CN1287176A

CN1287176A - Method of modifying and cloning nucleic acid target molecule

Info

Publication number: CN1287176A
Application number: CN 00111339
Authority: CN
Inventors: 吴昌; 吴明
Original assignee: 吴昌; 吴明
Current assignee: QINGDAO JING PU BIOLOGY TECHNOLOGY LTD.
Priority date: 2000-09-12
Filing date: 2000-09-12
Publication date: 2001-03-14

Abstract

The method of the present invention features that the complementary target molecule has a 3' specific end part and non-specific 5' end part. The specific 3' end part has specific complementation with the target molecule to be cloned and is made to hybridize with target molecule in set conditions. The 5' end part is used as the template of common sequence and is replicated by the 3' end of the target molecule. The PCR cloning utilizes non-specific primer. The present invention can simplify molecule testing process.

Description

The method that nucleic acid target molecule is modified and increased

The present invention relates to a kind of method of modification and amplification of nucleic acid target molecule.

When nucleic acid is comprised that thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA) carry out Molecular Detection, need the target molecule in the nucleic acid samples is selectively increased (copying a plurality of same a part).Target molecule is the nucleic acid fragment that must detect, the hereditary feature of organism under its constitutional features can be represented.Target molecule can be DNA, RNA or at the external DNA that is become through reverse transcription by RNA.After amplification obtains a large amount of target molecules, can measure the constitutional features of target molecule with Protocols in Molecular Biology, thereby the hereditary feature of the organism (patient) of the nucleic acid samples that is provided is provided.At present, the target molecule in the nucleic acid is increased to adopt polymerase chain reactions (PCR) more.When a double stranded DNA target molecule is carried out polymerase chain reaction, two the single-chain nucleic acid small molecules narrow spectrum to the target molecule tool, that direction is opposite, i.e. introductions be arranged.Introduction is the dna molecular by chemical process synthetic strand, can carry out molecular hybridization with target molecule, and its length is generally the 10-50 base.Molecular hybridization is to be undertaken in conjunction with the process that forms double chain acid molecule by the base pairing principle between the single stranded nucleic acid molecule, and the relation of two single chain molecules on structure (sequence) that can carry out molecular hybridization is complementary relationship.The complementary target molecule be meant can with the nucleic acid molecule of target molecule hybridization.A molecular chain in introduction and the target molecule is hybridized, and duplicates target molecule selectively under the effect of polysaccharase, and this process can obtain a large amount of target molecules repeatedly.Know that now causing a kind of gene locus of inherited disease to change can be more than one, that is to say, in the time of detecting one or more inherited diseases, the target molecule kind number of required amplification can reach hundreds of more than.Described gene locus is meant base position specific in the nucleic acid construct.Gene is the division of nucleic acid function or the nucleic acid fragment of the certain function of tool, tool certain structure feature.As with conventional PCR method, need synthetic a large amount of introduction when carrying out Molecular Detection, and respectively target molecule is screened and increase with a large amount of reactions.The needed man power and material of this way, expense are all very big.When a plurality of introductions being combined in PCR reaction, the interaction between these introductions can produce by product, thereby influences the amplification of target molecule.

The purpose of this invention is to provide and a kind of a plurality of different nucleic acid target molecules are optionally modified the method that is increased then, it can overcome the above-mentioned deficiency of prior art.

The method that a kind of nucleic acid target molecule is modified and increased, comprise that nucleic acid molecule is carried out fragmentation to be handled, make fragmentation 5 ＇ of molecular chain terminal coupled with known nucleic acid molecule chain, nucleic acid target molecule after coupled is under the effect of complementary target molecule and polysaccharase, with its 3 ＇ end is the 5 ＇ end parts that introduction duplicates the complementary target molecule, last target molecule with the effect of the corresponding introduction of the new sequence in two ends under increased through polymerase chain reaction, it is characterized in that the complementary target molecule has specific 3 ＇ end parts and non-specific 5 ＇ end parts, 3 ＇ terminal specifics part has single-minded complementary relationship with the target molecule of required amplification, under the reaction conditions of setting, hybridize with target molecule, 5 ＇ ends of complementary target molecule are divided into consensus sequence and are duplicated by 3 ＇ ends of target molecule as template, target molecule through with the complementary target molecular hybridization before and after secondary modification after, increase through PCR with nonspecific introduction.

Advantage of the present invention is to carry out selective modification to a large amount of target molecules simultaneously, increase with the target molecule of a small amount of several general introductions then to a large amount of kinds, can simplify the Molecular Detection process, use manpower and material resources sparingly, reduce and utilize nucleic acid to carry out the cost of Molecular Detection.After with present method target molecule being increased, the constitutional features of target molecule can be by hybridization, sequencing, and molecular mass, molecular size, the quantity of electric charge and molecular weight ratio etc. is measured, and is specially adapted to the specimen preparation in the biochip use.Present method can be used for measuring animal, plant, the genotype or what of its nucleic acid amount of microorganism and virus.Use present method and can make the test kit of commercial value, or be engaged in the molecular diagnosis service.

Below by embodiment the present invention is described.

But a suspect is carried out genotype identification, and compare with the genotype of the blood sample of collection in worksite.From these two samples, extract nucleic acid respectively, with restriction enzyme the nucleic acid of 0.1 microgram in each sample being carried out fragmentation handles and obtains small molecules, make it to carry out coupling reaction with known method with a double-stranded nucleic acid, thereby obtain single chain molecule fragment at the terminal lengthening of 5 ＇, be target molecule fragment, this is to modify for the first time.Kind of complementary target molecule surplus adding 100 is then hybridized itself and target molecule, makes complementary target molecule 3 ＇ end keep length constant with known chemical process simultaneously.Target molecule is relevant with sex, the colour of skin, color development, face type etc.The forebody of complementary target molecule (5 ＇ extreme direction) has about 20 bases of a common sequence long, and its latter half of (3 ＇ extreme direction) has narrow spectrum with specific target molecule complementary sequence (about 20 bases) mutually.3 ＇ of the target molecule after hybridization end can be removed 3 ＇ terminal portionss of strand under the effect of the 5 prime excision enzyme activity of polysaccharase, duplicate 5 ＇ terminal sequences of complementary target molecule then.This is to modify for the second time.Through as above process, have only target molecule possessed two modified and common sequence.At last the target molecule of kind surplus 100 is carried out pcr amplification with two public introductions simultaneously.Target molecule after the amplification is labeled, and hybridizes with molecular probe on gene chip then, and the position that records signal at last is with strong and weak.By the signal that records examinee's genotype as can be known, thereby learn its morphological specificity.But the gene as on-the-spot blood sample is consistent with suspect's genotype, but can conclude that then the suspect must be at the scene; If genotype is inconsistent, can go to seek related person by the form that genotype is inferred.

Claims

1. a nucleic acid target molecule is modified and the method for amplification, comprise that nucleic acid molecule is carried out fragmentation to be handled, make fragmentation 5 ＇ of molecular chain terminal coupled with known nucleic acid molecule chain, nucleic acid target molecule after coupled is under the effect of complementary target molecule and polysaccharase, with its 3 ＇ end is the 5 ＇ end parts that introduction duplicates the complementary target molecule, last target molecule with the effect of the corresponding introduction of the new sequence in two ends under increased through polymerase chain reaction, it is characterized in that the complementary target molecule has specific 3 ＇ end parts and non-specific 5 ＇ end parts, 3 ＇ terminal specifics part has single-minded complementary relationship with the target molecule of required amplification, under the reaction conditions of setting, hybridize with target molecule, 5 ＇ ends of complementary target molecule are divided into consensus sequence and are duplicated by 3 ＇ ends of target molecule as template, target molecule through with the complementary target molecular hybridization before and after secondary modification after, increase through PCR with nonspecific introduction.