Manipulating genomes

• DNA profiling produces an image of the patterns in the DNA of an individual. It involves the identification of repeating patterns in the non-coding regions of DNA
• DNA profile is a genetic fingerprint that is unique to each person, often formed from sections of DNA called variable number tandem repeats.
• To form a DNA profile you need to:

  1. extract DNA and make copies using PCR
  2. digested/fragmented using restriction endonuclease
  3. separate fragments using electorophoresis
  4. separate into single strands using alkaline solution
  5. transfer from gel to nylon by Southern blotting
  6. Hybridised with probes
  7. development/ visualisation on x-ray film
• Restriction endonuclease are enzymes found in bacteria, each cuts DNA at specific base sequences
• A PCR machine is called a thermocycler
• Stage 1 of PCR is 95 degrees when hydrogen bonds are broken
• Stage 2 of PCR is 55 degrees when primers anneal to DNA strand
• Stage 3 of PCR is 72 degrees when Taq DNA polymerase joins free nucleotides to each strand
• Taq DNA polymerase is more tolerant to heat and will not denature so is better than human DNA.
• Electrophoresis

  1. DNA fragments in wells at top of agar gel plate
  2. Positive electrode at opposite end
  3. current applied, -ve DNA move towards positive electrode
  4. Longer fragments move slower
• Profiling is useful for forensics, paternity tests, disease risk analysis and classification
• 2 ways nucleotides can be modified for sequencing is terminate polymerisation and fluorescently coloured
• The first step in DNA sequencing is PCR
• DNA sequencing helps disease anaylsis, classification, synthetic biology, genotype-phenotype relationships
• Sticky ends are produced when DNA is cut with the same restriction endonuclease
• Reverse transcriptase is an enzyme used to convert mRNA to cDNA
• DNA ligase is an enzyme that joins sugar-phosphate backbones
• Transgenic is an organism that carries a gene from another organism
• The polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences and produce a billion copies of the target sequence in just a few hours and an important tool in forensic DNA analysis and genetic disease DNA analysis.
• Electrophoresis is a laboratory technique used to seperate DNA, RNA, or protein molecules based on their size and electrical charge
• A DNA probe is a short single stranded piece of DNA that is complemenry to a section of the DNA being investigated whcih can be labelled with either a radioactive or fluorescent marker
• DNA is mixed with Primers, Taq polymerase, normal nucleotide bases and terminator bases
• DNA sequencing show patterns of inherited DNA, show diseases that we are vulnerable to and affects epidemiology. This has helped allow for genome wide comparison
• DNA sequencing compares the sequences from different organisms, rate of mutation used to figure out when the organisms had a common ancestor. This helps allow for the study of evolutionary relationships