A New Method For Detecting Single Base Variation Of DNA Based On Molecular Beacon Fluorescence

The human is a complex open system and disease is the disaster of life. Human diseases are often due to their genetic factors, environmental factors, or is the result of a combination. If we can determine the cause of disease and its property, it can be prevent and treat early. The molecular biological technologies for locating and identifying human genes is creating new opportunities for advances in our understanding of human disease. Single nucleotide polymorphism is at the genetic level the variation of the DNA sequence polymorphism caused by a single nucleotide mutation, it belongs to one of the most common human genome variation types. SNPs with the advantages of high accuracy, large amount of information and easy detection in human evolution, migration, disease research and other fields shows important application value. At present, the SNPs detection methods include mass spectrometry, surface plasmon resonance method, fluorescent method and electrochemical biosensor method, etc. At home and abroad in recent years, it can use the base analogues to recognize dsDNA’ mismatch bases, such as some molecule, because these small molecules themself has fluorescence which can be tested, and can be used as a potential targeting drug to predict disease.Molecular beacon in accordance with the principle of complementary base pairing and fluorescence resonance energy transfer is cleverly designed, combined with fluorescence method has achieved significant results in different fields.This paper consists of two parts:the first part is the review, and the second part is the research report. The first part is review which mainly introduces the concept of single nucleotide polymorphisms and detecting methods, molecular beacon, some properties of graphene oxide and the way of interaction of DNA with small molecules, and finally introduces the research purpose and content of this paper.The second part is the research report which consists of two parts. The first part is to use small molecule ATMND combined with fluorescence method to identify the G-C single-base mutation. We set up a fluorescence analysis method which combines conbines small molecule ATMND and molecular beacon to identify G-C mutation in the specific sequence form P53gene. The5’and3’end of classic molecular beacon are tagged fluorescent group of FAM and quencher of TAMRA respectively, Because of fluorescence resonance energy transfer, the fluorescent of fluorescence group was quenched. When the target sequence is added to a system, the circular part of the molecular beacon Interact with target sequence which leads to confromnational change. Because the distance between fluorescence group and quencher increases, the fluorescent of fluorescence group recovery. The target sequence will hybrid partly with molecular beacon when the target sequence contained G-C mutation is added to the system, and the fluorescent of fluorescence group also recovery. The fluorescent intensity of system is weaker than the target sequence which does not contain G-C mutation for its double-stranded DNA structure is not relatively stable. On this basis, a certain concentration of ATMND that can specificitily recognize C bases is added to the system so as to improve the stability of the DNA double helix structure in the system, so that the molecular beacon’s fluorescence intensity is further increased and may even reach to the fluorescence intensity of the system. It is based on the change trend of fluorescence intensity when small molecule is added before and after. We have realized specific G-C single-base mutation from the P53gene in the sequence.The second part of the paper introduces that a small molecule and abasic site of molecular beacon fluorescence method are used to identify MicroRNA. Work molecular beacon containing abasic site and small molecules Amiloride together to identify T bases and be further used oidentify MicroRNA. Molecular beacon of annular part comes from a complementary sequence of P53DNA sequence and in the annular part we design abasic site. The5’and3’end of classic molecular beacon are tagged fluorescent group of FAM and quencher of TAMRA respectively. Due to molecular beacon and target sequence hybrid, fluorescent group FAM and quencher TAMRA are separated when four P53target sequences which contained A, T, C, G bases of abasic site are added so that the fluorescent of fluorescence group recovery. On this basis, a certain concentration of Amiloride that can specificitily recognize T bases is added to the system When forming double-stranded DNA’s abasic site across the site is T base, fluorescence intensity of system is higher than abasic site across A, C, G base. Finally, we use the method to identify MicroRNA associated with cancer and diseases of the let-7in a family of let-7a, let-7f, let-7g and set up a new method to distinguish different MicroRNA which is used small molecular specific recognition for bases.