manipulating genomes

• dna sequencing


  finding the nucleotide sequence for a gene or a whole genome
• sanger sequencing 1

  1. create copies of DNA fragments-extract dna sample -add heat-to seperate two dna strands- then cut dna strands into dna fragments which then can create many copies
• sanger sequncing 2


  create complimenatry strand for each dna fragment

  1.place dna fragments with, dna nucleotides, dna polymerase,dna primer,terminating dna nucletides

  -dna polymerase use dna primers to attach to dna fragments

  -then make complimentary dna fragments with dna nucletides

  -terminating dna nucletides stop it from adding further nucleotides

  = so they all have different terminating nucleotides
• sanger sequencing 3


  analyse complementary fragments by seperating using agrose gel

  and length

  and because we know the base we can workout the original strand as it is complementary

  
• high throughput 


  is automated

  very rapid

  cheaper
• benefits of dna sequencing

  1. genome wide comparison between individuals and species which reveals how closely related species are
  2. predicts amino acid sequence of genes-allows prediction of Tertiary structure of polypeptide
  3. used for synthetic biology which modifies existing dna sequences
• gel elctrophoresis


  which is to seperate DNA, RNA, proteins

  dna and rna are seperated by mass

  shorter fragments have a lower mass

  proteins can be seperated by mass or dterminied by the size of their R groups or number of amino acid present

  or by charge determined by the R groups
• stages of elctrophoresis


  1.get agar gel amd cut holes called wells

  2.submerge in a buffer soulution

  3.load molecules in a well and place negative elctrode next to the well

  4. and positive at the opposite side of the gel

  5.apply electric current moving from negative to positive lighter molecules can move across the gel and alsoif it is negative

  -so molecules with more light and negative can move further

  diffrent bands are seen by adding a flurecent dye to the gel which glows under uv light
• use of gel electropheresis


  genome sequencing

  dna profiling
• uses of genetic engeneering

  gene therapy is when a patient dna is altered to cure a disease

  somatic -targets tissues that need treatment, short lived effects

  germ line -long term effects

  inherited

  can modify plants for insect resistance

  modify pathogens for new medical treatment
• pharming


  animals dna is altered to produce human protein for medicine or to develop illness to test on them
• ethical issues


  animals

  patents-prevents other companies, can charge large amounts of money
• genetic engeneering 


  isolating a gene from one organism and placing it into another and then can translate the gene as genetic code is universal
• producing dna fragments


  restriction endonuclease- which is an enzyme that cuts specific set of bases known as a recognition sequence or site and if there is two it results in a dna fragment can produce sticky or blunt ends
• producing dna fragments


  reverse transcriptase- which dna nucletides to an mrna molecule which forms a strand called cDNA and an enzyme destroys original strand and then dna polymerase builds the second strand
• genetic engeneering

  1. prepare dna fragment by using restriction endonuclease to create sticky ends
  2. insert dna into vector usually a plasmid use same restriction endonuclease so it is complimenmtary
  3. and use dna ligase to join the plamid and dna fragment
  4. which forms a recombinant plasmid
  5. recombinant plasmid is inserted into bacteria this process is called transformation and requires calcium ions and heat shock and makes it easier for plasmid to pass through bacteria cell membrane