topic 7

• PCR: used to amplify the DNA by making millions of copies of given DNA samples
• The process of PCR is:

  1. a reaction of a mix of the DNA sample is added to primers, free nucleotides and DNS polymerase
  2. this mix is then heated to 95 degrees to break the hydrogen bonds between the complementary base pairings and separating the two strands
  3. the mixture is then copied to a temperature between 50 and 60 degrees so the primers can bind to the stands
  4. the temp is then increased to 70 degrees, at this temp the DNA polymerase works and creates a copy of the sample by complementary base pairings using free nucleotides
• PCR can by used in both DNA sequencing and DNA profiling
• DNA sequencing is used to predict the amino acid sequences of proteins and determines the links to genetically determined conditions
• DNA profiling is used to identify criminals and test paternity
• In DNA sequencing:

  1. The sample is split into 4 sections which have been fluorescently labelled
  2. a terminator nucleotide is added to a growing chain, replication is terminated
  3. DNA fragments of different lengths are produced across the reaction vessels
  4. High-resolution gel electrophoresis is used to separate the fragments by size
  5. these fragments are visualised under UV light enabling the base sequences to be read from the bottom of the gel
• DNA profiling is:

  1. fragments of DNA are cut with the restriction endonuclease enzymes
  2. these fragments are separated and visualised using gel electrophoresis
  3. Southern blot (a buffer solution) is added which draws the solution containing the DNA fragments to the surface
  4. these are then compared and the number recorded is produced on a graph
• multipotent cells are cells which can differentiate into multiple cell types, such as neurons, muscle cells and blood cells
• pluripotent cells can differentiate into all types of cells but not placenta cells
• totipotent cells can differentiate into all cell types including placenta cells
• transcription factors:

  proteins that bind to DNA base sequences and help regulate transcription
• enhancer sequences:

  regulate DNA activity by changing the chromatin structure, making it less or more open to DNA polymerase
• promoter sequences:

  enable the binding of RNA polymerase and promote transcription
• the promoter sequence is located upstream from the gene being transcribed
• RNA splicing is the process of removing introns and joining exons to form mature mRNA
• The process of RNA splicing is

  1. the gene is transcribed which results in pre-mRNA
  2. all introns (non-coding DNA) and some exons (coding DNA) are removed
  3. the remaining mix of genes are then joined together by enzyme complexes, splicosomes. The same exons can be joined to produce several different versions of RNA
• Gene expression can be affected by: epigenetics, DNA methylations, Histone modification and Non-coding DNA
• epigenetics are reversible modifications of DNA
• DNA methylation prevents transcription and affects histone structure to make more or less DNA accessible to RNA polymerase
• IPS cells are reprogramed adult stem cells which become pluripotent again
• The process of IPS cells is:

  1. connective tissue is taken from skin samples
  2. viruses can be used as vectors to induce 4 genes for transcription factors (these factors activate specific genes in adult cells to become pluripotent)
  3. Cells behave similarly to embryonic cells - their effectiveness long term is not known and cells show a tendency to become cancerous
• Recombinant DNA - isolation of the gene through:

  cutting out the gene with a restriction endonuclease - leaving sticky ends

  reverse transcriptase-making DNA from MRNA
• In recombinant DNA:

  1. you cut plasmids with the same restriction endonuclease enzyme to leave complementary base pairs
  2. join plasmids and genes with DNA ligase to form the recombinant DNA
  3. incorporate plasmids into host nucleus
• the plasmid (aka the vector carrying the recombinant DNA) is:

  • gene guns (metal pellets with DNA)
  • viruses
  • microinjections
  • liposome wrapping (DNa wrapped in liposomes which fuses with the cell membrane)
• a gene marker is a gene that is transferred with the desired gene to identify which cells have been successfully altered and now contain recombinant DNA
• using gene markers:

  • bacteria is transferred from a "master plate" onto the plates with antibiotics using a sterile block - if the bacteria doesn't grow the gene was inserted properly
• genetic engineering of crops: removing a gene with the desired characteristics from one organism to another, transferring the gene into another organism where the desired gene is then expressed
• genetic engineered crops:

  1. the plasmid which transfers the bacterial genetic information directly to plant DNA is extracted
  2. bacterial genes are inserted into the plasmid via genetic modification
  3. plasmid is returned to the bacterium and the plant is now infected
  4. plant grows a cluster of cells which contain the inserted gene - these cells can be isolated and cultured to grow a whole new plant
• soya beans - adv:

  herbicide resistance

  changes in nutrient values

  cheaper
• soya beans dis:

  increase in abiotic resistance
• knockout mice:

  have genes swapped or changes inside the embryo of the mouse by "homologous recombination"
• knockout mice:

  by removing a specific gene you can see what the effect on the organism that gene has, for example, the influence on diseases caused by mutations
• knockout mice:

  some of the offspring will consist of some tissues where the desired gene is removed, originating form the special ES cells
• knockout mice: drawbacks

  • essential genes can be chosen to remove - leading to the death of the embryo
  • multiple genes could cause diseases - removing a single gene may not be enough
  • there are still genetic differences in humans and mice - not all affects seen in mice may be seen in humans
• introns are:

  non-coding sequences of DNA, which are removed by RNA splicing before translation
• exons are: Coding areas of the DNA which are present for transcription from RNA
• alternative splicing introduces variety
• promotor sequences:

  bases at the beginning of genes which pinpoint the gene to be transcribed by the RNA polymerase
• IPS stem cells: advantages over embryonic stem cells

  • more ethical to produce
  • less risk of rejection when transferred as they are from your cells
  • can be created in large numbers
• how are IPS cells produced:

  • from fully differentiated cells such as muscle cells
  • genes for transcription factors are inserted
  • the cell regains the ability to differentiate into other cell types