Manipulating genomes

• What is gene therapy

  altering alleles in cells to cure genetic disorders
• What could gene therapy be used for

  Cure genetic disorders
• What are genetic disorders

  inherited disorders caused by abnormal genes/chromosomes
• What does gene therapy depend on

  if genetic disorder is caused by a mutated dominant allele or two mutated recessive alleles
• What would gene therapy do if disorder was caused by a mutated recessive allele

  add a working dominant allele-supplement a faulty one
• What would gene therapy do if disorder was caused by a mutated dominant allele

  silence dominant allele by sticking DNA middle so it doesn't work by using vectors
• What is somatic therapy

  altering alleles in body cells particularly in cells most affected
• Disadvantage of somatic therapy

  doesn't affect sex cells so offspring inherit disease
• Germ line therapy 


  altering alleles in sex cells every cell of any offspring produced from cells will affect gene therapy
• Advantages of germ line therapy

  offspring won't inherit disease
• Disadvantages of germ line therapy

  currently illegal in humans

  may kill embryos
• Disadvantages of gene therapy

  -body may identify vector as foreign and trigger an immune response

  -allele may be inserted in the wrong place and cause more problems e.g cancer

  -allele may be overexpressed too much protein cause other problems

  -effects in somatic may be short lived and patients require multiple treatments

  -difficult get allele specific body cells
• Positive ethical issues with gene therapy

  -prolong lives people life-threatening genetic disorders

  -give genetic disorders better quality of life

  -decrease sufferers of genetic disorders

  -allow genetic disorder carriers to conceive baby
• Negative ethical issues


  -potential risk more harm than good e.g. overexpression

  -technology used other treatments e.g. cosmetics and ageing

  -expensive
• What is PCR


  Technique used to select a fragment of DNA (containing a gene/section DNA interested in) and amplify to produce millions of copies and is repeated
• PCR Step 1


  Reaction mixture set up with DNA sample, free nucleotides, primers, DNA polymerase
• What are primers in PCR

  Short pieces DNA complementary to bases at start of fragment
• DNA polymerase in PCR

  Enzyme that creates new DNA strand
• Step 2 in PCR

  DNA mixture heated to 95 degrees to break hydrogen bonds between 2 DNA strands

  DNA polymerase doesn't denature-many cycles of PCR carried out without using lots of enzymes

  Cooled to 50-65 degrees so primers bind or anneal to strands
• Step 3 in PCR

  Mixture heated 72 degrees so DNA polymerase can work-line up free nucleotides along template strand

  Complementary base pairing-new complementary strands are formed
• Step 4 in PCR

  2 new copies of fragment DNA are formed and ne cycle of PCR is complete

  Cycle starts again this time with 4 strands
• What can restriction enzymes be used for

  get DNA fragment from an organisms DNA
• What is a palindromic sequence

  anti-parallel base pairs that can be read the same in opposite directions
• How do restriction enzymes work within DNA

  recognise recognition sites and cut the DNA
• What are recognition sites

  specific palindromic sequences
• Why do different restriction enzymes cut at different recognition sites


  as the recognition site is complementary to the enzymes active site
• Restriction enzymes when recognition site either side of DNA

  1. DNA sample incubated with specific restriction enzyme (cuts DNA via hydrolysis)
  2. Leaves sticky ends-unpaired bases
  3. Sticky ends anneal (bind) DNA fragment with complementary sticky ends
• What is electrophoresis 


  procedure uses electrical current to separate DNA/ RNA fragments or protein depending on size
• Step 1 electrophoresis 


  using agarose gel create rows of wells one end gel

  put gel tray into box/tank ensuring end gel with wells closest negative electrode on gel box

  add buffer solution
• Electrophoresis step 2


  take fragmented DNA sample and micropipette add same volume of loading dye to each-samples sink and more visible

  add set volume DNA sample in well ensuring micropipette doesn't pierce
• Electrophoresis step 3


  put lid on and connect power supply DNA moves anode as DNA slightly negative

  small fragments move quicker

  stain DNA so bands more visible
• How is RNA electrophoresis carried out 


  same way as DNA
• How is protein electrophoresis carried out

  Before undergoes it is mixed with chemical to denature protein so it has the same charge as can either be positive or negatively charged
• What can protein electrophoresis be used for

  identify protein in urine or blood to diagnose diseases
• What are DNA profiles

  some of an organism genome, repetitive non-coding base sequence
• Why is the number of times repeated differs person to person

  lengths of nucleotide differs
• How can sequence repeats be analysed

  Gel electrophoresis-repeats at a specific loci creates DNA profile
• Why is the probability 2 individuals having same DNA profile low

  probability same number of repeats at each locus in DNA is low
• DNA profiling in forensic science

  Compare samples of DNA collected crime scenes

  PCR amplify multiple areas of repeats-primers bind to either side

  Products run on electrophoresis gel and DNA profiles produced and compared to see if match
• DNA profiling in medical diagnosis

  Used to analyse risk of genetic disorders

  Useful mutation isn't known identifies mutation causes disorder