Module 6.1.3- Manipulating genomes

• Genome


  All the genetic information in an organism; all of an organism's chromosomes.
• Introns


  Noncoding region of nucleic acid that lie between coding sequences.
• DNA Profiling


  producing an image of the patterns in the non-coding DNA of an individual
• Polymerase chain reaction (PCR) ingredients


  -DNA sample

  -DNA primers

  -Free nucleotides

  -Taq polymerase

  -Thermocycler

  -Buffers
• DNA Primers in PCR


  short sequences of DNA nucleotides that are complementary to the opposing 3' to 5' ends of the DNA target sequence that is to be replicated
• Taq polymerase in PCR


  A DNA polymerase enzyme that can withstand the high temperatures of PCR
• PCR- Step 1 at 95 degrees celcius


  temperature is increased to 95 degrees Celcius

  • this denatures DNA by breaking the hydrogen bonds separating the strands
• PCR- Step 2 at 55 degrees celcius (after strands separate)

  temperature is decreased to 55 degrees Celcius

  • primers bind to ends of the DNA strands
  • hydrogen bonds are reformed
• PCR- Step 3 at 72 degrees celcius (after primers bind to DNA)

  temperature is increased to 72 degrees Celcius

  • this is the optimum temperature for Taq polymerase to work best
  • Taq polymerase binds to primer-free nucleotides join together to primer through the formation of phosphodiester bonds building up the complementary strand of DNA
• PCR- Overall summary

  1) 95 degrees - separating strands by breaking H-bonds

  2) 55 degrees - primer binds to ends of DNA strand through reformation of H-bonds

  3) 72 degrees- optimum temp for Taq polymerase which binds to primer. Free nucleotides join together to primer through phosphodiester bonds to build the complementary strand of DNA
• Gel electrophoresis


  Procedure used to separate and analyze DNA fragments (of different lengths) by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel
• Gel electrophoresis components

  -Restriction enzymes: cut/digest DNA at a certain sequence

  -Buffer: ions which carry a current and to maintain pH

  -Wells: DNA sample is injected into the well

  -Gel: made up of agarose

  
• Process of gel electrophoresis

  1. A DNA sample is obtained and PCR is used to amplify the DNA (VNTR sections of DNA)

  2. They areplaced in a well in a gel slab covered in buffered solution at the cathode end

  3. An electrical charge is passed through the gel and DNA moves to the Positive electrodes

  4. Smaller fragments move further than large fragments as there is less resistance.
• In gel electrophoresis, why is DNA placed in the cathode end (negative) rather than the anode end (positive)

  
  

  DNA is negatively charged due to the phosphates group and therefore it repels towards the positive anode side separating the fragments
• Producing a DNA profile

  1. extract DNA, make copies using PCR

  2. digested/fragmented using restriction endonuclease

  3. separate fragments using electrophoresis

  4. separate into single strands using alkaline solution

  5. transfer from gel to nylon by Southern blotting

  6. hybridised with probes (fluorescent/radioactive)

  7. development/visualisation on x-ray film
• Restriction endonucleases


  an enzyme that cuts DNA at specific sites (restriction sites), producing small fragments used in genetic engineering.

  
• Role of alkaline solution in DNA profiling


  Separates the DNA double strands into single strands
• Southern Blotting


  A hybridisation technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA.

  Strands are transferred to a nylon membrane which is placed over the gel. The membrane is covered with several sheets of dry adsorbent paper, drawing the alkaline solution containing DNA through the membrane by capillary action

  
• Hybridisation in DNA Profiling

  the blot is subjected to hybridisation using a labelled DNA probe

  
  

  -the probe may be labelled with a radioactive marker visualised through trough photography

  -labelled with fluorescent marker and visualised

  -multiple different probes can be used at once on a fixed surface known as microarray
• DNA sequencing


  the process of determining the precise order of nucleotides within a DNA molecule
• DNA Sequencing process (first steps only which are the same as PCR)

  1. Take DNA sample and mix with primers, DNA polymerase, excess free nucleotides and terminator bases with coloured fluorescent marker attached.

  2. Temperature is increased to 96 C using a thermal cycler separating the double-stranded DNA into single strands.

  3. Temperature is then decreased to 50 C so that primers anneal to the DNA strand.

  4. Temperature is then increased to 60 C to allow DNA polymerase to build up new DNA strands by adding nucleotides with complementary bases to the single-strand DNA template
• DNA sequencing process (after using thermal cycler/changing temperature)

  4. terminator bases terminate the synthesis of DNA and so adding no more bases. this results in many DNA fragments of different lengths

  5. the DNA fragments are then separated by capillary sequencing (similar to gel electrophoresis). fluorescent markers on the terminator bases are used toidentify the final base on each fragment and lasers detect colours.

  6. order of bases in the capillary tube shows sequence of the new complementary DNA strands. a computer is used
• Rapid advancements of DNA sequencing

  
  

  • New automated high through put sequencing
  • Sequencing reaction takes place on a plastic slide known as a flow cell
  • Entire genomes are sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences reducing cost as well
• Suggest why a genome has to be fragmented before sequencing

  
  

  • Genome too big
  • There will be fewer errors with smaller fragments
  • Divide the job between different labs
• Bioinformatics


  the development of the software and computing tools needed to analyse and organise raw biological data
• Computational biology


  The study of biology using computational techniques to analyse large amounts of data e.g. analysing DNA sequencing information for identifying genes linked to specific disease
• Analysing the genomes of pathogens


  
  

  • find outsource of infection
  • identify antibiotic
  • resistant strains of bacteria
  • track progressof an outbreak of a potential serious disease
  • identify regions in genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines
• Identifying species (DNA barcoding)


  a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species
• Proteomics


  the study and amino acids sequencing of an organism's entire protein complement
• Spliceosome


  A large complex made up of proteins and RNA molecules that splices RNA by interacting with the ends of an RNA intron, releasing the intron andjoining the two adjacent exons (using spliceosomes)

  Spliceosomes may join the same exons in a variety of ways and therefore a single gene may code for different mRNA and so different proteins
• Synthetic biology


  ability to sequence the genome of organisms and understand how each sequence is translated into amino acids along with the increasing ability of computers to store, manipulate, and analyse the data forming a new field of biology
• Example of different techniques in synthetic biology

  -genetic engineering

  -use of biological systems or parts of biological system in industrial contexts

  -synthesis of new genes to replace faulty genes

  -synthesis of a new entire organism
• Describe how X gene could be removed from the DNA of an organism and inserted into bacteria:

  
  

  • Restriction endonuclease enzymes
  • Cut the specific base sequence of the X gene
  • Creating sticky ends
  • Use the same restriction enzymes to cut the plasmid
  • Creates complementary sticky ends
  • DNA ligase is used to seal the X gene into the plasmid
  • Plasmid is transported into bacteria using a vector, such as electroporation/shock treatment
• What 2 cuts can restriction endonuclease enzymes create


  Staggered sticky ends and straight blunt ends

  
• Isolating desired gene using the enzyme....

  Using the enzyme reverse transcriptase to produce a single strand of complementary DNA. (cDNA)

  Advantage is that it is easier to identify the desired gene

  
• Recombinant DNA


  DNA produced by combining DNA from different sources
• Electroporation


  A technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing the cells. The pulse creates temporary holes in the cells' plasma membrane, through which DNA can enter fusing with nuclear DNA
• Disadvantages of electroporation


  -power of electric current can cause cell death

  -has to be carefully controlled

  -muscle contraction

  -less useful in whole organisms
• Electrofusion


  tiny electrical currents are applied to the membrane of two different cells. this fuses the cell and nuclear membranes of the two different cells together to form a hybridor polyploid cell , containing DNA from both
• Pros of genetically modified crops

  
  

  • Pest-resistant
  • GM crops
  • Increased yield
  • Disease resistance
  • Herbicide resistance
  • Extended shelf-life
  • Can be flood/drought resistance
  • Increased nutritional value
  • Medical uses