Definition
4-Dimension Hybridization(4DH) technology of nucleic acid[ 1 ], namely is the hybridizations technique established on the 4 dimensional space with temperature parameter, which was set up by introducing temperature factor (attributed to environment requirement) onto the basic of 3 dimensional space for conventional nucleic acid hybridization (XYZ attributed to the sizes of DNA fragments, buildup and sequence of basic groups) as a 4th parameter.
Background
Genome refers to the summation of all genetic information in human cells. Genome information is carried by deoxyribonucleic acid (also known as DNA). DNA of genome is a double-chain polymer composed of 4 types of basic groups (A, G, C and T). Every single chain of nucleotides of this double-chain structure has ribose phosphates as its backbone. The sequence of its basic groups is referred as the first level of sequential structure of DNA. The two chains of DNA of Genome are joined by the hydrogen bonds between interactions of complementary bases. The chemical structure of basic groups has shown that the interaction between A and T forms two pairs of hydrogen bonds (do not interact with other basic groups), and that the interaction between G and C forms three pairs of hydrogen bonds (do not interact with other basic groups). The intrinsic internal double-chain of DNA and the accuracy of interactions among basic groups are utilized during the process of hybridization. Hybridization is a process in which two complementary single-chain nucleotides forming a stable double-helix double-chain structure. Hybridization reaction can occur between two complementary chains in solution, or it can occur between a chain in a solution and a complementary chain fixed in a solid phase carrier. DNA chip analysis utilizes the single-strand chain with fluorescent marking to undergo hybridization reaction with the other single-strand chain sequence on the DNA chip and is a biochemical process of nucleic acid hybridization, which is the principle basis of DNA chip (or genetic chip and also known as DNA micro array or genetic micro array).
Genetic Chip (or also known as DNA chip) technology is based on the principle of complementarity of basic groups, hybridization reaction with testing DNA (or gene) is carried out on solid chip surface (several square millimeters) according to a certain permutations and combinations of array to integrate large amount of DNA probes, which is a technology undergoing parallel and fast analysis testing on large amount of DNA (or genes).
PCR technique is the thermocycle process that according to complement sequence of the target nucleic acids the new oligonucleotide strand is being made by the polymerase from one by one bases on the primer 3’, after the artificial oligonucleotide primers have hybridized with the target single-strands under the optimal annealing temperature or hybridizing temperature. After several tens cycles of amplification the target nucleic acids chains will be increased by millions times.
Therefore the hybridization reactions of nucleic acids(namely Southern hybridization between DNA and DNA strands, Northern hybridization between DNA and RNA strands) are the basic platform of nucleic-acid Molecular Biology and Gene Diagnostics technology.
Problem
The traditional concept thinks Southern or Northern hybridization of nucleic acid is base pairing with absolutely specific one vs one, however, it is not so perfect in fact. When there is deviation of hybridization temperature, one or two bases mismatch, or the condition of degenerate energy level, the oligonucleotide strands can also form the chains of hybridization pair.
Research and Development of PCR reagent kit are usually done by optimized experiments according to pure and single sequence DNA. While facing the coverall clinic samples, because the extracted nucleic acid is often the total nucleic acid, thereinto if the clinic samples present nucleic acid sequences which are isogenous sequences or approximate isogenous sequences with primers and probes (mismatch one or two bases) or provided with degenerate energy level, PCR will generate non-specific false positive signal.
DNA Chip technology is another hot one in connection with Gene after PCR. There are more than the decade for DNA Chip technology developing and but it still can not be utilized on clinical diagnosis up to now. Why? By the clinical testing experimenter’s saying, very poor reduplications of the results. Why very poor reduplications? It is cause that the traditional hybridization or Southern hybridization——by single hybridization temperature (often at 40ºC or 60ºC), are utilized on the common DNA Chip technology. There is a large number of probes with different permutation and combination bases on DNA Chip. One kind of probes with specific permutation and combination is equivalent to the sole Tmp——relevant to its most optimal hybridization temperature of perfect match double-chain complex. Tmp can not be able to be calculated with definitiveness(the quantum mechanics equation has no ratiocinative solution) and can be measured by the experiment optimal screening on the traditional method. That the hybridization temperature can not match with Tmp may cause that there may be one, two or more mismatch bases between the probe and sample single-strand chain. On common DNA Chip sample single-strand chain first is marked with fluorescein and then hybridized with the probe under homogeneous temperature. The judgement of positive or negative signals is by the measured intensity of fluorescence that is higher than certain budgetary fluorescence intensity for positive signal and lower than one for negative signal. Whereas, if there are enough number of sample single-chain with one or two mismatch bases and the hybridization temperature is lower than normal one, the result may have some false-positive signals. If the hybridization temperature is highter than normal one some probes with the lower Tmp can not be able to hybridized with sample single-chain, the result may have some false-negative signals. It is the defective limitation for the Southern hybridization and also for the Northern and in-situ conventional nucleic acid hybridization.
Just because of there is a number of false-positive signals in the traditional hybridizations technique it results in that there is a number of false-positive signals in PCR analysis and also in DNA finger-print analysis. These defective problems can be able to be overcome by the 4 dimensional hybridizations technique (4DH).
Solving Method
From the standpoint of basic theory study, due to no ratiocinative solution of quantum mechanics formula, there is no definitive formula capable of calculating the temperature value of melting point (Tmp) of DNA fragment. The approximate estimated value is generally very different than the actual value. In the actual application, accurate value must be known in order to determine the most optimal hybridization condition. Therefore, many manufacturers and companies developing products have to do one after one of optimized experiments to obtain the most optimal condition. On the other hand, 4DH DNA Chip presents a convenient method of determining the melting point temperature for large amount of oligonucleotide fragments, which brings great convenience and efficiency not only for scientific research personnel but also for diagnostic reagent manufacturers. Now, all genetic diagnostic reagents required probes and primers (PCR technology requires primer, and the existing fully automatic genetic sequencing apparatus needs primer as well)——These are all oligonucleotide fragments, and their melting point temperature (Tmp) are very important to the genetic diagnostic reagent kits.
The application of 4DH technology must be used by the 4DH reagents[ 2 ]. The last used method that the melting curve was analyzed immediately by the temperature scanning after real-time PCR process results in that the reduplications of the characteristic melting point temperature (Tmp) scanning process for the single same reaction vessel are very poor. But the new method that the temperature scanning is made by adding 4DH reagent after real-time PCR procedure results in that the reduplications of the characteristic melting point temperature (Tmp) scanning process for the single same reaction vessel are very good and the analysis of the melting curve for the results of PCR TaqMan Probe reagents could be made in order to solve the problem of non-specific false positive signals. Because the reagents of the generic commercial PCR kits are optimal for the amplifying reactions of the polymerase by the optimizing reaction conditions for the specificity of the polymerase(Taq enzyme), and maybe not optimal for the one of the hybridization reaction of nucleic acids. The 4DH reagents are optimal for the hybridization reaction of nucleic acids by the optimizing reaction conditions for the specificity of the oligonucleotide fragments hybridization reaction and more suitable for the hybridization reaction of nucleic acids. The application of the 4DH reagents to the analysis of Gene Chip(or DNA chip) may be the better solving for the problem that the reduplications of the results of the traditional hybridization Gene Chip for the single same reaction vessel are very poor. The 4-Dimension Hybridization DNA Chip system from the designing according to the reference patent may analyze the clinic sample by not marking target oligonucleotide chains and it simplifys the processing of sample, and more suitable for the clinic applications. About the more details of it, please see the literature of the patent(US Patent: US7,101,671).
Between Traditional 3DH and Innovational 4DH
If someone says that the hybridization technology of nucleic acid is the basic platform of Molecular Biology it is the truth. If building the house of Molecular Biology on the technical platform for the traditional 3D hybridization of nucleic acid it would like to making the house on the sand beach. It is no problem to build the flat house or even the two and three storied building. But if one wants to build the skyscraper of Molecular Biology on the basic platform of the sand beach it will collapse and it must be making on the massiness basic platform of the granite. The innovational 4DH of nucleic acid is the platform of the granite that will be need for building the skyscraper of Molecular Biology. The traditional 3DH is the especial example of the 4DH technology. The reduplications of the results of the 4DH are more excelled than the ones of the 3DH. This reduplication means that the reduplications between the results on different times, or at different laboratorys, or by different manipulators or in different kinds of reactions and does not mean that the reduplications between the results on the same time, or at the same laboratory, or by the same manipulator or in the same kind of reactions. Our meaning of the reduplications is the repeats of the objective rule of the hybridization reaction. This is the commercial and technological value of the 4DH technique. Whether or not one kind of technology is a mature one the reduplication of its results is a very important criterion. The high technology may be changed to the repeat-used productivity on the daily life or industry and could be benefit for humanity only under the reduplications of results on the practice application are very good.
Note:
1.China Patent: CN1164769; US Patent: US7,101,671; Internation Patent: PCT/CN02/00751.
2.4-Dimensional Hybridization Reaction Reagent Kit manual.