Biology OCR - Gene technology

general notes on gene technology

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  • Created by: sophie
  • Created on: 24-04-12 17:04

Sequencing the genome of an organism

  • Operates on a length of DNA of 750 base pairs so the genome must be broken up and sequenced n sections. to ensure the code is accurate, sequencing happens on overlapping fragments which are more easily put back together
  • genomes are mapped to identify which part of the genome they come from. Microsatalites are used - easily identifyable
  • Genome samples are sheared to smaller seections
  • The sections are placed into seperate bacterial artificial chromasomes (BACs) and transfered to bacteria cells. As bacteria multiply, clones are produced - clone libraries

To sequence a BAC section: 

  • Cells conaining specific BACs are cultured. the DNA is extracted and restriction enzymes cut it into smaller fragments. different restriction enzymes give different fragment types
  • Fragments are separated by elecrophorises, sorting them into size order
  • Each fragment is sequenced using an automated process
  • Overlapping regions due to different cuts by different restriction enzymes are compared and ordered to resemble the BAc segment sequence
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Manipulating DNA and comparing genomes

  • DNA has been used for:
  • DNA profiling: forensic crime scene analysis and paternity/maternity testing
  • Genomic sequencing: used in research in gene function and regulatory DNA sequences
  • Genetic engineering: production of pharmaceutical chemicals and GMOs
  • Gene therapy: used to treat conditions like cystic fibrosis

Comparing Genomes

Compaative gene mapping: comparing genes fr the same proteins across a ange of organisms. The comparing of genomes:

  • Identification of genes for proteins in many organisms gives relative importance of the genes to life
  • Shows evolutionary relationships. more shared DNA sequences,, the closer related
  • Modelling the effects of changes to DNA can be carried out
  • Comparing genomes from pathogenic and similar non-pathogenic organisms can identify genes/base pair sequences that are important in causing the disease
  • DNA of individuals can be analysed to reveal mutant alleles
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Electrophoresis separates DNA fragments

Separates DNA fragments based on their size

  • A gel slab containing agergose is coveerd with buffer solution
  • Electrodes are attached: cathode near the sample, anode opposite so current passes through gel
  • DNA samples are treated with restriction enzymes and put into wells into neg. end of the gel
  • Gel is immerised in buffer solution and electric current passed through for set time
  • DNA is neg charged so its attracted and diffuse through the gel to the anode
  • Shorter lengths move faster so move further in the fixed time
  • The position of the fragment is shown using a dye
  • The fragments are lifted from the gel onto for analysis: Southern blotting
  • A nylon/nitrocellulose sheet are placed over the gel, covered with paper towels, pressed and left overnight
  • The fragments transferred to the sheet can be analysed
  • The samples can be labeled with a radioactive marker and seen using photo film
  • If a particular fragment is being looked for, the probe can be used to check for it
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DNA Probes

A short single stranded piece of DNA complementary to the section of DNA being investigated. They are labelled in 2 ways:

A radioactive marker so the location is revealed by exposure to photographic film

A florescent marker: emits colour in exposure to UV light. Also used in automated DNA sequencing

Copies of the probe can be added to a sample of DNA fragments. Because they're single stranded, they bind to any complementary strand. this binding is called annealing

Examples of probes

  • Locate specific desired gene wanted for genetic engineering
  • Identify the same gene on different genomes for genome comparison studies
  • Identify presence/absence of allele for particular genetic disease

Diagnosis of some symptomless genetic diseases can be made my analysng the patients DNA. Probes are complementary to sequence of faulty alleles

In order to anneal, the sample DNA but be in smaller fragments and amplified by PCR

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Polymerase Chain Reaction (PCR)

PCR is DNA replication carried out on tiny samples of DNA to generate multiple copies of the sample. Primar molecules have to be added to initiate the process. 

The sequencing relies on the fact that DNA: 

  • is made of anti-parallel backbone strands
  • is made of strands that have a 5' and and 3' end (both prime)
  • grown only from the 3' end
  • complementary base pairing occurs: A-T, C-G

PCR is a cyclic reaction

  • DNA sample is mixed with DNA nucleotides and DNA polymerase
  • Mixture is heated to 95deg. to break H bonds between bases. Samples are single-stranded. Primers are added to mixture
  • Temp is reduced to 55deg so primers bond and form small sections of DNA 
  • DNA polymerase bonds to the double stranded sections
  • Temp raised to 72deg (optimum for enzyme). Enzyme extends double stranded section by adding free nucleotides to the DNA
  • A double stranded sample is produced. The process is repeated so amount of DNA increases exponentially
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Sequencing Genomes

Automated DNA sequencing is based on an interrupted PCR and electrophoresis

The reaction mixture contains: many copies of the DNA fragment (single stranded); DNA polymerase, primers and free DNA nucleotides. Some of the nucleotides are modified, carring a florecent marker and if added to the growing chain the DNA polymerase is ejected so no more nucleotides can be added

  • Primer anneals at the 3' of template strand so DNA polymerase can attach
  • DNA polymerase adds free nucleotides (base pairing) to extend strand
  • If a modified nucleotide is added, DNA polymerase is ejected and reaction stops
  • Many DNA molecules are made. The fragments vary in size, depending when modified nucleotide is added. The nucelotides are tagged with spsecific colours
  • In a machine (like electrophoresis) a laser reads the colour sequence and the base sequence
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Sequencing a genome - Human Genome Project

Set its aim in 1990 to determine the whole human genome squence

DNA from volunteers all round the world controbuted. In 2004 the completed genome sequence was published. Each lab worked on a different part on the genome to build the whole sequence. By 2004 the estimate was that humans only had about 25000 genomes, not much more than a worm. Human complexity is more to do with the regulation of gene expression than the number of genes

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Genetic Engineering

A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from 2 different sources

Obtaining a gene to be engineered

  • mRNA from transcription is obtained from cells where the gene is expressed. E.g nRNA for isulin is obtained from beta-cells in pancreas. The mRNA is a template to make gene copies
  • The gene can be synthesised using automated polynucleotide sequencer
  • DNA probe is used to locate a gene on DNA fragments and the gene can be cut out of the fragment using restriction enzymes

Placing a gene in a vector

  • Gene is sealed into the bacterial plasmid using DNA ligase - most popular
  • Genes may be sealed into virus genomes and yeast cell chromasomes
  • Vectors often have to contain  regulatory sequences of DNA. These ensure the inserted gene is transcribed i the host cell
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Genetic Engineering

Getting the gene into the recipient cell

  • The newly made plasmid cant easily cross a membrane into the recipient cell
  • Electroporation - high-voltage pulse to disrupt membrane
  • Microinjection - DNA injected into host nucleus via micropipette
  • Viral transfer - Vector is a virus; method uses viral mechanism for infected cells by inserting DNA directly
  • Liposomes - DNa wrapped in lipid molecules so cross lipid membrane via diffusion
  • Ti plasmids used as vectors can be inserted into soil bacteria. DNA can me placed in a plant genome from the soil
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Genetic Engineering - enzymes

Restriction enzymes but through DNA at specific points. A particular restriction enzyme cuts DNA only when a specific base sequence occurs. The sequence is a restriction site, and is a few bases long. The enzyme catalysis the hydrolysis reaction whch breaks the sugar-phosphat backbone at a specific point to give a staggared cut, leaving exposed bases - sticky ends

DNA fragments are stuck together by DNA ligase, catalysing a condensation reaction, joining sugar-phosphate backbones of a double helix together. To join 2 fragments from different sources, they must have complementary sticky ends. The ends are joined by DNA ligase

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Why have GMOs?

Improving a feature of the recipient organism

Inserting a gene into crops to give herbicide resistance ao farmers can use herbicied abnd not kill the crops

Inserting a growth-gontrol hormone to promote muscle growth in animals

Organisms that synthesise useful products

Inserting gene for human hormone eg insulin into bacteria. They grow and produce large quantites of the hormone

Inserting gene for pharmaceutical cheicalks into female sheep so the milk contain the chemical - easy to collect

Golden rice: Inserting genes for Beta-carotene prodution into rice so peopkle eat it. B-carotene is converted to Vitamin A

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Bacterial cells ad plasmids

Genetic engineering usually uses bacteria plasmids as vectors. They carry codes for resistance to antibiotic chemicals

Plasmids are cut with the same restriction enzyme to give complementary sticky ends. DNA ligase seals the new plasmid to form a recombinant plasmid

Bacterial cells take up plasmid DNA – they become transformed and transgenic

Large quantities of plasmid are mixed with bacteria cells. Adding Ca2+ salts to ‘heat shock’ the culture so increase the rate of plasmid uptake. However the process is very inefficient.

The ones that take up the plasmid are transformed bacteria. This means they contain the new DNA and are transgenic

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Bacteria exchanging DNA

Bacteria can exchange genetic material by conjunction. Copies of the plasmid DNA are passed between bacteria. Because plasmids often carry antibiotic resistance, there is a spread of antibiotic resistance between bacterial populations – BAD

Resistant strains of bacteria cause healthcare problems. MRSA is found on skin. If it transfers into a wound, it leads to a serious infection. Scientists continuously look for new antibiotics

Advantage: Conjunctions may contribute to genetic variation and, in the case of antibiotic resistant genes, survive in the presence of the antibiotics

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Case study: Bacterial transformations

Bacterial transformations and pneumonia in mice

Pioneering work was done to demonstrate that bacteria can take up DNA from their surroundings and incorporate it into their genome. They used 2 strains of the same bacteria:

S-strain- quickly kills mice on infection, R-strain – doesn’t kill mice on infection

Only S-strain mice were killed by toxic protein. S-strain must have instructions to make protein, R-strain cant

Injecting a mix of living R and dead S killed the mice. The mice contained living S. The S bacteria were transformed from R bacteria.

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Genetic Markers

Radio-labelled antibodies that bind specifically to insulin are used to identify transformed bacteria

Original plasmids are chosen if they carry genes that make any bacteria receiving them resistant to 2 antibiotic chemicals (ampicillin and tetracycline) The resistant genes are genetic markers

Plasmids are cut by restriction enzymes that have a target site (restriction site) in the tetracycline gene, so if the required gene is taken up, the gene for tetracycline is broken up and doesn’t work. The ampicillin gene still works

Replica plating

* Bacteria is grown on nutrient agar to form colonies

* Some cells are transferred to agar with ampicillin, so only those that have taken up the plasmid grow

* Some of these cells are transferred to agar with tetracycline so those that took up the plasmid without insulin grow

* By noting which cells are from which colony, we know that cells that grow on ampicillin but not tetracycline have taken up to recombinant plasmid

* We can identify and grow these colonies to produce more insulin

Some viruses – retroviruses, carry genetic material as RNA. When infecting a cell they reverse transcriptase to be synthesised, which copies the viral RNA to DNA

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Case study: Human insulin

Human insulin

The DNA code for insulin is very small so could not be mapped. Scientists focused on finding the mRNA for the gene.

* Centrifuge separated mRNA of the right length from pancreas tissue

* Reverse transcriptase was used to synthesis a complementary single DNA strand to the mRNA

* DNA polymerase and free nucleotides are added. cDNA strands are formed – a copy of the original mRNA. Unpaired nucleotides are added to each end to give complementary sticky ends to the cut plasmid

* Plasmids are cut open with restriction enzyme and mixed with the cDNA. Some plasmids take up the gene and are sealed by DNA ligase to form recombinant plasmids

* Plasmids are mixed with bacteria, some take up the recombinant plasmids

* Bacteria are grown on an agar plate to produce a colony of cloned bacteria cells

3 possible colonies are produced:

* Bacteria that didn’t take up the plasmid

* Bacteria that took up an original plasmid

* Reformed bacteria that took up the recombinant plasmid

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Case study: Golden rice

  • People in the developing world have a vitA deficiency, causing night blindness and weakened bones. These peoples staple food is rice. Without access to meat, they cannot get vitA.
  • Rice plants contain genes that code for Beta-carotene. These are in the green parts so aren’t eaten, it is swiched off in the endosperm (grain)
  • Scientists have GE’d rice plants so B-carotene is accumulated in the grain. The rice became gold-coloured.
  • The insertation of 2 genes into the rice genome was needed to activate the metabolic pathway in the endosperm cells: Phytoene synthase (from daffodils) and Crt 1 enzyme (from soil bacterium)
  • The genes were inserted into the rice genome near a specific promoter sequence to switch on genes associated with endosperm development so they were expressed as the gene grew
  • However this gives a small concentration of B-carotene, not very useful
  • Golden ride was cross bred with normal rice. The offspring produced more B-carotene than before
  • Goldenrice 2 had 20x more B-carotene in endosperm than original
  • Cons: It could lead to reduced biodiversity, its human food safety is unknown, it could breed with wild rice and contaminate the population
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Gene therapy

Techniques of molecular gene technology that can be used to treat some genetic disorders. Cant treat recessive conditions

Somatic cell gene therapy

* Adding genes (augmentation): some diseases are caused by inheriting faulty alleles leading to loss of a polypeptide. Engineering a functioning copy of the gene into the relevant specialised cell means the polypeptide can be synthesised

* Killing specific genes: Cancers treated by eliminating certain pops of cells. Genetic techniques can make cancerous cells express genes to produce proteins to make cells vulnerable to immune system

Germline cell gene therapy

* Engineering a gene into sperm, egg or zygote so as organism grows all cells contain a copy of the gene. The functioning allele is onto offspring

Germline therapy is illegal and unethical in UK and USA:

Inadvertent mods could create a new human disease or interfere were human evolution

Moral, ethical and social issues

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Somatic Cell and Germline Cell therapy

Somatic Cell  Germline Cell

Techniques to get gene to target location are needed/ cells removed, treated and reinserted (ex vivo)

Functioning allele into germline cells, straightforward delivery techniques

Treatment is short-lived. Specialised cells wont divide to pass on allele

All cells will derive from germline cells and contain functioning gene copy. Offspring may get copy

Difficult getting functioning allele to genome. Host becomes immune to GMviruses so cells won’t accept vector 2nd time. Liposomes are inefficient

Unethical to engineer human embryos. Not possible to know if allele is successful w.o any unintentional changes to it, which can damage the embryo

Genetic manipulations restricted to patient

Genetic manipulations passed to offspring

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Case study: SCID


10 different types of SCID

One had defective enzyme ADA – leads to buildup of toxic matabolites so loss of T-lymphocytes

Gene therapy trials:

Retrovirus (able to transfer its DNA to eukaryotic cells) engeneered to contain notmal ADA gene

Bone marrow containing T cells is removed and exposed ti retrovirus. Infection leads to uptake of ADA gene

Transgenic cells formed are placed into patients bone marrow to astablish a line of cells with functional ADA

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Transplantation of cell tissues o irgans between anomals of different species. There is no rejection

Pigs were engineered to lack enxymes α-1,3,-transferase – the ke trigger for graft rejection in humans

Engineering human nucleotidase enzymes into pig cells in clutire reduced immune activity involved in xenotrandplantation rejection


  • Organ size
  • Pigs have short lifespan so xenograph ages fast
  • Pigs have higher body temp – chemical reactions are slower
  • Opposed to killing animals for organ use
  • Religis regions – Jewish and Muslim don’t eat pork
  • Possible disease transfer between organisms
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Ethical concerns and risks

The capacity to move genes between molecules is 'unnatural'

Humans have produced 'unnatural' organisms for centuries: breeding valuble traits into animals and crops

There is a hype around GM foods

Microorganisms: can produce useful products eg insulin.

May escape containment and transfer and mutate other genes. GE uses antibiotic resistance genes as merkers. Genes could be passed on - more widespread resistance

Plants: Beta-carotene for goldenrice, resistance to pests/pesticied for higher yield.

Genes may pass to wild relatives - less variaion. Genes passed to weeds/other species reducing stability. GM plants toxic to other organisms

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Ethical concerns and risks

Animals: Pharmaceutical chemicals producied in milk. Increased milk/meat production. Production of organs for human transplant.

Animal welfare issues. Religious reasons

Humans: Gene therapies treat some genetic disorders.

Germline cell gene therapy is illegal in UK and USA. Eggects of gene transfer and unpredictable. Defects introduced to embryo and offspring. Germline individuals have no say in whether they want the therapy. Can be used to enhance favourable characteristics - designer children.

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