Gene Cloning Technology Also known as: Genetic engineering or Genetic manipulation (GM) technology implies precision engineering being applied to DNA molecules Recombinant DNA technology - implies that new combinations of DNA molecules can be made i.e. recombinant DNA molecules Overview of Genetic Engineering procedure 1. Making recombinant DNA molecules that can replicate in bacterial cells 1
Genetic engineering applications A tool kit for recombinant DNA technology 1. Tools to cut DNA molecules into large gene-sized fragments 2. Vector DNA molecules that can replicate and into which foreign DNA can be inserted 3. Tools to join vector DNA and foreign DNA together to create recombinant DNA molecules 4. Methods for introducing recombinant DNA molecules into bacterial cells where they can replicate 5. A method for identifying bacterial cells that have taken up the recombinant DNA molecules = a method of selecting for transformants 2
1. Tools for cutting DNA molecules into gene-sized fragments A typical chromosome might contain more than 5 million base pairs Molecules of this enormous size cannot easily be cloned and they contain 1000 s of genes A single gene might typicallly contain a few thousand base pairs of DNA sequence. Molecules of this size can be easily cloned For this reason, we need to be able to cut the very large chromosome-sized DNA molecules into smaller gene-sized fragments This can be done using a cutting tool called a restriction enzyme Restriction enzymes Restriction enzymes are made by bacteria to protect themselves incoming virus DNA They act as molecular scissors to cut DNA into large fragments They have a unique property: they cut DNA in a sequence-specific manner by recognizing a specific sequence of bases e.g. The restriction enzyme EcoRI will only cut a DNA molecule if it contains the following sequence: G-A-A-T-T-C C-T-T-A-A-G 3
Restriction enzymes cut DNA infrequently The frequency with which this particular sequence of 6 base pairs will occur in a very large DNA molecule can be calculated on the basis of: the probability of occurrence of any specified base at each position: G A A T T C ¼ ¼ ¼ ¼ ¼ ¼ = 1/ 4 6 = 1 in 4,100 bases pairs Because this enzymes cuts DNA only once in every 4,000 base pairs (on average), the fragments are sufficiently large to contain an entire gene DNA fragments have single-stranded sticky ends after being cut by a restriction enzyme. The sequence of bases at the ends of the fragment are unique for each type of restriction enzyme G-A-A-T-T-C C-T-T-A-A-G G A-A-T-T-C C-T-T-A-A G single-stranded 4 base sticky ends The single-stranded ends can anneal or base pair with the same type of end on a DNA molecule from any source e.g. animal 4
The DNA polymerase used for PCR is Taq polymerase. Taq polymerase is derived from a heat-stable bacterium. It works in a similar way as the polymerase found in human cells. www.odec.ca/projects/2005/anna5m0/public_html/methods.htm 5
2. DNA Cloning Vectors A cloning vector is a DNA molecule that can contains information for replicating itself a so-called origin of replication If a piece of foreign DNA is incorporated into the vector, then the foreign DNA will be replicated along with the vector DNA The most commonly used type of cloning vector is a small (4000 bp) circular DNA molecule called a plasmid Plasmids are found naturally in various types of bacteria Plasmids can replicate independently of the bacterial chromosome because they have an origin of replication A typical plasmid DNA might have the following features Antibiotic resistance gene Amp r Any bacterial cell that takes up the plasmid will be resistant to ampicillin lacz gene for colony colour selection EcoRI restriction site The plasmid DNA circle can be opened up at this site and foreign DNA can be inserted into the plasmid 6
3. Making recombinant DNA molecules A recombinant DNA molecule is made by joining together (a) a piece of foreign DNA (e.g. human DNA) and (b) vector DNA (e.g. a plasmid) The two DNAs can be joined together by an enzyme called DNA ligase 4. Cloning the recombinant DNA molecules The recombinant DNA molecules will replicate if they are taken up by a bacterial cell and the bacterial cell will be transformed to antibiotic resistance Cells that have not taken up a recombinant DNA molecule will be killed by the antibiotic Cut plasmid DNA Cut human DNA 7
Transformation of bacteria with recombinant DNA Selection of transformed bacteria on agar plates containing ampicillin and X-Gal X-Gal is a b-galactosidase chromogenic substrate. In the presence of b-galactosidase X-Gal is hydrolysed and forms a blue precipitate. Cloned human DNA click4biology.info/c4b/4/gene4.4.htm 8
www.nba.gov.au/publications/0708report/part3-4.html Factor IX : A human clotting factor is produced by genetically modified sheep. The protein(factor IX) is expressed in milk from which it must be isolated before use byhaemophiliacs. click4biology.info/c4b/4/gene4.4.htm 9
Factor IX protein purified from CHO cells (a mammalian cell line) engineered to express human Factor IX using cloning technologies described in this lecture. 10
In the 1980s, researchers used genetic engineering to manufacture a human insulin. In 1982, the Eli Lilly Corporation produced a human insulin that became the first approved genetically engineered pharmaceutical product. Human insulin is grown in the lab inside common bacteria. Escherichia coli is by far the most widely used type of bacterium, but yeast is also used. cited www.madehow.com/volume-7/insulin.html fhs-bio-wiki.pbworks.com/w/page/22957102/recombinant DNA (plasmids) 11
Genetic Manipulation and Agriculture Rice is the major food source for more than a billion people The problem: Rice has two major nutritional problems if it is the major (or only) part of a person s diet: - it is a poor source of iron - it contains no vitamin A or provitamin A A diet deficient in Vitamin A causes irreversible blindness in children and weakens their immune systems A diet deficient in iron leads to anaemia which affects growth potential and weakens the immune system The solution: The Golden Rice project The project was led by a Swiss Government Research centre not by a Biotechnology Company Aim: to genetically engineer rice (i) to produce provitamin A (ii) to accumulate iron from the soil more efficiently 1. Genetic engineering for provitamina production Inserted genes from daffodil and bacteria that enable rice to make carotenoids = provitamin A (yellow orange pigments) human metabolism can convert carotenoids into Vitamin A 12
2. Genetic engineering for increased iron accumulation Introduced 3 genes - a gene for increased iron uptake - a gene for iron storage - a gene that increases iron bio-availability Golden Rice has been donated royalty-free to the International Rice Research Institute in the Phillipines for use in third world countries Crops as biotechnology factories: BioPharming 1. Plants can be genetically engineered to produce important therapeutic proteins e.g. vaccines and proteins like insulin 2. There are major advantages in using plants for this purpose: - plants are a very inexpensive production system - very easy to scale-up the process - alternative production methods are very expensive e.g. production in animal cell cultures or bacterial cultures 3. The recovered protein is free from animal viruses and other pathogens 13
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We sometimes refer now not only to the genetic code (the nucleotide sequence of DNA) but also the histone code www.naturalproductsok.com/sites/natural/uploads/images/header_images/epigenetic_markers.jpg http://sciblogs.co.nz/code-for-life/files/2010/08/epigenetics-400px.png 15
The chemical modifications to DNA and histone proteins tend to show significant differences between healthy and diseased cells see the example of cancer above. abnormal epigenetic patterns are also found in, e.g, ageing cells etc. A cardiogenic diff B myogenic diff C neuronal diff 16
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www.eurostemcell.org/files/images/ips_scheme.preview.jpg 18
www.liferesearch.com/media/cms/stem.jpg web-japan.org/trends/07_sci-tech/images/l_scia080214.gif www.umassmed.edu/uploadedimages/umscr/images/ipsderivation copy.jpg 19
www.jci.org/articles/view/40553/files/jci40553.f2/medium http://www.cira.kyoto-u.ac.jp/e/research/images/osafune/img02.gif 20
ScienceDaily (Mar. 5, 2009) Whitehead Institute researchers have developed a novel method to remove potential cancer-causing genes during the reprogramming of skin cells from Parkinson's disease patients into an embryonic-stem-cell-like state. Scientists then used the resulting induced pluripotent stem (ips) cells to derive dopamine-producing neurons, the cell type that degenerates in Parkinson's disease patients. DNA fingerprinting applications 1. Definitive linking of victim with crime scene and with suspect(s) 2. Creation of a DNA fingerprint database 3. Has had major application in successful criminal prosecutions across Europe and the USA 4. Has played a major role in freeing wrongly convicted prisoners 5. Used to identify victims of disasters that cannot be identified by other means e.g. airplane crash victims 6. Used in paternity disputes: positive identification of fathers 21
www.scq.ubc.ca/fingerprinting www.scq.ubc.ca/fingerprinting 22