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DNA manipulation - separating and probing 1

Electrophoresis     is used to separate DNA fragments based on their length to an accuracy of 1 base length.


  • DNA samples are treated with restriction enzymes (cut into fragments)
  • Samples are placed into wells in the agarose gel at the negative end
  • Gel immersed in a tank of buffer solution
  • Electrical current passed through solution for 2 hours
  • DNA negativity causes it to move towards the positive electrode (Due to phosphoryl groups)
  • Shorter fragments of DNA move quicker and so move further in a fixed time period
  • Position of fragments identified with a DNA staining dye
  • Fragments lifted from gel with nylon or nitrocellulose sheet, blotted, left to dry and then analyzed
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DNA manipulation - separating and probing 2

A DNA probe is a short single-stranded piece of DNA  (Between 50-80 nucleotides long) which is complementary to the section of DNA being investigated

Labeling of the probe

  • Using a radioactive marker (32P) in the probes phosphoryl groups. Location is then revealed by photographic paper
  • Using a fluorescent marker, emits a colour under UV light

When using a DNA probe, copies of the probe are added to a sample of DNA and due to their single stranded nature, they bind to any fragment where a complementary base sequence is present (Annealing)

The use of probes

  • useful in in locating a desired gene for genetic engineering purposes
  • To identify a particular gene in a range of genomes (Genome comparison studies)
  • To identify the presence of an allele for a genetic disease
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Sequencing and copying DNA - 1

The polymerase chain reaction (PCR) is artificial DNA replication which amplifies samples of DNA possibly for forensic use

PCR is not the same as natural DNA replication

  • It can only replicate short lengths of DNA
  • Addition of primer molecules is necessary 
  • Heating and cooling is used to separate and join strands of DNA (Naturally DNA Helicase is used)

The Procedure

  • DNA sample mixed with DNA nucleotides and DNA polymerase
  • Mixture heated to 95°C, breaks H bonds, creates single strands.
  • short (10-20) single stranded lengths of DNA added (Primers) 
  • Temperature is reduced to 55°C, primers H-bind, small DNA lengths made
  • DNA polymerase bonds to small double-stranded sections
  • Temp raised to 72°C (optimum temp for DNA-P), Enzyme extends double stranded section by adding free nucleotide bases
  • This process is repeated and DNA quantity increases exponentially (2,4,8,16,32) 
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Sequencing and copying DNA - 2

Interrupted PCR and Electrophoresis are used to sequence the genome of an organism by identifying the order of bases

How the order of bases is determined - (take note that modified nuclotides are also added during this process which contain a flourecent marker specific to the type of base, when the modified nuclotide is added DNA-P is thrown off)

  • Primer is added and anneals at the 3' end of template strand
  • DNA Polymerase can now attach
  • DNA-P adds free nucleotide bases according the A-T G-C rules
  • When/if a modified nucleotide base is added DNA-P is thrown off and reaction stops
  • As the reaction proceedes many differing lenghts of DNA are made due to the random probability that a modified base may be added and thus DNA-P thown off (The final nucleotide base is always flourecently marked)
  • These strands are run through a machine (same way that DNA moves in electrophoresis a lazer reads the colour sequence.
  • A strand with only 1 nucleotide is read first compared with a strand with 2
  • This sequence of colours relates to the sequence of bases (Due to the assortment by size) and so they can be displayed.
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An introduction to genetic engineering

Genetic engineering involves the extraction of genes from on organism and placing them into another organism

Extraction of desired gene from donor organism using restriction enzymes

  • Restriction enzymes (RE) (restriction endonucleases) are used to cut DNA at specific locations (50 common RE's)
  • RE's will cut the DNA only where a specific base sequence is present (called the restriction site)
  • The RE breaks the sugar-phosphate backbone by catalysing hydrolysis reaction
  • A sticky end is made when DNA is cut as some bases are exposed

The role of ligase in inserting DNA fragments into plasmids

  • DNA ligase is used to catalyse a condensation reaction which joins the sugar-phosphate backbone of DNA together
  • To join the DNA fragments together they both must have been cut with the same RE (So that the sticky ends are complementary
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An introduction to genetic engineering continued +

Vectors into which DNA is incorporated

  • Sealed into a bacterial plasmid (common method)
  • Sealed into virus genomes or yeast cell chromosomes

Ways in which to insert the gene into the recipient cell

  • Electroporation - High voltage shock disrupts the membrane
  • Microinjection - DNA injected using a fine micropipet
  • Viral transfer - This method uses the virus mechanism for infecting cells
  • Ti Plasmids
  • Liposomes

A sticky end

 is formed when DNA is cut using a restriction enzyme. It is a short run of unpaired, exposed bases seen at the end of the cut section. Complementary sticky ends can anneal as part of the process of recombining DNA fragments

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Genetic engineering and bacteria

Advantages of genetic engineering

  • Improving features - giving plants resistance to herbicides, insertig a growth-control gene so that livestock have more muscle
  • Synthesizing useful products -Insering hormone genes to produce insulin, inserting gene for pharmeaceutical chemicals, inserting genes for beta-carotene into rice

How plasmids are taken up by bacterial cells

  • large quantities of plasmid are mixed with bacterial cells
  • Calcium salts and heat shock (Temp lowered then increased to 40°C)  used to increase the rate at which plasmids are taken up bacterial cells
  • Process is very inefficient
  • Bacteria that take up plasmids are called transformed bacteria and therefor the bacteria are transgenic

The advantage of bacteria conjugation is that it may contribute to genetic variation and for example, the gain of antibacterial genes would increase its chance of survival

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Engineering case studies - 1:human insulin

Genetically engineering bacteria to produce human insulin (The process)

  • mRNA for human insulin found in pancreatic tissue via centrufugation
  • Reverse transcriptase used to form a complementary DNA strand
  • DNA polymerase and DNA nucleotides to the single DNA strand prduces an original copy of the gene (Called a cDNA gene)
  • Unpaired nucleotides are added to the end to give complementary sticky ends to those on the cut plasmid
  • Plasmids cut open with restriction enzymes and mixed with cDNA genes
  • DNA ligase enzyme seals up the plasmids
  • The recombinant plasmids are then mixed with bacteria (Some take up recombinant plasmid)
  • Bacteria are grown on agar to produce a mound of cloned cells (colony)
  • (Three typs of colon could grow, bacteria that did not take up plasmid, bacteria which have taken up plasmid but the plasmid sealed up on itself, Some which have taken up the recombinant plasmid
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Engineering case studies - 1:human insulin

The use of genetic markers

  • Original plasmids contain two antibiotic resistant genes
  • The target site of the restriction enzyme is in the middle of one gene
  • therefor if the required gene is take up, antibiotic resistance is broken
  •  (however other antibiotic resistance gene does work)
  • Bacteria are then grown on agar to form colonies
  • Cells taken and placed on agar with 1 antibiotic
  • only cells with plasmid will survive (They have the resistance)
  • some cells from colonies on this agar are then transfered onto the next antibiotic, only the bacteria that have non-recombinant plasmids will therefor survive
  • By keeping track of which colonies are which it is possible to identify which bacteria have the recombinant plasmid (Therefor insulin gene)

Replica plating - refers to the process of growing bacteria on an agar plate, then transferring a replica of that growth to other plates, usually containing different growth promoters or inhibitors . Analysis of growth patterns on the replica plates gives information about the genetic properties of the growing bacteria.

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The growth curve

The three stages to the growth curve in a close system

  • Lag phase - Organisms are adjusting to envirnoment (activating genes, taking in water) therefor the organsms are not reproducing and the populating remains fairly constant
  • Log (Exponential) phase - Population size doubles each generation as the organisms have enough nutrience and space to reproduce
  • Stationary phase - Nutrient level decreases and waste products build up. the rate of death compared to rate of new organisms is the same
  • Decline phase - nutrience exhaustion and the increase in water products increase the death rate so that it is higher than the 'birth' rate and population declines

Primary and secondary metabolites

  • Primary metabolites (amino acids, enxymes, proteins) are substances that are produced as part of an organisms normal growth
  • Secondary metabolites (Antibiotic chemicals) are substances produced that are not part of an organisms normal growth. It occurs after the main growth period and so does not match the growth inpopulation.
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3- Cloning animals

Producing clones through splitting embryos or nuclear transfer

  • Splitting embryos - cells from an embryo are seperated out, each group going onto produce a seperate organism
  • Nuclear transfer - differencated cell from adult taken and its nucleus is placed inside an egg cell which has had its nucleus removed (Enucleated cell). This cell then undergoes the stages of development (Using DNA of inserted nucleus). Cell/group of cells is placed in uterus of surrogate mother.

Advantages and disadantages of cloning

  • + High value (Desired) animals can be produced in large numbers
  • + Rare animals can be clones to maintain their species
  • + Genetically modified animals can be produced quickly
  • - Not always produced with animals welfare in mind (Some produced chickens cannot walk)
  • - Due to the lack of genetic diversity it is unlikely that they would be able to adapt to changes in the environment
  • - It is not clear if cloned animals are as healthy in the long term as un cloned animals.
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3- Cloning animals definitions

Non-reproductive cloning involves the use of cloned cells for the developement of organs or tissues which could replace those which have been damaged

Reproductive cloning is cloning a whole organism

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2 - Artificial clones and agriculture

Micropropagation by callus tissue culture

  • A small piece of tissue (Explant) is taken from the plant to be cloned
  • The explant is placed on a nutrient growth medium
  • Cells in the tissue divide and form a mass of undifferenciated cells called a callus.
  • single callus cells are removed and placed on a growing medium containing shoot growth hormones
  • These cells are moved onto a growing medium containing root growth hormones
  • These plants are then transfered to a greenhouse to become acclimatised

The advantage and disadvantage of plant cloning in agriculture

  • + Farmers know what the planted crop will be like (Cloned from plants of known charecteristics)
  • + Costs are reduced as harvest will be ready at the same time
  • + Quicker than selective breeding
  • - Plants are equally suseptible to disease or pests
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Engineering case studies - 2: Golden Rice

Production of Beta-carotene in the rice endosperm

  • Two genes were inserted into the rice's genome in order to activate a metabolic pathway (These coded for enzymes involved in the manufacture of Beta-carotene)
  • The two enzymes are, Phytoene synthetase - extracted from daffodil plants, Crt 1 enzyme - extracted from soil bacterium
  • These genes were inserted near a specific promoter sequence which switches on genes associated with endosperm development
  • Therefor when the endosperm develops these genes were expressed
  • The amount of beta-carotene produced was increased by 4 times when bred with natural rice varieties, then they later developed Golden Rice 2 which contains 20 times the amount
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Gene therapy

Gene therapy is any therapeutic technique where the functioning allele of a particular gene is placed in tge cells of an individual lacking functioning alleles of that particular gene. Can be used to treat some recessie conditions such as huntingdon's disease

Somatic cell gene therapy

  • Gene therapy by augmentation - a copy of the functional allele is added to the cell which would produce the correct polypeptide and relieve symptoms
  • Gene therapy by killing specific cells - Genetic techniques are used to make cancerous cells produce proteins such as antigens which make these cells vunerable to attack by the immune system

Germline cell gene therapy

  • is the posibility of engineering a gene into a sperm, egg or zygote or the germline cells of an embryo so that when the organism is fully developed all its cells contain the functional allele
  • Further more, it is possible that the offspring will also have this (Functioning) allele
  • This therapy however is considered unethical as whether or not the allele has been take up by the cells is not known and these genetic variations would be passed on to its offspring
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The rights and wrongs of genetic manipulation

Engineered pigs as organ donors

  • Pigs are engineered to lack an enzyme (alpha-1,3-transferase)
  • Addition of an enzyme into the pigs genome (human nucleotidase enzyme)
  • These genetic changes reduced the rejection of the pigs heart by the human immune system

Genetic conscerns surrounding the manipulation of animals, humans, plants and microorganisms

  • (Microorganisms) - May escape from culture, transfer genes to pathogens and these genes could then mutate to produce undesired effects. Genetic engineering also uses antibiotic resistance genes as markers, these could escape and pass genetic information onto pathogens providing them with antibiotic resistance
  • (Plants) - Genes could pass onto wild relatives, reducing variation. Genes could pass to weeds giving them herbicide or pesticide resistance. Genes for pest resistance could pass onto other plant species, destabilizing biological communities. Plants resistant to pathogens could stimulate more rapid evolution of attack mechanisms
  • (Animals)  - Animal welfare issues could arrise from genetic engineering. Strong views are held about animals in some religious groups
  • (Humans) - Most objections are against germline cell therapy. Gene transfer effects are unpredictable.Individuals haveing germline therapy would have no say in the matter. Germline therapy could be used not only to rid of genetic disorders but to enhance favourable characterstics (Hence this involves eugenics)
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Studying whole genomes

An outline of sequencing the genome of an organism

  • Sequencing reaction operates up to 750 bases, DNA must be broken up
  • Genomes mapped (to identify location on chromosome) microsatellites
  • Samples of the genome are mechanically sheared into smaller pieces (100,000 base pairs long)
  • These sections are placed into bacterial artificial chromosomes and transfered to E.coli.
  • As these cells crow in culture many copies of these lenghts of DNA are produced. The cells are called clone librarys
  • Cells containing specific BACs taken out, cultured, DNA extracted, restriction enzymes used to cut these piecs of DNA into smaller pieces.
  • The use of different restriction enzymes gives different fragment types
  • Fragments seperated by electrophoresis, and automatically sequenced
  • Computer programmes compare overlapping regions in order to reassemble the BAC segment

The use of genome sequencing for genome-wide comparions between species

  • shows importance of some genes for life, they show evolutionary relationships, mutations can be identified, used to identify the disease causing lenghts of DNA, show the likely hood of a disease occuring (Heart disease etc..)
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industrial enzymes

Ways to immobilize enzymes

  • (Adsorption) - Enzymes are mixed with immobilizing support and bind to it due to hydrophobic interactions and ionic links. Enzymes can become detached called leakage, but reactions rates are high
  • (Covalent bonding) - Enzyme molecules are covalently bonded to a support. A cross linking agent is used to bind enzymes together. process does not immobilize a large number of enzyme but there is very little leakage
  • (Entrapment) - Enzymes are trapped in a gel bead. Exist in their natural state so active site is not affected but reaction rates can be reduced because substrate molecules must get through the trapping barrier.
  • (Membrane separation) - Enzymes are phisically separated from the substrate via a membrane. Substrate molecules can pass throughthe membrane as well as the products.

Why are immobilized enzymes used?

  • Enzymes are not present in the products (Low cost as no extraction)
  • Enzymes are immediatly available for re-use
  • Immobilized enzymes are more stable because the immobilising martix protects the enzymes
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Commercial applications of biotechnology

Compare and contrast the processes of continuous and batch culture

  • (Growth rate) Batch - slower. Continous - fast.
  • (Maintanace) Batch - easy. Continouse - hard.
  • Contamination) Batch - only 1 batch lost. Continouse - huge volumes lost
  • (Efficiency) Batch - less efficient. Continous - more efficient.
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Comprehensive, detailed and relevant resource for this topic. Gives a well rounded set of revision cards for Biotechnology that could be used to create a set of flashcards for the key terms and their definitions.

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