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.
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
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.
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.
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.
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;
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.