What is gene cloning and production of a recombinant molecule?

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1 Recombinant DNA o Recombinant DNA technology brought a huge boost to the study of genetics, because it allowed manipulation of the genetic material DNA. o The ability to directly analyse DNA and manipulate it has increased the power of Genetics immensely. The main problem is that any particular DNA sequence is only present at very low concentration in the total DNA of an organism. E. coli has a genome size of 4.6 Mb and a typical gene is around 1kb (i.e. about 0.02% of total DNA). The situation with the 3000 Mb human haploid genome is even worse. However, it is possible to purify some sorts of small DNA molecule e.g. plasmid or phage DNA. In a 5 kb plasmid, a 1 kb gene is 20% of total DNA and thus amenable to analysis. If a gene from another organism can be incorporated in a plasmid it will be amplified and can be analysed. o It is possible to transfer recombinant DNA into almost any organism and this is the basis for a lot of modern biotechnology. o Much of the insulin used now is produced from recombinant E. coli bacteria. Hepatitis B vaccine is a protein produced in recombinant yeast cells. In the last few years, almost all babies born in Germany have been injected with this vaccine, both to protect from the acute disease and from the chronic problems ending in liver cancer. Erythropoetin (EPO) has been in the headlines because of doping in sport, but is a major treatment for some sorts of anaemia. It is produced from recombinant animal cells. These products have important medical uses, which can not be met by other methods and do not seem to be very controversial now.

2 What is gene cloning and production of a recombinant molecule?! A fragment of DNA, containing the gene to be cloned, is inserted into a second DNA molecule called a vector, to produce a recombinant molecule. (Fig1)! The recombinant DNA molecule is introduced into a host cell.! Within the host cell the vector directs multiplication of the recombinant DNA molecule, producing numerous copies of the required gene.! Copies of the recombinant DNA are passed to the progeny during host cell division.! A large number of cell divisions results a clone formation. Requirements for gene cloning Vector A vector is the vehicle for the genes in gene cloning. It should display several features: " It must be capable of replication within the host cells. " It should be relatively small, ideally less than 10 kb in size. " It must contain at least one gene that facilitates the selection e.g. antibiotic resistance. " It must carry at least one specific recognition site for restriction endonucleases.

3 Fig 1 the basic steps in gene cloning

4 The most important type of vectors are plasmids (yeast or bacteria) and viruses (bacteria or mammalian) Plasmids - It is small, usually circular, extra-chromosomal DNA. - It is capable of autonomous replication as it possesses an origin of replication (ori). - Sometimes it carries antibiotic resistance genes, which can be used as selectable markers e.g. pbr322 and puc18 (fig2). Fig 2 a map of pbr322 showing the positions of the ampicillin resistance (amp r ) and tetracycline resistance (tet r ) genes, the origin of replication (ori) and some of the most important restriction sites. - Some plasmids can integrate into the bacterial chromosomal DNA and hence replicate with the bacterial chromosome. - Size and copy number are particularly important for cloning. Generally a good cloning vector should present in the cell in multiple copies, so that large quantities of the recombinant molecule can be recovered.

5 - Conjugative plasmids can promote sexual conjugation between bacterial cells, so it facilitate the transfer of a recombinant molecule from one bacterium to another. - Small vectors such as puc18 are very convenient for manipulating DNA, but are less convenient for constructing new gene banks of organisms. - COSMIDS are suitable vectors for this purpose. A cosmid is a plasmid that carries the cos sequence of phage λ. - Often vectors designed for cloning very large pieces of DNA are called BACTERIAL ARTIFICIAL CHROMOSOMES. They are capable of cloning inserts of larger than 100 kb. - Another vectors called yeast artificial chromosomes (YACs). These are vectors that are used in Saccharomyces cerevisiae. They allow the cloning of large inserts (100kb - 1 Mb) and are often used for cloning large genomes (e.g. the human genome). - Phages have also been used as vectors. They can carry kb inserts. However, nowadays such vectors are not used as much as earlier. - Another important class of vectors are EXPRESSION VECTORS. These are vectors in which the cloned gene is expressed from a strong promoter so that large quantities of protein are produced.

6 A cloning experiment needs several steps: 1. Purification of DNA from the organism of interest 2. Purification of DNA from a suitable vector (e.g. plasmid) 3. Cutting of the DNAs 4. Mixing of the DNAs 5. Ligation of the DNAs (i.e. sticking together) 6. Introduction of the ligated DNA into a suitable host (e.g. E. coli) 7. Selection of the desired clone as this procedure produces a gene bank in which random pieces of the organism's DNA are cloned Introduction of the ligated DNA into a suitable host It is now necessary to transform this DNA into a host cell. - E. coli does not have a natural transformation system, but if you incubate cells on ice in the presence of calcium ions (e.g. 0.1M), add the DNA and then give a short heat shock (e.g. 42 C, 90 s) some of the cells take up DNA. - Electroporation is another method of promoting transformation. In this method the cells are briefly shocked with an electric field of kv/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After

7 the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms. - It is also possible to introduce DNA into mammalian cells. One method is to use microinjection. The DNA usually integrates fairly randomly into the chromosome. If a fertilised egg is used, it is possible to get integration in the germ line so that the progeny carry the cloned genes. However, if there is a large region of homology it is also possible to get integration by homologous recombination. - Another possibility of introducing DNA is by encapsulating into membranes (liposomes), which are then fused with the cells. - In some cases viral vectors are used that introduce the DNA into cells via infection (e.g. retroviral vectors that allow integration into the chromosome have been used). Note that the term transformation is usually used for introduction of DNA into bacterial cells, however the term transfection is used for introduction of DNA into mammalian cells. 5. Selection of the desired clone (white/blue selection)

8 Vectors 1. Plasmids A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. Plasmids as cloning vector Plasmids are the most-commonly used bacterial cloning vectors. These cloning vectors contains a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker, which has several commonly-used restriction sites to which DNA fragments may be ligated. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation. These plasmids contain a selectable marker, usually an antibiotic resistance gene, which confer on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive. In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA. The vector may also contain other marker genes or reporter genes to facilitate selection of plasmid with cloned insert. Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of plasmid preparation. A plasmid cloning vector is typically used to clone DNA fragments of up to 10 kbp.

9 To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used. 2. Cosmids A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda phage. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. Cosmids can contain 37 to 52 (normally 45) kb of DNA. 3. Bacterial artificial chromosome (BAC) A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is kbp. A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid. E. coli bacteriophage P1 is similar to phage lambda in that it can exist in E. coli in a prophage state. It exists in the E. coli cell as a plasmid, NOT integrated into the E. coli chromosome. P1 cloning vehicles have been constructed that permit cloning of large DNA fragments- few hundred kb of DNA. Cloning and propogation of the chimeric DNA as a P1 plasmid inside E. coli cells

10 BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism. BACs were replaced with faster and less laborious sequencing methods like whole genome shotgun sequencing and now more recently next-gen sequencing. 4. Yeast artificial chromosome Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC). The inherently fragile chromosome was stabilized by discovering the necessary autonomously replicating sequence (ARS); a refined YAC utilizing this data was described in 1983 by Murray et al. The primary components of a YAC are the ARS, centromere, and telomeres from S. cerevisiae. Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector.

11 Gel Electrophoresis Basic tool in molecular biology Use electric field to separate macromolecules: DNA, RNA and proteins Vertical electrophoresis for the separation of DNA and RNA. Horizontal electrophoresis for protein separation. Horizontal Gel Electrophoresis DNA and RNA Tris-EDTA (TE) buffer or Tris-Borate-EDTA (TBE) buffer. Agrose or polyacrylamide Molecules are negatively charged Separation according to size Vertical Gel Electrophoresis Separation according to size (SDS-PAGE) Separation according to size and charge (2D-electrophoresis) SDS PAGE Polyacrylamide matrix SDS for protein denaturation Separation according to Size Stain: Coomassie blue, Silver nitrate or fluorescent stain OR Western blotting

12 See related animation here: Two-dimensional electrophoresis Analysis of complex protein mixtures 2 methods: size and charge The first dimension: ph gradient separate proteins according to its charge (IP) Second dimension is typical SDS gel Proteomic research See related animation here: