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58 Cards in this Set
- Front
- Back
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Genome Includes |
All genetic material an organism contains Non coding regions, regulatory genes, mitochondria, telomeres, centromere |
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Exon and intron |
Exon - codes for proteins Intron - non coding regions |
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Telomeres |
Found at end of chromosomes to protect from deterioration |
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Satellite DNA Where are they found ? |
Short sequences of DNA repeated multiple times Found within telomeres, centromeres and introns |
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Mini satellite Another name |
Sequence of 20-50 base pairs will be repeated from 50 to several hundred times Variable number tandem repeats |
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Micro satellite Another name |
sequence of 2-4 base pairs repeated 5-15 times Short tandem repeats |
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Satellites properties |
Always appear in sane position of chromosomes Number of repeats of each mini or micro satellite varies between individuals Different length of repeats are inherited from both parents |
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DNA profiling |
Producing image of patterns in DNA of an individual |
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Stages of DNA profiling |
DNA is extracted from tissue sample DNA is digested into small fragments using restriction endonuclease DNA is amplified in polymerase chain reaction to generate multiple copies and provide enough DNA to make the band visible Fragments are separated by gel electrophoresis and fixed by southern blotting Small dna probes complementary to micro satellite regions are added ( process is hybridisation) Evidence is observed using X-ray images or UV light |
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How is DNA extracted from tissue sample? |
By adding salt so water diffuses out of cell by osmosis Add soap to dissolve membrane and ethanol to isolate DNA from water and make it insoluble |
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How is DNA digested into smaller fragments? |
Cut DNA at restriction site on introns and leave repeats intact Break phosphate sugar backbone and weaken H bonds and create sticky ends Site is palindromic |
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How is DNA amplified in PCR? |
Temperature in PCR is increased to 95 degree c so denature DNA by breaking H bond so strand separate Temperature decrease to 55 degree and primers bind to end of DNA strands which are needed for replication Temperature increased to 72 degree and DNA polymerase add bases to primer building up complementary strands |
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Formula for finding the number of molecules in PCR after set time |
Back (Definition) |
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Reason for representing large fragments of DNA number using logs |
Easier to plot on graph Test for logarithmic growth Reducing number varying by magnitude of 10 |
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Limitations of PCR |
Need to know sequence of target DNA to make primers size of DNA fragment is limited Taq polymerase is not completely accurate Contaminating of DNA will be copied |
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How are fragments separated by gel electrophoresis? |
Create agarose gel plate in a tank with electrodes at either end DNA samples added to wells in the gel Elective current is passed through electrophoresis plate DNA fragment in wells of cathode end move through gel towards anode Compare to marker DNA ladder and rate of movement depends on length of DNA |
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How is southern blotting done? |
Fix DNA in its place by immersing gel into alkali to separate DNA strands and transferring by assisted diffusion to nylon or nitrocellulose membrane |
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Uses of DNA profiling |
Forensics Paternity test Identifying species Diseases as particular satellites with mutation |
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dNTP dATP Without OH AT 3’ end |
Deoxynucleotide Adenine Dideoxynucleotide |
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DNA sequencing |
Process of determining the precise order of nucleotides within a DNA molecule |
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Sanger sequencing techniques |
Involve radioactive labelling of bases and gel electrophoresis on a single gel Reads sequence of 500-800 bases at a time |
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Stages of DNA sequencing capillary method |
DNA extracted from cell DNA digested into small fragments using restriction endonuclease DNA amplified in polymerase chain reaction to generate multiple copies Each fragment is sequenced into short fragment Data is fed into computer and compared sequenced fragment to find areas of overlap |
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How is each fragment sequenced in short fragments? |
DNA for sequencing is mixed with primer, DNA polymerase , excess of normal nucleotides and dideoxynucleotide Mixture placed in thermal cycles, which changed temperature from 95 to separate DNA fragments to 55 for annealing primers to DNA strand Heated to 60 degree for DNA polymerase to build up new DNA strand by adding complementary bases Dideoxynucleotide is added randomly to terminate synthesise of DNA strand All possible DNA chain lengths produced and fragment separated according to length by capillary sequencing Flourescent markers on terminator base used to identify final base of each fragment sequence of new complementary strand of DNA is used to build up sequence of original strand |
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Why Dideoxynucleotide added randomly ? |
Get DNA fragments of different length After many cycles , all possible lengths produced with reaction stopped at every base |
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Next generation sequencing |
Reaction take place on a flow cell DNA fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments Use coloured terminator base to stop reaction All clusters being sequenced and imaged at same time , technique called massive parallel sequencing |
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Advantage of next generation sequencing |
Efficient and fast Cheap Used in analysis of pathogen genome, identification of species and synthetic biology |
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Bioinformatics Computational biology |
Development of software and computational tools to organise and analyse raw biological data Use data to make theoretical models and make predictions Predict 3D model of protein, identify disease genes, understand gene regulation pathways |
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Whole genome sequence benefits But why is it not a solution to everything? |
Easy and cheap to obtain and can isolate malfunctioning genes Because diseases polymorphic, influenced by environment and cannot predict how genetic diseases behave |
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Why sequence genomes of pathogens? |
To find source of infection To identify antibiotic-resistance strains of bacteria To track spread of transmission and plan suitable treatment options Identify regions that may be useful targets in development of new drugs and vaccine |
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How to identify species using genome? Challenge |
By comparing to standard sequence for particular species But the challenge is produce stock sequence for all different species |
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DNA barcoding |
Identify particular section of genome common to all species but vary between them Animals - cytochrome c oxidase Plants - two regions in DNA of chloroplast No suitable region for fungi and bacteria |
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How to do phylogeny using whole genome ? |
Comparing number of changes in DNA Basic mutation rate can estimate time between species diverging |
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Proteomics |
Study of complete complement of all amino sequence of an organism |
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How is pre mRNA modify? |
Remove introns Exons join tgt by spliceosome |
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Synthetic biology Example |
Construction of artificial biological pathways, organism , genes Genetic engineering, creating whole organism |
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Genetic engineering |
Combining DNA fragment of one organism with DNA of another separate organism |
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Process of genetic engineering |
Obtain DNA from an organism Place gene into a vector to make recombinant dna Recombinant dna with gene enter recipient and replicates |
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How to genetically engineered plants? |
Cut leaf Expose leaf to bacteria carrying weed killer and antibiotic resistance gene to allow bacteria to deliver genes into leaf cells Expose leaf antibiotic to kill cells that lack new genes Wait for gene altered cell to multiply and form callus Allow callus to sprout shoot and roots to develop into plant |
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Gene therapy |
Replace faulty alleles with normal alleles, add a useful gene, treatment of infectious diseases |
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Somatic cell gene therapy |
Temporary Manipulation of gene expression in body cells that will be corrective to patient but not inherited by offspring |
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Germ line gene therapy |
Permanent Gene manipulation of germ cells and alteration is passed onto offspring |
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Why does DNA not pass easily through plasma membrane? |
Because it’s negatively charged |
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Vectors Examples |
Carrier molecule used to deliver genes into target cells Virus and liposomes |
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Problem with virus as vectors |
Toxic Trigger immune response Could recover its ability to cause diseases Disrupt host genome by inserting it to wrong position |
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What is cystic fibrosis caused by? |
Mutated recessive allele located on chromosome 7 Cystic fibrosis transmembrane regulator fails to transport chloride ions out of cells and no inhibitory effect on sodium cells so water move into cells and result in viscous mucus Block airway and Unable to clear airway so lead to infections and prevent sufficient enzymes entering intestine |
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Use of genetically modified bacteria Ethical concerns |
Produce insulin and vaccines Medical and epidemiological research Beneficial in producing medicine and used safely for many years GM pathogens used as biological warfare Develop antibiotic resistance |
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GM plants uses and ethics |
Higher yield as resistance to pests and weed killers Reduction in genetic variation and all genetically identical so susceptible to environmental change and new diseases Gene spread to wild population Does not produce seeds |
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How to obtain gene for genetic engineering? |
Cut gene with restriction enzyme , break hydrogen bonds and phosphodiester bonds Reverse transcribe single stranded mRNA to gene using reverse transcriptase |
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Genetically modified animal uses and ethics |
Disease resistance farmed animals , create animal models with certain diseases by removing or adding genes, create human protein Compromise animal welfare |
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How to insert gene into vector? |
Plasmid cut with same restriction so have complementary sticky needs Use ligase to create recombinant DNA |
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How does recombinant DNA enter recipient? Why is it an inefficient process? |
Plasmid mix with bacterial cell Calcium salts are added Heat shock is applied - temperature lowered to freezing and quickly raised to 40 degrees Because only 1% transformed as some don’t take up palmed |
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The old way of identifying transformed bacteria |
Replica plating Plasmid have marker gene Restriction enzyme cuts right through tetracycline-resistance gene so inactivate it Bacteria grown in agar jelly containing ampicillin so cells that survive have taken up plasmid Each colony grown on plate with tetracycline so those cannot grow must have taken up gene |
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New way to identify transformed bacteria |
Plasmids given second marker gene that produces fluorescence or enzyme that causes colour change Desired gene placed in middle of fluorescent gene so bacteria that doesn’t fluorescent has been genetically engineered Observed under UV light |
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The problem with placing vector inside bacteria an the solution |
Vector is a large molecule and doesn’t easily cross cell membrane Solution is electroporation, micro injection, liposome |
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What is electroporation? |
Small electric current applied to bacteria and membrane become porous so plasmid move into cells |
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What is Electrofusion ? |
Small electric current applied to membrane of two different cells so fuses cell and nuclear membrane Hybrid is formed with DNA from both |
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How is human insulin genetically engineered? |
Remove mRNA from beta cells in islets Incubate mRNA with reverse transcriptase to produce complementary single stranded DNA and convert to double stranded insulin gene Put gene into vector such as plasmid using DNA ligase Use vector to transfer gene into bacteria Bacteria multiply in fermented and produce insulin |
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Why non coding DNA used for DNA profiling? |
Genome is very similar in most people So using coding regions would not provide unique profiles Non coding DNA contain variable number of short tandem repeats |
