6.3 Manipulating Genomes

  • Created by: elbungay1
  • Created on: 08-06-19 17:16
What is DNA Sequencing?
A technique that allows the polynucleotide sequence of a gene to be isolated and read
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What is the DNA mixed with in Sanger Sequencing?
DNA added to a mixture containing a solution of each nitrogenous base, DNA polymerase and modified 'terminator bases', that stops further bases being added by DNA polymerase after the complementary bind, labelled by radioactive/fluorescent marker
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How is DNA sequenced in Sanger Sequencing?
All lengths of DNA generated and separated by capillary electrophoresis, shorter lengths travel fastest and terminator base marked read by laser, computer builds up sequence
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What modern techniques are there for DNA sequencing
High Throughput Sequencing (Pyrosequencing)
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What is Bioinformatics?
Store of large amounts of data from DNA sequencing. Uses include medical, phylogeny
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What are the applications of gene sequencing?
Comparisons between species, evolutionary relationships, variation between individuals, predicting amino acid sequence of proteins
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What is Synthetic Biology?
Science concerned with designing and building useful biological devices and systems. Applications include information storage, medicine production, nanotechnology, biosensors
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What are Tandem repeats?
Repetitive segments of DNA that do not code for proteins. Number of tandem repeats can show family resemblance
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How is DNA obtained for DNA Profiling?
Mouth swab, saliva from toothbrush, blood or hair, or for ancient remains, bone
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Describe the process of DNA profiling
DNA digested with restriction enzymes, size will vary per individual, fragments separated by gel electrophoresis. Banding pattern can be seen, DNA compared treated with same enzymes and banding patterns compared
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What type of DNA is analysed in DNA Profiling?
Short Tandem repeats, highly variable short repeating lengths of DNA. Exact number varies per individual. Highly sensitive
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What are the applications of DNA Profiling
Forensic Science (establish innocence or guilt), Maternity and Paternity disputes (half STRs from mother and father), analysis of disease (protein electrophoresis can detect and diagnose sickle cell anaemia)
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What is the Polymerase Chain Reaction (PCR)?
Biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
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What principles does PCR rely on?
DNA only grows 5' to 3' direction, bases pair up using complementary pairing rules, DNA made of two antiparallel strands
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In what ways does PCR differ from Semi-conservative replication?
In PCR, only short lengths can be replicated, not whole chromosomes. PCR requires addition of Primer molecule for process to start, a cycle of heating and cooling is requires to separate strands, primer anneal and for replication.
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What does the PCR mixture contain before the process starts?
The sample of DNA to be replicated, free DNA nucleotides, primers, magnesium ions and the enzyme Taq Polymerase
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Why is the mixture heated to 94-96°C?
Break the hydrogen bonds between complementary nucleotide base pairs and thus denature the double-stranded DNA into two single strands of DNA.
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What happens when mixture is cooled to 68°C?
Primers can anneal to one end of each single strand of DNA. This provides a short strand of double-stranded DNA at each end. Taq polymerase can only bind to double stranded DNA
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Why is the mixture then heated to 72°C?
Optimum temperature for Taq Polymerase and keeps DNA as single strands. Enzyme catalyses addition of DNA nucleotides to single stranded DNA molecules, starting at primer in 5' direction. When enzyme reaches other end, new strand of DNA synthesised
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What are the applications of PCR?
Tissue typing (lowers risk of rejection in transplant), detecting mutations for genetic diseases, identifying viral infections (detect viral genome), forensic science, research
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What is Electrophoresis?
Process used to separate proteins or DNA fragments of different sizes
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Describe how an electrophoresis gel plate is set up
Agarose gel is covered by buffer solution. Electrodes are placed at each end. DNA has overall negetive charge (phosphate groups) so wells placed at cathode and DNA travels towards anode
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How are fragments of DNA separated?
DNA fragments have the same overall charge regardless of size, but smaller fragments experience less resistance in the agarose gel matrix so are able to travel at a faster rate
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How is protein electrophoresis different?
Needs charged detergent (SDS) to equalise surface charge on molecules, allows proteins to separate as they move through gel according to molecular mass. Uses include diagnosis of sickle cell anaemia
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What is a DNA probe?
Short length of single stranded DNA complementary to section of DNA being investigated. Fluorecent or radioactive marker. Uses to locate genes for genetic engineering, genome comparisons, find alleles for genetic diseases in diagnosis
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What is a Microarray?
Surface with a number of different fixed probes, applying DNA can reveal presence of mutated alleles that match the probes, as sample will complementary anneal
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What are the main stages of genetic engineering?
1. Required gene obtained. 2. a copy of the gene is placed inside a vector. 3. Vector carries gene into recipient cell. 4. recepient expresses the novel gene
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How can the required gene obtained in genetic engineering?
mRNA obtained from cells where gene is expressed. Reverse Transcriptase can catalyse formation of single strand of complementary DNA (cDNA), addition of primera and DNA polymerase can catalyse formation of double stranded DNA, base codes for protein
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How is the gene placed into a vector?
Plasmids obtained from bacteria and mixed with restriction enzymes, cut at recognition sites, creating sticky ends, nucleotide bases added to gene to create complementary sticky ends and DNA ligase catalyses annealing. May use attenuated virus
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How is the vector placed into recipient cell?
Heat Shock (alternating 0°C and 42°C in calcium chloride, increase fluidity and reduced repulsion of DNA). Electroporation (high voltage disrupts membrane). Transfection (bacteriophage transfects host cell)
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How is recombinant DNA detected in GM E.Coli in insulin production?
Plasmids have ampicillin and tetracycline resistant genes, insulin gene splcied into tetracycline resistance gene, disrupting it. In amplicillin agar, all colonies grow, in tetracycline, only non-recombinant colonies grow.
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What are potentials risks of Genetic Manipulation?
Micoorganisms escaping and spreading antibiotic resistance, GM crops toxic to natural species, herbicide resistance passed to weeds, people won't consume food with foreign DNA, welfare of animals used to testing
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What is Somatic Cell Gene Therapy?
Gene therapy by inserting functioning alleles into body cells. Only affects certain cell types and alternations not passed onto offspring. Cystic fibrosis treated by inhaling liposomes with CFTR gene, affects cells in respiratory tract.
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What are the potential risk of using Viruses as vectors in gene therapy
May provoke immune or inflammatory response, patient may become immune, virus may place allele in wrong location disrupting genome, increasing risk of cancer. Virus may insert allele into DNA that disrupts gene regulation
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What is Germ Line gene therapy?
Involves altering the genome of gametes or zygotes. Offspring may also inherit functioning alleles and all cells of body is altered. Risk of changing genetic makeup and wrong insertion can increase risk of cancer
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Card 2


What is the DNA mixed with in Sanger Sequencing?


DNA added to a mixture containing a solution of each nitrogenous base, DNA polymerase and modified 'terminator bases', that stops further bases being added by DNA polymerase after the complementary bind, labelled by radioactive/fluorescent marker

Card 3


How is DNA sequenced in Sanger Sequencing?


Preview of the front of card 3

Card 4


What modern techniques are there for DNA sequencing


Preview of the front of card 4

Card 5


What is Bioinformatics?


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