Molecular cloning technology

A typical gene cloning experiment consists mainly of the following operations and results:

(1) PIECEs of DNA, including the gene of purpose, are inserted into another DNA molecule (clone vector, usually ring-shaped) to form a recombined DNA molecule.

< "9101" > (2) recombined DNA molecules are imported into the subject cells by conversion or other similar methods. E. coli is a more used subject cell.

< "9101" > (3) in the subject cells, clone vectors guide the replication of recombined DNA molecules, producing many identical copies.

(4) When the subject cells divide, copies of the recombined DNA molecules enter the child cells, and replication of the clone vector continues in the subcells.

< a name, "9101" > (5) A large number of divided subject cells form clones: a group of cells, each of which contains copies of many recombined DNA molecules.

< is a name " 9101" > it is obvious that gene cloning is a more intuitive and simple procedure. It is of great biological significance because it can provide us with a pure genetic specimen. Usually, one gene is always with the other genes in the cell. Before gene cloning, we simply couldn't purify individual genes, which meant that we could only study and exploit gene groups, not the structure and function of specific genes.

< name"9101" > 1, recombination of DNA molecules

Building recombined DNA molecules is the first step in a gene cloning experiment, in which a ring-shaped vector is cut off at a specified location and then the DNA molecule containing the gene of destination is inserted into it, linking the two. This process requires two dna-operated enzymes: the restrictive endonucleases and the ligases.

< a name"9102" > restrictive endoenzymes are able to identify specific nucleotide sequences on DNA molecules and specifically cut off DNA molecules there. For example, Pvu I (Bacteria Protus vulgaris separation) identifies and cuts off only 6 nucleotide sequences CGATCG; Many restrictive endoenzymes identify six nucleotides, but there are also restrictive endoenzymes that identify four or five, or even eight, nucleotide sequences. In addition, some restrictive endoenzymes may not be unique in their order of identification, for example, Hinf I can identify and cut off GAATC, GATTC, GAGTC, and GACTC. Therefore, the of Hinf I is also usually recorded as GANTC, N represents any nucleotide in A, T, G, and C.

< name""9102" > There are two types of DNA molecular breaks after restrictive endoenzyme treatment: flat and sticky ends, and their properties have important effects on the experimental design of gene cloning. Wherein, restrictive endoenzymes with different identification points can produce the same sticky ends. For example, Bgl II (AGATCT) and Bam HI (GGATCC) produce the same GATC sticky ends as Sau 3A. Obviously, the DNA molecular fragments treated by the above three enzymes can form complementary double strands at the corresponding break end.

< "9102" > DNA molecular fragments formed by the sticky end of the base complement each other can not be connected, the refore process requires the catalytic action of the connecting enzymes. Connectivity enzymes are produced in all biological cells, but the most commonly used in gene cloning _T4 bacterphages. The connecting enzyme catalytics the formation of phosphate phosphate bonds between adjacent nucleotides. Since the flat end cannot keep the DNA fragments close to each other, the catalytic efficiency of the connecting enzyme to the connection reaction between the flat-end DNA molecules is less efficient than that of the sticky end.

< name" "9102" > 2, clone vector and its main function

vector is the key part of the cloned gene, the vector enables DNA molecules to be copied in the cell. Prolitons and phages are two natural DNA vectors. Currently, there are hundreds of vectors that can be used in different subject cells, of which the largest number can be used in E. coli.

proton pBR322 is a typical E. coli clone vector with a total length of only 4.3kb (usually, it is difficult to completely isolate and purify LARGE MOLECULEs of DNA longer than 50kb). pBR322 comes with two > antibiotics resistance genes: the b-endamidease gene and the tetrycline resistance gene, which modify and eliminate the toxicity of ampicillin to E. coli. In general, the gene of the destination is inserted into the vector prosurfic mass, which destroys the tyrocytin resistance function. Therefore, usingculture base containing ampicillin and tectocycline, we can identify the converted cells of E. coli: conversion cells with recombinative primers can only be found inother hand, primary subject cells cannot grow on media containing ampicillin and tycycline, while transformational cells with carrier primer but no purpose gene can grow on media containing ampicillin and tycycline. In addition, pBR322 is a relaxing type of primer, in the culture of chloramphenicillin can be added to the conversion cells from the usual number of copies of the primer from the usual 15 to 1000-3000, during which time, E. coli chromosomes do not replicate.

In fact, many of the prosulte vectors that are often used today are different from pBR322, and in addition to antibiotic resistance genes, other genes in these prosultes can also be used as select genes. For example, pUC8 carries the ampicillin resistance gene and LacZ ^/ gene. Because the insertion site of the gene of destination is located within LacZ ^/ gene, the operation of screening the transformed cells becomes more intuitive and simple. The primer enables the transformational cells to grow on a culture containing ampicillin, and if the LacZ ^/ gene expression-induced substances IPTG and LacZ ^/ enzyme (b-semi-lactose glycosidease) are added at the same time, then the converted cells with the destination gene appear blue and the converted cells of the destination gene appear white.

< "9103" > In bacteria, phage vectors are another commonly used clone vector. Different from the protons, phage vectors are transducted into the host E. coli cells through the infection process. Usually, phages, as clone vectors, are treated with certain mutations and deficiencies. Therefore, when these phages enter E. coli cells, they do not integrate on the host chromosomes as normal phages do, but go directly into the lysis cycle: a large number of replication phages, cleavage host cells, and eventually the formation of phage spots on the media containing a large number of copies of phages.

screening for phages with the destination gene in a variety of ways, for example, when using a phage vector with the LacZ ^/ gene, the color of the phage spot can be formed on the X-gal media, distinguishing between the recombined phage (recombination) with the destination gene and the vector without the destination gene. Sometimes, recombiners can also be identified simply by the phage plaque pattern formed before and after transducting.

< vectors are able to clone longer pieces of DNA than >"9103" vectors and proton vectors. For example, pBR322 and pUC8 can insert up to 8kb pieces of DNA in the protons, and vectors and other phages can clone up to 25kb of DNA fragments.

< a name," "9103" > 3, the clone carrier of the uerical organism

Generally, E. coli and its protons or phage vectors can fully meet the purpose of isolating and purifying certain experimental genes. However, we sometimes need to use etonucleotic cells instead of E. coli cells as a subject, for example, using gene cloning to control and promote the synthesis of important metabolites (insulin, etc.), changing the specific characteristics of the subject organism (importing insect-resistant properties into food crops, etc.), and so on. At this point, we must choose a clone vector suitable for the nucleocytes.

yeast are commonly used in gene cloning experiments in the uterine bioreceptor cells, yeast culture is as convenient as the cultivation of E. coli. There are also many kinds of yeast cloning vectors. Among them, free plasmid YEps (yeast episomal plasmids), integrated vector YIps (integrative yeast vectors) and artificial chromosomes (yeast artificial chromosomes) are the three most representative yeast cloning vectors. YEps is a rare etonyr cell granules that are 2mm in size and about 6kb in length. The number of copies of YEps in cells is 70-200. The nature of YEps is very similar to that of bacterial granule vectors, the only difference is the screening method for transforming cells. When using YEps, the transformational cells and the affected cells are identified mainly by changes in the nutritional requirements of the subject cells. Because genes cloned from YEps are easily lost in cell generation, YIps are often used instead of YEps. However, as a cloning vector of yeast, YIps is very low in conversion frequency. On the other hand, a typical YACs consist of a silk dot, two telomeres, a replication starting point, and several selective marker genes, which are microsomes. YACs are mainly used to clone long genes or genomic DNA fragments that include several gene sequences. Many important animal genes often contain multiple inclusions that occupy a considerable area of DNA, and it is often difficult to obtain complete gene sequence cloning using common vectors.

< a name " 9104" > In some special cases, we also need to use animal or plant cells as cloned subject cells. For example, cloned genes are imported into food crops to improve their nutritional quality. The commonly used plant cloning vectors are mainly Ti protons and their derivatives, and the commonly used mammalian cloning vectors are mainly carriers altered by E. coli granules or mammalian viruses.

Gene Library is a set of DNA sequences containing all the genes of a particular organism, in which different fragments of DNA sequences are cloned on appropriate vectors. For example, the human gene library is a group of E. coli cells with human gene clones from which we can screen, identify, and study any human gene. The gene library includes a genome library of genomic DNA and a cDNA library of DNA complementary to mRNA. The cDNA library does not contain non-tweed genome sequences (repeated sequences, etc.). The main method of screening and identifying the gene of destination from the genome library is to use various molecular probe means and DNA side sequencer.

< a name is "9105" > 1, build a gene library