gene technology

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  • Created by: Emma
  • Created on: 05-04-13 17:10

recombinant DNA technology by in vivo cloning

  • in vivo = in living organisms
  • allows genes to be manipulated, altered and transferred from one organism to another, eg. insulin
  • techniques have been developed to isolate genes, clone them and transfer them into microorganisms

the stages:

1. isolation and identification of the DNA fragments that have the gene for the desired protien

2. insertion of the DNA fragment, into a vector (plasmid)

3. transformation- the transfer of DNA into suitable host cells

4. identification of the host cells, that have sucessfully taken up the gene, uses gene markers

5. growth/cloning of the population of host cells

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1. isolation of the DNA fragments

identifying and isolating the gene from the rest of the DNA

potentially difficult as there are 1000s of genes, and millions of bases in a genome

uses: restriction endonucleases and reverse transciptase

using restriction endonucleases (genetic scissors, cut out the DNA)

each one reconises specific palindromic sequence of bases, cuts and leaves 'sticky ends'

using reverse transcriptase (conversion of mRNA to cDNA)

mRNA -> RNA strand + DNA strand join by reverse transcriptase -> RNA/DNA strands seperate by an enzyme -> 2 DNA strands by complementary base pairing and DNA polymerase = cDNA

cDNA is complementary to the mRNA used to create it

an advantage os using this method compared to restriction endonucleases:

here the isolated genes can be put into bacteria, as it doesn't contain introns, bacteria can't code introns

its also quicker, less different bits of DNA to look for

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2. insertion of the DNA fragment into the vector &

a vector is a carrier of DNA into a host cell organism... often uses a plasmid and bacterial cells

plasmid = small circular bit of DNA that can replicate independantly, move easily between

             = contains genes that act as markers (resistance genes)

need to use the same restriction enzyme = complementary 'sticky ends', so can pair up and form a hybrid vector (of bacterial and foreign DNA)

after pairing up- DNA ligase is used to join the sugar-phosphate backbone

'marker genes' - later used to identify the correct hybrid vector

3. transformation: if it has worked, the 'sticky ends' will interupt one of their anitbiotic resistance genes, so it will only be resistant to one, not two..

.. the plasmids not have recombinant DNA, when they are reintroduced to the bacterial cells the cells are said to be transformed

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4. indenfication of the host cells using genetic m

only a small number of bacterial cells take up the plasmids, they are called 'genetically modified organisms'

plasmids contain two different marker genes, which are needed to identify the required cells

the 1st marker gene = distinguishes between cells that have taken up a plasmid from those that haven't

the plasmid contains a gene for resistance to an antibiotic, so if the plasmid is taken up it will be resistant, growing on a medium: untransformed cells will be killed, transformed cells will survive, can then be grown and cloned on another plate

the 2nd marker gene = distinguishes between cells that have taken up the recombinant plasmid from those that have taken up the original plasmid

foreign DNA is inserted inside the second marker gene

this marker can be a gene for resistance to another antibiotic, in this case cells with the recombinant plasmid are not resistant to this antibiotic, on a 2nd plate, cells that are killed (but survive the 1st plate) are the ones we want

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uses of recombinant DNA technology to benefit huma

genetic engineering in agriculture:

  • adding genes improving the nutrient content of foods
  • adding genes to develop tolerance to extreme environmental conditions eg. temp./drought
  • adding genes coding for resistance to herbicides, insect pests and viral disease

direct uses of genetically modified bacteria:

  • to increase the quantity of antibiotics and the rate in which they are made
  • to produce hormones such as insulin or oestrogen, avoids killing animals and fewer side effects
  • to produce many enzymes used in the food industry

genetically modified animals:

  • transfer of genes for disease resistance from one animal to another
  • introducing growth hormone genes so animals grow larger faster, improves food prod.
  • introduction of genes which code for important human proteins into milk-producing proteins
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the use of gene therapy to supplement defective ge

the gene is inserted into a plasmid which is used to transform affected cells in a body

plasmid is contained in a liposome or a virus- delivers the gene into the nucleus of a cell following transformation

the cell will transcribe the gene into mRNA- then translated into a protein by the ribosomes

the cell can now produce a healthy protein that can function correctly, disease is cured

     in theory, there are two possible methods:

1. germ cell therapy of sperm, egg or early embryo= mutated cell in an embryo is replaced with a healthy gene

2. somatic cell therapy= healthy gene inserted into mature cells, by a vector, gene only expressed in the lifetime of a cell

gene therapy will only work when the disorder is caused by recessive alleles= because the existing allele will stay in the cell and if it was dominant it would prevent the added gene from being expressed

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effectiveness of gene therapy and ethical issues

  • the effect is short-lived- semotic cells don't reproduce
  • can induce and immune response- vector might stimulate an antibody reaction
  • viral vectors present problems- virus might mutate, back to being a pathogen
  • genes are not always expressed- no guarantee genes will reach the nucleus, or then be expressed
  • not effective in treating conditions that arise in more than one gene- too many changes would need to be made

ethical issues:

  • is it immoral to tamper with genes? if we can treat disabilities we should
  • a responsibility to develop crops to survive drought / a danger of big companies having too much control
  • GM crops can help prevent disease / genes could mutate into harmful genes
  • impossible to predict the risks when releasing GM organisms into the environment
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polymerase chain reaction (PCR) and in vitro gene

in vitro = in glass       DNA fingerprinting        rapid and efficient      can amplify a small amount

a method of directly cloning fragments of DNA making multiple copies

seraration of DNA strand- DNA fragments (to be amplified), primers (allow polymerase to bind) and DNA polmerase are placed in the vessel in the thermocycler (a machine where the temperature will vary over a number of cycles). temperature increase to 95C

addition (annealing) of primers- mixture cooled to 50C, causing primers to join to their complementary bases on the DNA fragment. Primers provide the starting sequenced for DNA polymerase to begin copying the DNA

synthesis of DNA - temperature is increased to 72C, optimum for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. begins at the primer on both strands and adds nucleotides in sequence until it reaches the ends of the chain

this process allows billions of copies to be made in only a few hours... the DNA polymerase is thermostable- won't denature until way above 100C

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genetic fingerprinting

a diagnostic tool, used in forensic science, based on DNA being unique to an individual...

used to determine: criminality, paternity, medical diagnosis                                                           

stages of genetic fingerprinting:

1. extraction of DNA- a sample of tissue, DNA is extracted, PCR will increase the quantity

2. digestion- DNA is cut into fragments using restriction endonucleases- will cut close to groups of core sequences

3. separation- gel electrophoresis separates DNA fragments based in size (the technique depends on the movement of charged molecules across a gel in an applied electrical field). secondly- southern blotting, separates into single strands before being transfered to a nylon memrane

4. hybridisation- radioactive probes bind with the core sequences, probes have complementary base sequences to the core sequences

5. development- an Xray film is placed over, radioactive probes will cause bars to be seen on the film, the pattern of bands is unique to every individual

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determining the base sequence of a gene

once a gene has been identified, scientists will want to know the exact base sequence of the gene and the position of restriction endonuclease recognition site, which can be manipulated during in vivo cloning by either: 1. DNA sequencing or 2. restriction mapping

1. DNA sequencing: using modified nucleotides that can't attach to the next nucleotide in the sequence when they are joined together- called terminator nucleotides, they end the synthesis of a DNA strand. 4 different terminator nucleotides are used, each with one of the 4 bases.

shortest fragments move the further distance- read from shortest first, original strand would be complementary to what is read

2. restriction mapping: uses restriction enzymes to cut the gene into smaller sections, as most genes are too long to sequence in one go. DNA sequencing can then determine to order. also be used to help identify the position of restriction sites, if not known. gel electrophoresis separates the DNA fragments based on size, the start of the fragment is radioactively labelled.

sometimes involves using pairs of restriction endonucleases, rather than each one separatly

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