Genetic fingerprinting (BIOL5)

Question 1

Genome contains many repetitive non-coding base sequences…

Answer 1

• Probability of 2 individuals having same repetitive seq v low
• chances of having same no seq repeat at each place is v low

Question 2

Electrophoresis separates DNA fragments to make genetic fingerprint…

Answer 2

1. DNA sample obtained (saliva?)
2. PCR used to make copies of areas containing repeated seq; primers used to bind at either side, so repeat amplified
   
   • DNA fragments where length corresponds w/ no. repeats person had at each spec position
3. Fluorescent tag added to fragments to be viewed under UV light
   
   • fragments undergo electrophoresis
     
     • DNA mix placed in well in slab of gel, covered in buffer solution to conduct e-
     • e- current passed through gel - DNA fragments negatively charged, move towards +ve electrode
     • small DNA fragments move faster/travel further through gel, so fragments separate according to size!
4. DNA fragments views as bands under UV light - this is the gen fingerprint
5. 2 compared:
   
   • if both have band at same location on gel, means they have same no. nucleotides, so same no. seq at same place - MATCH!

Question 3

Genetic fingerprinting used to determine relationship/variability…

Answer 3

• Genetic relationship:
  
  • inherit repetitive, non-coding base seq fr parents
  • more bands match = more closely related
• Genetic variability within population:
  
  • greater no. of bands non-matched = more genetically diff ppl are!

Question 4

DNA probes are used to…

Answer 4

• Locate genes or see if person’s DNA contains mutated gene
• Short DNA strands:
  
  • have spec base seq. that’s complementary to base seq of part of target gene
    
    • DNA probe will bind (hybridise) to target gene if present in sample
    • DNA probe has label attached, to be detected
      
      • radioactive/fluorescent

Question 5

How are DNA probes used to locate genes?

Answer 5

1. Sample DNA digested into fragments by restriction enzymes, separated using electrophoresis
2. Separated fragments transferred to nylon membrane, incubated w/ fluorescently labelled DNA probe
3. If gene present, DNA probe will hybridise (bind) to it
4. Membrane exposed to UV light; if gene present, will be a fluorescent band

Question 6

Features of restriction mapping

Answer 6

• Determine base seq. of gene
• Diff restriction enzyme used to cut labelled DNA into fragments
• Size of fragments prod. used to determine relative locations of cut sites
• Restriction map of original DNA made

Question 7

Features of gene sequencing

Answer 7

• Determine order of bases in DNA
• Carried out by chain termination method:

1. Mix added to 4 separate tubes:
   
   • single-stranded DNA - to be seq
   • polymerase - enzyme joins DNA nucleotides
   • primer - short pieces DNA
   • free nucleotides
   • fluorescently-labelled modified nucleotide
     
     • once added to DNA strand, no bases can be formed after
     • diff modified nucleotide added to each tube (A*, T*, C*, G*)
2. Tubes undergo PCR, producing many strands of DNA
   
   • strands diff lengths as each terminates at diff points depending on where modified nucleotide added
     
     • e.g tube A w/ A*, added to DNA at point 4 of A, stopping addition of bases
3. DNA fragments in tubes separated by electrophoresis and visualised under UV light
4. Complementary base seq read from gel
   
   • smallest nucleotide is at bottom of gel
   • each band after represents one more base added

Question 8

Use of DNA probes in medical diagnosis

Answer 8

• Used to screen mutated genes
• Probe can be labelled to look for single gene
• Can be used as part of DNA microarray, which screens lots of genes at a time:
  
  1. DNA microarray is glass slide w/ microscopic spots of diff DNA probes attached to it in rows
     
     • sample of labelled DNA washed over array
     • if labelled DNA contains any DNA seq matching probes, will stick to array
  2. Array washed to remove any labelled DNA that hasn’t stuck
  3. Array visualised under UV light - labelled DNA attached to probe will show
  4. Spots that fluorece mean DNA contains spec gene

Question 9

Features of gene therapy

Answer 9

• Used to treat/cure genetic d/o/cancer
• Involves altering defective genes inside cells:
  
  • D/o caused by 2 mutated recessive alleles:
    
    • add working dominant allele - supplement faulty
  • D/o caused by dominant allele:
    
    • ‘silence’ domiant allele - stick DNA in middle of allele so doesn’t work anymore
• ‘New’ allele is inserted into cells using vectors
• There’re 2 types of therapy - somatic and germ line

Question 10

Somatic gene therapy involves…

Answer 10

Altering alleles in body cells

Question 11

Germ line therapy involves…

Answer 11

Altering alleles in sex cells

So line of offspring made from cells will be affects and won’t suffer disease

Question 12

Advantages of gene therapy

Answer 12

• Prolong lives
• Give better quality of life
• Carriers may be able to conceive baby w/o d/o (germ line)
• Could dec. no. of sufferers (germ line)

Question 13

Disadvantages of gene therapy

Answer 13

• Effects may be short-lived (somatic)
• Patient may undergo multiple treatments (somatic)
• Hard to get allele into spec body cells
• Body could see vectors as foreign bodies, start immune response against em!
• Inserted DNA may get overexpressed, prod too much of missing protein
• Ethical concern - may be used for things other than treatment e.g. cosmetic effects of aging