Manipulating genomes

• 3 hydrogen bonds form between C and G in DNA
• 2 hydrogen bonds form between A and T in DNA
• in a nucleotide, the phosphate group attached to the 5' carbon on a deoxyribose sugar
• the Meselson and Stahl experiment is evidence for semi-conservative replication
• Meselson and Stahl experiment:

  1. Bacteria were grown in a medium containing heavy isotope N15 for many generations.
  2. Some bacteria were moved to a medium containing light isotope N14. Samples were extracted after 1 & 2 cycles of DNA replication.
  3. Centrifugation formed a pellet. Heavier DNA (bases made from N15) settled closer to bottom of tube.

  • shows that all DNA is different so semi-conservative DNA replication occurred
• a genome is all of the genetic material an organism contains
• a chromatid is one of the two identical strands of DNA that make up a chromosome
• a telomere is molecular protective caps of repetitive DNA at the ends of chromosomes
• genomic DNA includes all DNA- nuclear DNA, mitochondrial DNA, all coding and noncoding DNA
• 2% of the genome are exons
• satellite sequences are tiny non-coding sequences repeated, they are found in the same location in everyone but the number of repeats varies between individuals
• satellite sequences are useful for identifying people e.g. forming a DNA profile of someone
• DNA profiling is looking at patterns in the DNA of an individual (genetic fingerprinting)
• PCR stands for Polymerase Chain Reaction
• PCR enables scientists to amplify sequences of DNA when small recovered samples would otherwise be impractical to use
• PCR is the artificial replication of DNA
• PCR relies on:

  • DNA is made of 2 antiparallel backbone strands
  • each strand of DNA has a 5' end and a 3' end
  • DNA grows only from the 3' end
  • base pairs pair up according to complementary base pairing
• PCR differs from DNA replication as:

  • only short sequences of up to 10,000 base pairs of DNA can be replicated in PCR, not entire chromosomes
  • it requires the addition of primer molecules to make the process start
  • a cycle of heating and cooling is needed to separate DNA strands, bind primers to the strands, and for the DNA strands to be replicated (its a cyclic reaction)
• the enzyme Taq DNA Polymerase is obtained from a bacteria that lives at high temperatures (thermophilic bacteria) so it doesn't denature at high temperatures
• in PCR the amount of DNA increases exponentially
• in PCR the sample of DNA is mixed with DNA nucleotides, primers, Mg ions, and the enzyme Taq DNA Polymerase
• in PCR the mixture is heated to 94-96 degrees to break the H-bonds between complementary nucleotides, denaturing the double stranded DNA into 2 single strands of DNA and then is cooled to approx 68 degrees so the primers can bind by H-bonding to one end of the single strand of DNA. this gives a small section of double stranded DNA at the end of each single stranded molecule
• in PCR the enzymes bind to the end where there is double stranded DNA and then the temperature is risen to 72 degrees which keeps the DNA as single strands
• in PCR the enzyme Taq DNA polymerase catalyses the addition of DNA nucleotides to the single stranded DNA starting at primer in 5' to 3' direction and when the enzyme reaches the other end of the DNA molecule, a new double strand of DNA has been generated
• the process of PCR is repeated for many cycles
• uses of PCR:

  • amplify DNA sampling for sequencing
  • tissue typing - donor tissues for transplantation
  • detection of oncogenes - find type of mutation in specific cancer for tailored medicines
  • detection of mutations - could lead to genetic diseases
  • identifying viral infections
  • monitoring spread of infectious disease
  • forensic science
  • research - dna from extinct sources
• electrophoresis is a method of separating DNA fragments or proteins using an electric charge
• separating proteins using electrophoresis:

  • the principle is the same as for separating DNA fragments, but it is often carried out in the presence of a charged detergent such as sodium dodecyl sulphate (SDS), which equalises the surface charge on the molecules and allows the proteins to separate as they move through the gel, according to their molecular mass
  • in some cases the proteins can be separated according to mass and then, without SDS according to their SA
• separating proteins by electrophoresis can be used to analyse the types of haemoglobin proteins for diagnosis of conditions such as sickle cell anaemia and aplastic anaemia and thalassaemia
• DNA probes are a short (50-80 nucleotides) single stranded length of DNA that is complementary to a section of the DNA being investigated
• DNA probes are labelled using:

  • radioactive markers
  • a fluorescent marker that emits a colour on exposure to UV light
• DNA probes are useful in locating specific DNA sequences

  e.g. locate a specific gene needed for genetic engineering
• microarrays are a number of different probes on a fixed surface
• how a microarray works:

  • DNA sample must first be broken down into smaller fragments
  • microarray can be made with fixed probes specific for certain sequences found in mutated alleles causing genetic diseases
  • DNA samples are labelled with fluorescent markers
  • where a test subject and a reference marker both bind to a probe the scan reveals fluorescence of both colours indicating the presence of the particular sequence in the test DNA
• microarrays work as applying the DNA under investigation to the surface can reveal the presence of mutated alleles that match the fixed probes, because the sample will anneal to any complementary fixed probes
• the lagging strand in DNA replication is made in small pieces (Okazaki fragments) joined by ligase
• DNA sequencing involves reading the base sequence of a length of DNA

  based on a technique developed by Fred Sanger in 1975
• the first step in the procedure for chain termination sequencing:

  • label 4 test tubes A, T, C, G into each test tube add:

  a sample of DNA to be sequences, a radioactive primer, the 4 DNA nucleotides, the enzyme DNA polymerase
• the second step in the procedure for chain termination sequencing:

  • add a small amount of a special modified dideoxy nucleotide that cannot form a phosphodiester bond and so stops further synthesis of DNA
• the third step in the procedure for chain termination sequencing:

  • let the DNA polymerase synthesise many copies of DNA
  • a range of DNA molecules will be synthesised as stop at different times
  • in tube A all the fragments will stop at an A nucleotide etc.