using genome projects

• genome


  contains all of the genes within an organism
• • Genome sequencing can aid the understanding of gene function and interaction
  • Sequencing projects have read the genomes of a wide range of organisms from flatworms to humans
  • A genome project works by collecting DNA samples from many individuals of a species. These DNA samples are then sequenced and compared to create a reference genome
  • More than one individual is used to create the reference genome as one organism may have anomalies/mutations in their DNA sequence that are atypical of the species
• The Human Genome Project (HGP) began


  1990
• The Human Genome Project (HGP)


  • It was an international, collaborative research programme
  • It was publicly funded so that there would be no commercial interests or influence
• Creating the reference genome

  1. DNA samples were taken from multiple people around the world

  2. Samples were sequenced

  3. Used to create a reference genome
• Laboratories around the globe


  • They were responsible for sequencing different sections of specific chromosomes
• It was decided that the data created from the project would be made publicly available
• As a result, the data can be shared rapidly between researchers
• The information discovered could also be used by any researcher and so maximised for human benefit
• • Following the success of sequencing the human genome scientists have now moved onto sequencing the human proteome
  • The proteome is all of the proteins that can be produced by a cell
  • Although there are roughly 25,000 genes within the genome there are many more proteins within the proteome. This may is due to processes such as alternative splicing and post-translational modification
• • There is also work being done on the human epigenome
  • These are the inherited changes in DNA that do not involve a change in DNA base sequence
• Human Genome Project


  Project that generated information used to tackle human health issues and find cures for diseases
• Scientists have noticed a correlation between changes in specific genes and the likelihood of developing certain inherited diseases
• The mechanism which causes these inherited diseases to develop is not yet understood, but is being actively researched by thousands of scientists
• Genes linked to increased risk of certain cancers

  • BRCA1
  • BRCA2
• BRCA1 and BRCA2 genes


  If mutated, they substantially increase the risk of developing breast cancer
• Genes linked to the development of Alzheimer's disease


  • (specific genes not named)
• Once a genome is sequenced bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available. This can help to find the degree of similarity between organisms which then gives an indication of how closely related the organisms are and whether there are organisms that could be used in experiments as a model for humans
• • It can be highly difficult to translate the genome of complex organisms into their proteome
  • Determining the proteome of humans is difficult as large amounts of non-coding DNA are present in human genomes
  • It can be very hard to identify these sections of DNA from the coding DNA
  • The presence of regulatory genes and the process of alternative splicing in human genomes also affects gene expression and the synthesis of proteins
  • The proteome is larger than the genome due to:
  • Alternative splicing
  • Post-translational modification of proteins (often takes place in the Golgi apparatus)
• • DNA sequencing allows for the base sequence of an organism's genetic material to be identified and recorded
  • Sequencing methods are continuously evolving and becoming faster. Advances in technology have allowed scientists to rapidly sequence the genomes of organisms
  • Most sequencing methods used are now automated
  • The data obtained from sequencing can be entered into computers with specialised programmes that compare the base sequences of different organisms
• DNA sequencing

  All methods of DNA sequencing use dideoxyribose nucleotides
• Dideoxyribose molecule


  • Very similar in structure to ribose molecules and deoxyribose molecules
  • Has one less oxygen atom than a deoxyribose molecule
  • Has two fewer oxygen atoms than a ribose molecule
• Dideoxyribose


  Can form nucleotides in the same way that ribose and deoxyribose molecules do, by binding to a phosphate molecule and an organic base
• Dideoxyribose nucleotides


  Can pair with deoxyribose nucleotides on the template strand during DNA replication
• Dideoxyribose nucleotides


  Will pair with nucleotides that have a complementary base
• DNA sequencing

  1. When DNA polymerase encounters a dideoxyribose nucleotide on the developing strand it stops replicating

  2. This is the chain-termination technique that is used for DNA sequencing
• Automated DNA sequencing


  Makes use of the chain-termination technique
• Automated DNA sequencing

  1. Choose a short length of DNA

  2. Insert into a vector as a single strand

  3. Anneal a primer to the start of the recombinant DNA

  4. Add DNA polymerase and a mixture of deoxynucleotides containing all 4 bases

  5. DNA polymerase attaches to the primer and begins DNA replication

  6. Hydrogen bonds form between complementary bases on deoxynucleotides

  7. Add a mixture of dideoxynucleotides containing all 4 bases

  8. DNA polymerase can insert a dideoxynucleotide by chance, terminating DNA replication

  9. Produce complementary DNA chains of varying lengths

  10. Each dideoxynucleotide is labelled with a specific fluorescent dye

  11. Separate single-stranded DNA chains by size using capillary electrophoresis

  12. Use a laser to illuminate the dideoxynucleotides and a detector to read the colour and position of each fluorescence

  13. Feed the information into a computer for analysis
• An automated DNA sequencing machine can read roughly 100 different DNA sequences within 2 hours
• Manual DNA sequencing

  • Manual DNA sequencing follows a similar process to automated sequencing but there are some key differences:
  • A separate run is required for each type of dideoxynucleotide - ddNA, ddNT, ddNC and ddNG
  • The dideoxynucleotides are labelled using radioactivity instead of fluorescent dyes
  • After the incubation period, the four separate mixtures are added to separate wells in a gel and separated using gel electrophoresis
  • A Southern transfer is made using the electrophoresis gel and an autoradiograph is taken of the Southern transfer
• Interpreting the results from manual DNA sequencing

  1. The DNA sequence can be interpreted using the autoradiograph

  2. Below each well there is a track of bands (DNA fragments) produced from DNA replication in the presence of each type of dideoxynucleotide (ddNA, ddNT, ddNC and ddNG)

  3. The band that moves the furthest distance is the smallest DNA fragment

  4. The smallest DNA fragment that can be formed from the chain termination technique is one nucleotide long so whichever track this band is present in determines the first base in the sequence of the developing strand

  5. The second smallest DNA fragment that has travelled the second furthest will determine the second base in the sequence and the third smallest DNA fragment that has travelled the third furthest will determine the third base in the sequence etc.