whole biochip detection time. Therefore, how to shorten the amount of time consumed by these two techniques has become a key point. Presently, numerous researchers have put forth much effort on this issue, and the invention is focused on the most time-consuming nucleic acid hybridization technique.
The underlying mechanism for techniques such as Hybridization Helper is to utilize an oligonucleoride (i.e. Helper), which is complementary to the upstream or downstream region of the area where probing nucleic acid is hybridized to sample nucleic acid. Such hybridization between the nucleic acid Helper and sample nucleic acid is employed to stretch the sample nucleic acid (to eliminate the original cluttered circular conformation that is unfavorable for hybridization), which help the hybridization reaction.
In addition, another technique (called nucleic acid precipitating reagents) is available. The underlying mechanism for this technique is to utilize various buffered salt solutions to promote the sample nucleic acid precipitation at the nearby area of the probing nucleic acid. Such methodology increases the sample nucleic acid concentration in the local area to promote the processing of the hybridization reaction.
There is still another available technique called the branched oligonucleoride multimer technique, which uses probes immobilized on the surface of a chip for catching sample nucleic acid. Then, a branched oligonucleoride multimer, which is complementary to the sample nucleic acid, is linked to sample nucleic acid. Finally, fluor- or radio-labeled nucleic acid detectors are complementarily hybridized with the branched oligonucleoride multimer. Since tens or hundreds of detectors can hybridize onto a branched oligonucleoride multimer, the detection intensity can be greatly increased and the hybridization time can therefore be
Furthermore, a technique called electrically controlled hybridization takes advantage of the characteristics of negatively charged nucleic acid. By immobilizing the positive pole near the nucleic acid probe, nucleic acid in samples is lured near to the probe to make the sample nucleic acid highly concentrated in a small area and accomplish the goal of accelerating hybridization.
Another technique called volume exclusion agents employs organic molecules to form a reticular macro-structure, which can expel part of the hybridization buffer to increase the local concentration of sample nucleic acid and therefore promote the hybridization reaction.
Similar to the above technique, one technique called amphipathic hydrocarbon polymer (AHP) utilizes bipolar organic molecules (hydrophilic and hydrophobic) to form a reticular macro-structure. This macrostructure can expel part of the hybridization buffer to increase the local concentration of sample nucleic acid and therefore promote the hybridization reaction.
An apparatus called the highly parallel-integrated microfluidic biochannel array has integrated various functions, including sample pretreatment, PCR, hybridization, washing, and signal detection. However, the hybridization rate of this apparatus has not yet been improved.
Finally, in the apparatus called the dynamic hybridization system, a nucleic acid probe is fixed on a semipermeable membrane. Meanwhile, fluid (containing sample nucleic acid) is driven by air or vacuum compression to flow towards the semipermeable membrane. Sample nucleic acids are delayed when the fluid passes through the semipermeable membrane and sample nucleic acids accumulate around the membrane to yield a higher concentration of sample nucleic acid. The hybridization rate is increased because non-complementary nucleic acid is able to pass through the holes of the semipermeable membrane.
Most of the above mentioned methods increase the hybridization rate by increasing sample nucleic acid concentration, linearlizing samples, or employing branched structure. All these hybridization accelerating methods have limitations, such as only being operable on a large scale. Nevertheless, how to speed up nucleic acid hybridization while also making the process applicable on a small scale is still the focus of a great deal of effort in research.
SUMMARY OF THE INVENTION
In order to solve the problems of the above-mentioned known techniques, this invention provides a method and apparatus designed specifically for nucleic acid hybridization, which fulfills the aim of accelerating the nucleic acid hybridization rate by increasing kinetic energy and thermal energy of the nucleic acid-containing flow.
Introducing a novel nucleic acid hybridization method is the other aim of this invention. By increasing the thermal energy of nucleic acid-containing fluid, originally curled nucleic acid is linearlized and the hybridization rate is increased.
To accomplish these aims, the invention provides a method for nucleic acid hybridization. The method includes the following steps. Firstly, providing a nucleic acid hybridization area of the first temperature, which is the first channel immobilized with several nucleic acid probes. Secondly, providing a hydrogen bond denaturation area of the second temperature, which is the second channel attached with a nucleic acid hybridization area to form a connecting channel. Thirdly, guiding the nucleic acids-containing fluid into the connection channel. Finally, driving such fluid to repeatedly pass through the first channel and the second channel.
A two-way driving apparatus, such as a two-way air-driven or fluid-driven pump, is used to drive the fluid. The fluid is retained in the first channel for the first period (such as 3 minutes) and then driven to the second channel for the second period (such as 10 seconds). While being retained in the first channel, the kinetic energy of the fluid is increased by being driven to flow back and forth.
The invention further provides a nucleic acid hybridization apparatus, including a hydrogen bond denaturation area equipped with a first channel for denaturing hydrogen bonds of nucleic acids; and a nucleic acid hybridization area equipped with a second channel, which is immobilized with nucleic acid probes. A connecting channel is formed between the nucleic acid hybridization area and the first channel. A temperature control element is used for maintaining the temperature of the hydrogen bond denaturation area and nucleic acid hybridization area at the first and second temperatures, respectively. A two-way driving element is used for driving the flow of the nucleic acids-containing fluid that is infused in the connecting channel.
Nucleic acid probes for hybridization can be DNA, RNA, peptide, peptide-RNA complex or derivatives of peptide-RNA complex. The temperature control element can then keep a stable temperature for the hydrogen bond denaturation area 10 and the nucleic acid hybridization area 20. The energy source can be an electric heater, microwave, laser or light.
To achieve the above-mentioned and other objects, features and advantages of the invention clearer and easier to understand, several practical examples are described in detail below, together with attached figures: