Describe the use of recombinant DNA technology in the synthesis of human insulin by bacteria [8]

  •  mRNA coding for insulin/isolate gene for human insulin;
  •  from beta cells of islets of Langerhans/pancreas;
  •  reference to reverse transcriptase;
  •  to cDNA;
  •  reference PCR/DNA polymerase/double strand;
  •  reference sticky ends;
  •  use of vector/virus/plasmid;
  •  reference endonuclease/restriction enzymes;
  •  to cut plasmid;
  •  reference DNA ligase to join DNA;
  •  inserted into suitable host cell/E.coli/bacteria;
  •  reference method of insertion;
  •  identification of modified bacteria;
  •  reference growth/culture of engineered bacteria in fermenters

Describe how the gene coding for human insulin can be obtained and inserted into a plasmid vector. [8]

  • obtain mRNA from β cells (of islets of Langerhans of pancreas) ;
  •  reverse transcriptase ;
  •  make (single-stranded) cDNA ;
  •  DNA polymerase used to make cDNA double stranded ;
  •  sticky ends created ;
  •  (obtain) plasmids ;
  •  cut with restriction, endonuclease/ enzyme ; A named e.g. EcoR1
  •  ref. complementary sticky ends ;
  •  cDNA / insulin gene, mixed with plasmid ;
  •  DNA ligase ;
  •  seals nicks in sugar-phosphate backbone ;

Explain the advantages of treating diabetic people with human insulin produced by gene technology.[6]

  •  it is identical to human insulin ;
  •  (more) rapid response ;
  •  no/ fewer, immune response/ side effects/allergic reactions ;
  •  ref. to ethical/ moral/ religious, issues ;
  •  cheaper to produce in large volume/ unlimited availability ;
  •  less risk of, transmitting disease/infection ;
  •  good for people who have developed tolerance to animal insulin ;

Explain how bacteria can be genetically modified and then identified using antibiotic resistance genes.[8]

  • (recombinant) plasmids mixed with bacteria ;
  •  (some) bacteria, take up plasmids/ transformed ;
  •  heat shock / calcium chloride solution/Ca 2+ ions/electroporation ;
  • to identify bacteria containing plasmids
  •  grow on, agar/medium, containing antibiotic (A) ;
  •  plasmid contains, antibiotic (A)/ ampicillin, resistance gene(s) ;
  •  bacteria with plasmid survive ;
  • to identify recombinant bacteria
  •  replica plate ;
  • (onto) agar/ medium, containing second antibiotic (B) ; A tetracycline
  •  resistance gene inactivated (by insertion of new, DNA/ gene

Outline how genetic fingerprinting is carried out [8]
Describe how electrophoresis is used in genetic fingerprinting [8]

  • ref. to VNTR (sequences) ;
  • quantity of DNA increased by PCR ;
  •  DNA fragmented by, restriction enzyme(s) / endonuclease(s) ;
  •  loaded (into wells) in agarose gel ;
  •  (at) negative end/ cathode end ;
  •  ref. to buffer/ electrolyte ;
  •  direct current applied ;
  •  phosphate groups of DNA give negative charge ;
  •  (negatively charged) DNA attracted to, anode/ positive electrode ;
  •  short pieces/ smaller mass, move further/move faster ;
  •  (pieces) transferred to, membrane/ nylon/nitrocellulose/ absorbent paper or Southern blotting ;
  •  heated to separate strands ;
  •  probes/ fluorescent dye, added ;
  •  X-ray film/ UV light/ lasers ;
  •  pattern of stripes/ ref. banding pattern ;

Decsribe how genetic screening is carried out in an adult human [8]

  • samples of cells taken
  • e.g white blood cells
  • test for chromosomal abnormalities
  • e.g Down’s Syndrom
  • cells cultured so that they divide
  • stopped at metaphase
  • use of colchicines
  • cells made to take up water
  • chromosomes separated
  • karyotype produced
  • flourescent makers to help detect abnormalities
  • test for particular allele
  • DNA extracted
  • DNA cut with restriction enzyme
  • electrophoresis occurs
  • to separate DNA into strands
  • complementary/specific probe(for mutant allele)

Discuss the advantages of screening for genetic conditions[8]

  • information about the increased risk of person having genetic conditions ;
  •  ref. breast cancer/ named example ;
  •  allows people to prepare for late onset genetic conditions ;
  •  ref. Huntington’s disease/Alzheimer’s disease/named example ;
  •  identify whether fetuses are going to develop a genetic condition ;
  •  so can give early treatment when born ;
  •  allows parents to prepare for the birth of a child who will need treatment for a considerable time or even throughout life ;
  •  identifies carriers of genetic conditions ;
  •  helps to provide early diagnosis ;
  •  allows couples who are both carriers of a genetic condition to make decisions about starting a family/ having more children/ seeking IVF ;

Describe the ways by which gene mutations can occur. [6]

  •  change in, base / nucleotide, sequence (in DNA) ;
  •  during DNA replication ;
  •  detail of change ; e.g. base, substitution / addition / deletion
  •  frame shifts /;
  •  different / new, allele ;
  •  random / spontaneous ;
  •  mutagens ;
  •  ionising radiation ;
  •  UV radiation / mustard gas ;

Explain how genetic diseases may be treated using gene therapy. [6]

  • normal, gene/ allele ;
  • (insert into) vector ;
  • liposomes (as vectors) ;
  •  liposomes in, aerosol/ inhaler ;
  •  liposome fuses with host cell ;
  •  virus (as vector) ;
  •  virus vector harmless ;
  •  short term effect ;
  •  repeat treatments needed ;
  •  side effects ;

Explain how changes in the nucleotide sequence of DNA may affect the amino acid sequence in a protein. [7]

  •  code is three, bases / nucleotides ; triplet code
  •  (gene) mutation ; chromosome mutation
  •  base, substitution / addition / deletion ;
  •  addition / deletion, large effect (on amino acid sequence) ;
  •  frame shift ;
  •  completely new code after mutation / alters every 3 base sequence which follows ;
  •  (substitution) often has no effect / silent mutation ;
  •  different triplet but same amino acid / new amino acid in non-functional part of protein ;
  •  (substitution) may have big effect (on amino acid sequence) ;
  •  could produce ‘stop’ codon ;
  •  sickle cell anaemia / PKU / cystic fibrosis ;
  •  reference to transcription or translation in correct context ;

Explain the advantages and disadvantages of genetic screening in humans.

  • Advantges
    •  known risk for next generation;
    • allows possible abortion of affected fetus;
    • allows sorting of IVF embryos;
  • Disadvantages
    • loss of hope;
    • insurance company problems;
    • employment problems;
    • miscarriage risk;
    • trauma of knowledge;

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