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Genetic recombination and genetic engineering
 

Genetic recombination and genetic engineering

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  • 質體 可以由一種宿主傳到另一種宿主菌,因此抗藥性也可以因此傳播開來,使得很多細菌很快對抗生素產生抗藥性。 遺傳工程也是利用質體的這種傳播功能,把一個被修改過的重組質體,放到宿主細菌中去表現,生產我們所要的物質。
  • 質體 用限制脢切開後,可以插入各種外來的 DNA ,形成重組質體,後者可以轉形到宿主細胞中。 但是,一個細菌只能接受一種重組質體,而這個轉進去宿主的質體,可以在細菌中大量複製相同的質體。
  • 質體 在細菌中會大量複製,大約每一隻細菌可以累積 5~200 個質體 ( 以 100 計算 ) ;若把這些轉形菌途佈在培養皿上,則由單一細菌所形成的菌落,將只含有一種轉形菌,也就是只含有一種重組質體。 每一個菌落若有 1000 隻細菌,而每隻細菌含有 100 個重組質體,則每個菌落就有 1×100×1000 個均質的純系重組質體,沒有雜夾其他的質體;因為一個細菌只能包容一種質體,而一個菌落只有一種細菌。 因此這種 分子群殖 (molecular cloning) 手法,有點像在『 種植分子 』,把一個目標基因,經過上述的 純化 與 放大 的過程,得到數目眾多的複製 DNA ,可以抽取出來應用。 最後所得到的群落,可以用探針進行雜合反應,挑選出含有目標基因的菌落 (N3-13) 。

Genetic recombination and genetic engineering Genetic recombination and genetic engineering Presentation Transcript

  • Chapter 6 Genetic Recombination and Genetic Engineering
  • • In 1973, Herbert Boyer and Stanley Cohen successfully created the first recombinant DNA molecule. Their research work proved that it is possible to change the genotype of organisms artificially. In 1977, Walter Gilbert and Frederick Sanger worked out methods to determine the sequence of bases in DNA. All of these progresses make it possible to isolate and identify a gene, and induce the gene in host cells to express functional protein.
  • • The genetic endowment of organisms can now be precisely changed in designed ways. The development of recombinant DNA techniques has revolutionized biology and is having an increasing impact on clinical medicine. It offers a rational approach to understanding the molecular basis of a number of diseases, for example, new insights are emerging into the regulation of gene expression in cancer.
  • • Clinically useful proteins are now produced by recombinant DNA techniques. This powerful technique is also applied in diagnosis or therapy. And it paves the way to the modern fields of genomics and proteomics. The new opportunities opened by recombinant DNA technology promise to have broad effects.
  • 1. Concepts Involved In DNA Recombination
  • • A clone is a large population of identical molecules, cells or organism that is descended from a single individual through asexual reproduction and genetically identical to a single common ancestor. A DNA clone is a large number of identical DNA molecules produced from a single DNA molecule through an asexual process. Molecular clone in the genetics sense refers to DNA clone. • 1.1 DNA clone
  • 1.1.1 DNA Cloning • Cloning is the process of asexually creating identical copies of an original. DNA cloning(molecular cloning) refers to the production of large number of identical DNA molecules and usually involves the use of bacterial cells as host for the DNA.
  • • DNA cloning involves the preparation of a specific gene or DNA segment from a larger chromosome, insertion of it to a carrier DNA, and then introduction of the modified DNA to host cells, selection of the transformants containing the target genes. The result is selective amplification of a particular gene or DNA segment. So DNA cloning is also called gene cloning or DNA recombination.
  • • The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology or, more informally, genetic engineering. Genetic engineering, proteomic engineering, enzymatic engineering, and cellular engineering constitute biotechnology.
  • 1.2 Different enzymes are used in DNA recombination • The discovery of two types of enzymes permitted the now common technique of DNA cloning. One type of enzymes, called restriction enzymes, cuts the DNA from any organism at specific sequences of a few nucleotides, generating a reproducible set of fragments.
  • • The other type of enzymes , called DNA ligases, can ligate DNA restriction fragments, producing recombinant DNA. In different situations, some other enzymes may also be needed for DNA recombination (Table 6-1).
  • Enzyme( s) Function restriction endonucleases recognize specific nucleotide sequences and cleaves the DNA within or near to the recognition sequences DNA ligase covalently attaches a free 5'-phosphate group to a 3'- hydroxyl group DNA polymerase I synthesizes DNA klenow DNA polymerase proteolytic fragment of DNA polymerase that lacks the 5'-3' exonuclease activity reverse transcriptase CRT) synthesizeds DNA with RNA as template alkaline phosphatase removes phosphates from 5'ends of DNA molecules terminal transferase catalyzes the attachment of any dNMP to the 3'end of DNA Table 6-1 Common enzymes used in recombinant DNA technology
  • 1.2.1 Restriction enzymes • Restriction enzymes, also called restriction endonucleases, recognize, bind to specific sequences in double-stranded DNA, and cleave the DNA. They are usually isolated from bacteria. The role of these enzymes in bacteria is to "restrict" the invasion of foreign DNA by cutting it into pieces.
  • • Hence, these enzymes are known as restriction enzymes. The cell's own DNA is not degraded, because the sites recognized by its own restriction enzymes are methylated. Many restriction enzymes have been purified and characterized.
  • • The names of restriction enzymes consist of a three-italic-letter abbreviation for the host organism. For example, restriction enzyme EcoR is from Escherichia coli.Ⅰ The first three letters in the name of the enzyme consist of the first letter of the genus (E) and the first two letters of the species (co), which are followed by a strain designation (R) and a roman numeral ( )to indicate the order ofⅠ discovery.
  • • There are three types of restriction enzymes, designated , ,andIII. Types and containⅠ Ⅱ Ⅰ Ⅲ the activities of both the endonuclease and methylase. Type restriction enzymes cleaveⅠ DNA at random sites. Type restrictionⅢ enzymes cleave the DNA about 25 bp from the recognition sequence. Both types of enzymes require ATP for energy supply.
  • • Type restriction enzymes, require noⅡ ATP, and usually cleave the DNA within the recognition sequence itself. So typeⅡ restriction enzymes have extraordinary utility in DNA recombination.
  • • Many type restriction enzymesⅡ recognize specific sequences of 4 to 6 base pairs and cleave a phosphodiester bond in each strand in this region. One unique feature of restriction enzymes is that the nucleotide sequences they recognize are palindromic, or inverted repeats. It cuts one strand of the DNA double helix at one point and the second strand at a different, complementary point.
  • • For example, the sequence recognized by a restriction enzyme EcoR is GAATTC.Ⅰ In each strand, the enzyme cleaves the GA phosphodiester bond on the 5' side of the symmetric axis. The arrow indicates the cleavage site.
  • If the cleavage site is not at the center, the restriction enzyme ( e.g., EcoR ) will generateⅠ cohesive ends(sticky ends), which can base- pair with other DNA fragments cleaved by the same restriction enzyme. If the cleavage site is at the center, the restriction enzyme (e.g., Hpa ) will generate blunt endsⅠ
  • • Any two pieces of DNA containing the same sequences within their sticky ends can anneal together and be covalently ligated together in the presence of DNA ligase. Any two blunt-ended fragments of DNA can be ligated together irrespective of the sequences at the ends of the duplexes.
  • Table 6-2 Commonly used restriction enzymes The specificities of several of these enzymes are shown in Table 6-2.
  • 1.3 The interested gene • The purpose of recombinant DNA technology is to clone a gene or DNA fragment of interest or to obtain the product of the gene-protein. The interested gene in DNA recombination is the gene or a segment of a gene to be studied, or to be cloned, or to be expressed in recipient cells. The interested gene to be prepared could be either cDNA or genomic DNA.
  • • cDNA is the single strand DNA complementary to an mRNA, or double- stranded DNA with one strand complementary to an mRNA. • Genomic DNA refers to any DNA fragment coming from the genome of a cell or an organism. • In the process of DNA cloning, recombinant DNA is constructed by ligating a vector with a cDNA or genomic DNA, which is what we are interested in. Any interested gene can be cloned once it is introduced into a suitable vector for transforming a bacterial host.
  • 1.4 Vectors • Molecular cloning involves the amplification of a given DNA molecule by replication in a host cell. However, the DNA fragment to be amplified does not have an origin of replication, it must be inserted into a carrier which can be replicated in bacterial cells and passed to subsequent generations of the bacteria.
  • The ideal cloning vectors display the following properties: • (1) they are not integrated into the host genome so that they are easily isolated and purified; • (2) it is easy for them to enter into host cells because of their small size;
  • • (3)they contain an origin of replication that allows them to be replicated independently in host cells; • (4) they contain one or more selectable markers that allow the easy identification of the host cells harboring them; • ( 5)they have single restriction sites at which foreign DNA can be inserted.
  • • Such a carrier, termed “vector”, is usually a DNA molecule which has a suitable origin of replication. If a vector is used for amplification of a DNA fragment, it is called "cloning vector". If a vector is used for the expression of a given gene, it is called "expression vector". The vectors used for cloning are derived from naturally occurring bacterial plasmid and bacteriophage.
  • • Plasmids are autonomous extrachromosomal replicons found commonly in prokaryotes. They are circular duplex DNA molecules occurring naturally in some bacteria and ranging in size from 1 to 300kb. 1.4.1 Plasmids
  • • They carry genes for antibiotics- resistance, and can be replicated independently in host cells. Any DNA fragment can be inserted into a plasmid at one or two given restriction sites, and replicated along with the replication of the plasmid.
  • • Many plasmids have been ingeniously modified to enhance the delivery of recombinant DNA molecules into bacteria and to facilitate the selection of bacteria harboring these vectors. Most plasmid vectors in current use have a multiple cloning site, a cluster of unique restriction sites which provide a convenient position for the introduction of donor DNA prepared by using a variety of enzymes.
  • • One of the most useful plasmids for cloning is pBR322 (Figure 6-1), which contains genes for resistance to tetracycline and ampicillin. Different restriction enzymes can cleave this plasmid at a variety of unique sites, at which DNA fragments can be inserted.
  • Figure 6-1 Plasmid pBR322
  • • Transformation becomes less successful as plasmid size increases, and it is difficult to clone DNA segments longer than about 15kb when plasmids are used as the vector.
  • • Another group of plasmids in common use is pUC series(Figure 6-2), which contain a multiple cloning sites recognized by several different restriction enzymes, an ampicillin-resistance gene (ampr )for selective amplification, and a replication origin ( ori ) for replication in the host cell.
  • Figure 6-2 Plasmid pUC18
  • • Bacteriophage λvectors are used for DNA library construction because they have a greater capacity than plasmids. The middle segment of a phage DNA is not essential for productive infection and can be replaced with foreign DNA, thus λ phages designed for cloning have been constructed.
  • • M13 phage is another very useful vector for cloning DNA. It is especially useful for sequencing the inserted DNA. That is can be easily sequenced.
  • 2. Basic Principles Of DNA Recombination The basic process of DNA recombination includes several major steps: • (1) preparation of the DNA fragment of the interested gene; • (2)selection and preparation of a suitable vector;
  • • (3) ligation of DNA fragment with vector; • (4) transformation of host cells with recombinant DNA; • (5)screening the host cells harboring the recombinant DNA; • (6) identification of recombinant DNA. Figure 6-3 illustrates the process of DNA cloning using plasmid as vector.
  • Figure 6-3 The process of DNA cloning using plasmid as vector
  • 2.1 Preparation of the interested gene • The fragments of the interested gene for DNA recombination include genomic DNA, cDNA, PCR product and chemically synthesized DNA fragment. Different strategies should be chosen for the preparation of DNA fragments of the interested gene according to different purposes.
  • • If the sequence of the DNA fragment to be cloned is known, at least the flanking parts, the DNA fragment can be prepared using the polymerase chain reaction (PCR). The amplified DNA fragment can be cloned directly.
  • 2.2 Selection and preparation of vector • The selection of vector is dependent on the purpose of the construction of recombinant DNA. For the cloning of DNA fragment, a cloning vector should be chosen. For the expression of a gene in the given host cell, a suitable expression vector should be chosen according to the host cell. Many expression vectors have constructed for the expression of gene in different cells such as mammalian cells , bacterial cells, yeast and so on.
  • 2.3 Ligation of DNA molecules • After digestion with restriction enzyme(s) , different DNA fragments(including linearized vector) can be ligated by DNA ligase which fills in the nicks by catalyzing the formation of the phosphodiester bonds. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs.
  • • The ligation of DNA fragment with sticky ends is facilitated by the annealing of complementary sticky ends. The fragments digested by the same restriction enzymes generally will link together through base- pairing so that it is very easy for DNA ligase to fill the nicks.
  • • DNA fragments with same sticky ends(compatible ends) are ligated in this way, even if they are digested by different restriction enzymes. For example, a fragment generated by Mbo (▼ GATC)Ⅰ will link to a fragment generated by BamH (G▼ GATCC).Ⅰ
  • • The simplest strategy for cloning is thus to cut the donor and vector DNA with the same restriction enzyme and join them with DNA ligase. However, this allows the vector to reclose without an insert. There are one procedures which prevent vector self-ligation:
  • • the vector and donor can be prepared by digestion with same pair of restriction enzymes so that both vector and donor themself will have two incompatible ends, but compatible ends are existent between vector and donor. A further advantage of the second strategy is that the orientation of the insert can be predicted (directional cloning).
  • • Sticky-end ligation is technically easy, but sticky-end sites may not be available. To circumvent these problems, the strategy of ligation with blunt ends is used. This technique has the advantage of joining together any pair of DNA fragments without suitable restriction sites, albeit less efficiently and nondirectionally.
  • • Blunt ends can be generated by filling or trimming overhanging termini. The disadvantages are that there is no control over the orientation of insertion or the number of molecules annealed together , and there is no easy way to retrieve the insert.
  • 2.4 Introduction of recombinant DNA into host cells • The recombinant DNA molecules are then introduced into host cells, which amplify the DNA molecules in the course of many generations of cell division. Plasmids can be introduced into bacterial cells by a process called transformation. Bacterial cells are first treated with calcium chloride( CaCI2 )to make the cell wall more permeable to DNA. These cells are said to be competent, and some of them can take up DNA-Ca2+ complex.
  • 2.5 Screening and identification • Neither DNA manipulation nor gene transfer procedures are 100 efficient, so it﹪ is desirable to identify the recombinant population. A method is needed to select those that do. This process is termed screening or selection. There are direct and indirect selection systems employed depending on the vector.
  • • The gene or genotype can be detected directly based on some mark genes and target gene. The usual strategy is to use a plasmid that includes a gene that the host cell requires for growth under specific conditions, such as a gene that confers resistance to an antibiotic.
  • • Only cells transformed by the plasmid can grow in the presence of antibiotic. Cloning vector often contains genes that confer resistance to different antibiotics(tetr , ampr or kanr ), allowing the identification of cells that contain the intact plasmid or a recombinant version of the plasmid because those bacteria with the antibiotic resistance gene will survive, whereas those without it will die. antibiotics: a chemical substance derivable from a mold or bacterium that kills microorganisms and cures infections
  • • Ideally, cloning sites are placed within a selectable or visible marker gene to facilitate recombinant selection. The insertion of DNA into a functional region of the vector will interrupt an essential function of the vector. This concept provides a selection technique of insertional inactivation.
  • • For example, the common plasmid vector pBR322 has both tetr and ampr genes. Insertion of DNA at the Hind ,Ⅲ Sal , orⅠ BamH restriction site inactivates theⅠ tetr gene. Cells containing pBR322 with a DNA insert at one of these restriction sites are resistant to ampicillin but sensitive to tetracycline. Cells that failed to take up the vector are sensitive to both antibiotics, whereas cells containing pBR322 without a DNA insert are resistant to both. And so they can be readily selected.
  • • Specific DNA sequences are detectable by marker rescue. Insertional inactivation can therefore be used to distinguish clones of plasmid that contain an insert. A similar strategy is blue-white selection. Plasmids carry a nonfunctional, truncated allele of the LacZ gene, which encodes a N-terminal fragment of the β- galactosidase protein termed the α peptide.
  • • This can be complemented by an allele encoding the remainder of the polypeptide, which is found in specially modified host strains such as E.coli JM101. Functionalβ-galactosidase converts the colorless substrate X-gal into a blue precipitate. The lacZ gene contains multiple restriction site. Therefore recombinant cells form white colonies and non-recombinants form blue colonies on the appropriate detection media.
  • • Specific DNA sequences are detectable by PCR. If the sequence of the cloned DNA is known, the primers can be designed. Then the PCR product will ensure the foreign DNA in the transformants.
  • • Specific DNA sequences are detectable by hybridization. Cells containing particular DNA sequences can be identified by DNA hybridization. There are many variations of the basic method, most making use of a labeled probe, complementary to the DNA being sought. In one classic approach to detect a particular DNA sequence within a DNA library, nitrocellulose membrane is pressed onto an agar plate containing many individual bacterial colonies from the library.
  • • The membrane is treated with alkali to disrupt the cells and denature the DNA which remains bound to the region of the membrane around the colony from which it comes. The radioactive DNA probe anneals only to its complementary DNA. After any unannealed probe DNA is washed away, the hybridized DNA can be detected by autoradiography (Figure 6-5).
  • Figure 6-5 Use of hybridization to identify a clone with a particular DNA segment
  • • Specific DNA sequences are identified by DNA sequencing. The ultimate characterization of a cloned DNA is to determine its nucleotide sequence. Transformants can be identified by sequencing until a bacterial colony containing a plasmid with the foreign gene is found.
  • Drug Resistance Gene Transferred by Plasmid Plasmid gets out and into the host cell Resistant Strain New Resistance Strain Non-resistant Strain Plasmid Enzyme Hydrolyzing Antibiotics Drug Resistant Gene mRNA Juang RH (2004) BCbasics
  • Target Genes Carried by Plasmid 1 plasmid 1 cellRecombinant Plasmid Transformation Target Gene Recombination Restriction Enzyme Restriction Enzyme ChromosomalDNA Target Genes DNA Recombination Transformation Host Cells Juang RH (2004) BCbasics
  • Amplification and Screening of Target Gene 1 1 cell line, 1 colony X100 X1,000 Plasmid DuplicationBacteria Duplication Plating Pick the colony containing target gene =100,000 Juang RH (2004) BCbasics
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