Gene technology

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Genetic fingerprinting steps

Steps for genetic fingerprinting:

  • DNA sample is extracted and isolated e.g. blood or hair etc.
  • Restriction endonulceases are used to cut the DNA sample in fragments.
  • Fragments multiplied using PCR.
  • DNA fragments applied to a gel and voltage is applied.
  • Fragments separated by electrophoresis.
  • Fragments then transferred to nylon membrane through Southern Blotting.
  • DNA probes (radioactive or fluorescent) are attached to core sequences/tandem repeats.
  • Membrane placed under x-ray film or UV light to expose relative positions of fragments.

Short lengths of DNA travel further.

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PCR Technique

Stages of PCR:

  • A mixture is prepared that contains the DNA sample to be cloned, free nucleotides, primers and DNA polymerase.
  • The mixture is heated to 95 degrees to break the hydrogen bonds and cause the strands to unzip.
  • The mixture is then rapidly cooled to around 50 degrees so that the primers can anneal (bind) to the separated strands.
  • The mixture is then heated up to 72 degrees to allow the DNA polymerase to work - lining up free nucleotides alongside the original strands so they bind and create a hyrbrid strand.

The cycle is then repeated

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Stages of transcription:

  • DNA helicase unwinds a section of DNA that is required for synthesis by breaking hydrogen bonds.
  • One strand is used as a template to form mRNA. RNA polymerase attaches at the start of the strand.
  • It lines up free-floating RNA nucleotides alongside the template strand and base pairing means that the mRNA strand formed is complementary to original DNA template.
  • RNA polymerase moves along the DNA strand, assembling pre-mRNA. The hydrogen bonds reform and the original strands coil back into a helix.
  • When RNA polymerase reaches a terminator region and detaches from the DNA - stops making mRNA.
  • Spliceosomes remove introns from pre-mRNA to turn it into mRNA,
  • mRNA leaves via nuclear pores to be translated.
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Steps for translation:

  • mRNA enters the cytoplasm and attaches to a ribosome. tRNA molecules with complementray anticodons and amino acid attached arrive at the ribosome.
  • A tRNA molecule with an anticodon complementary to the first codon on the mRNA attaches to it via complementary base pairing.
  • A second tRNA molecule attaches itself in the same way.
  • The two tRNA molecules have amino acids attached, these two amino acids are joined by a peptide bond (condensation reaction). Once this occurs, the first tRNA molecule detaches as moves away.
  • A third tRNA molecule binds to the next mRNA codon. The amino acid binds to the second amino acid and the second tRNA molecule moves away.
  • The process is repeated, producing a polypeptide chain until a stop codon is reached.
  • Polypeptide chain detaches.
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Transcription model answer

  • DNA helicase unwinds the region of DNA to be translated - the DNA strands separate, exposing the promoter region.
  • RNA polymerase complex assembles onto the promoter region.
  • RNA polymerase then moves along the coding strand and selects complimentary free nucleotides to form the mRNA strand via polymerase condensation.
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Translation model answer

1. mRNA leaves (nucleus) through nuclear pore;
2. To ribosome;
3. tRNA molecules bring amino acids (to ribosome);
4. Specific tRNA molecule for specific amino acid;
5. Anticodon of tRNA corresponds / complementary to codon on mRNA;
6. Peptide bonds form between amino acids;
7. tRNA detaches and collects another amino acid;
8. Ribosome moves along mRNA;

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In Vivo Cloning

Steps for In Vivo cloning:

  • Vector DNA and the DNA required are cut using the same restriction enzyme. This results in the sticky ends of the vector and isolated DNA being complementary.
  • The vector DNA and DNA fragment are mixed together with DNA ligase which joins the phosphate backbones together (sticky end to sticky end)
  • This is called recombinant DNA.
  • The recombinant vector is now inserted into a host cell. With plasmids, host bacterial cells are placed in ice-cold Ca2+ solution. Plasmids are added and then mixture is heat-shocked to encourage bacteria to take up the plasmids. Bacteriophages will inject the recombinant DNA into the bacteria.
  • Marker genes are used to identify the cells that have taken up the plasmid. These can be inserted into vectors at the same time as the required gene.
  • This can be done by replica plating - where antibiotic resistance is tested - the plasmid often replaces a resistance gene. Or using a fluroresence marker - the gene will cause transformed cells to fluoresce under UV light. 
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