Chapter 21

• Genome 


  all the genetic material contained in an organism
• Exon

  DNA or RNA bases that code for proteins
• Introns

  non-coding bases removed before translation (lowercase)
• Satellite DNA

  • sections of DNA that are repeated many times
  • particularly in introns, telomeres & centromeres
• Minisatellite

  20-50 base pairs repeated from 50-100s times
• Microsatellite

  • 2-4 bases repeated only 5-15 times
  • known as short tandem repeats (STRs)
• Variation in satellites

  • locus for particular repeat is always same but number of repeats can vary in individuals
  • as diff lengths of repeats are inherited from both parents - unique to each person
  • more closely related you are, more similar patterns will be
  • only identical twins will have identical satellite pattern
• Stages of DNA profiling

  • extracting DNA
  • digesting sample
  • separating fragments
  • hybridisation - identifying microsatellite
  • viewing profile
• Stage 1 - extracting DNA

  • DNA extracted from tissue sample
  • polymerase chain reaction (PCR) is carried out to increase amount of DNA available
• Stage 2 - digesting the sample

  • restriction endonuclease (enzymes that cute DNA at specific base sequences or recognition sites) are used to cut DNA into smaller pieces
  • restriction enzymes chosen so that the satellite repeating regions are left intact

  
• Stage 3 - Separating DNA fragments 


  • electrophoresis separates DNA fragments:
  • DNA molecules are loaded onto a gel
  • an electric current is passed through the gel
  • DNA molecules are negatively charged so move towards positive electrode
  • small molecules move faster than large molecules
  • gel is immersed in alkali to separate DNA double strands into single strands
  • single stranded DNA fragments are transferred onto a nylon membrane by Southern blotting

  
• Stage 4 - Hybridisation (identifying microsatellite)


  • probes are used to visualise the DNA to identify satellites present
  • DNA probes are short single stranded pieces of DNA w complementary sequence to the piece of DNA you want to find
  • because DNA on membrane is single stranded they can bind together

  
• Stage 5 - viewing profile

  • DNA probe has a radioactive or fluorescent label attached so can be viewed using an x-ray image or UV light
  • the fragments give a pattern of bars - the DNA profile - which is unique to every individual

  
• Polymerase chain reaction (PCR)

  • temperature in PCR machine is increased, denaturing DNA so strands seperate
  • temp is decreased & primers bind (anneal) to the ends of the DNA strands
  • temp is increased to optimum for DNA polymerase to work best for DNA synthesis - double-stranded DNA identical to original sequence produced
• Uses of DNA profiling

  • forensic science
  • analysis of disease risk
  • paternity testing
• How is DNA profiling used in forensic science?

  • performed on traces of DNA left at crime scene
  • profile is compared to that of a sample taken from suspect
• How is DNA profiling used in analysis of disease risk?

  • certain non-coding micro satellites have been found to be associated w an increased risk of particular diseases
  • these specific gene markers can be identified & observed in DNA profiles
  • used together w more detailed info obtained from DNA sequencing to make more confident risk assessments for diff diseases
• History of methods of DNA sequencing

  1. radioactive gel based system
  2. fluorescent gel based system
  3. fluorescent capillary system
• Radioactive gel based DNA sequencing

  • first techniques involved radioactively labelled bases & gel electrophoresis
  • done manually so took a long time
  • now known Sanger sequencing
• Fluorescent gel based DNA sequencing

  • radioactive labels were changed for fluorescent tags which could be read automatically
  • a laser detected each tag on the DNA base as it ran through the gel past a sensor in the machine
• Fluorescent capillary DNA sequencing

  • later versions used a gel in capillary tube to run the sample through
  • Many samples could be prepared and sequenced in one go
  • Order of bases read by computer
• Principles of DNA sequencing

  • DNA mixed w primer, DNA polymerase, excess of nucleotides & terminator nucleotides - placed in thermal cycler where DNA strands separate, primers anneal & nucleotides are added
  • each time a terminator base is added, strand terminates until all possible chains produced
  • DNA fragments separated by electrophoresis in capillary tubes by mass & lasers detect colours and sequence of new complementary DNA strand
  • Data analysed by computer to reconstruct original DNA sequence by comparing all fragments & finding areas of overlap between them
• Next generation sequencing

  • Millions of DNA fragments are attached to a slide
  • Terminator nucleotides and PCR are still used to create new fragments
  • Images are taken so the DNA is sequenced and read at the same time
  • Using these methods the 3 billion base pairs of the human genome can be read in days
  • High-throughput sequencing also reduces cost so more genomes can be sequenced 
• Bioinformatics

  development of software & computing tools needed to organise & analyse raw biological data
• Computational biology

  study of biology using computational techniques to analyse large amounts of data
• Why is it useful to compare genomes of diff individuals?

  • computers can compare genomes to reveal patterns in the DNA we inherit & the diseases to which we are vulnerable - allowing for better diagnosis, treatment & medicine tailored to individuals genetic makeup
  • (However, scientists increasingly recognise our genes work together w the environment to affect our physical characteristics, our physiology, & our likelihood of developing certain diseases)
• Why is sequencing the genomes of pathogens useful?

  • allows doctors to find out the source of an infection
  • helps indetify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective
  • allows scientists to track progress of an outbreak of a potentially serious disease & monitor potential epidemics
  • allows scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs & to identify genetic markers for use in vaccines
• DNA barcoding

  • used to identify diff species
  • identifies particular sections of the genome that are common to all species but vary enough to give clear differences between species so comparisons can be made
  • (system not perfect - barcode areas for animals and plants are different, and there are no suitable regions for bacteria or fungi yet)
• How does genome sequencing help understand evolutionary relationships between organisms?

  • the basic mutation rate of DNA can be used to calculate how long ago 2 species diverged from a common ancestor
  • DNA sequencing enables scientists to build up evolutionary trees with increased accuracy
• Proteomics

  the study & amino acid sequencing of an organisms entire protein complement
• Why is the sequence of amino acids not always what would be predicted from the genome sequence alone?

  • Spliceosomes - Before translation, pre-mRNA undergoes splicing where introns and sometimes exons are removed. Exons are then joined forming spliceosomes - Spliceosomes can join exons in diff ways, producing variation in types of mRNA -  result in different proteins & phenotypes
  • Protein modification - some proteins modified by other proteins after they are synthesised - may be shortened or lengthened to give a variety of other proteins
• Synthetic biology

  design & construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems
• Techniques of synthetic biology

  • genetic engineering - single change in biological pathway or major genetic modification of entire organism 
  • Use of biological systems or parts of systems in industrial contexts - e.g. production of drugs from microorganisms
  • Synthesis of new genes to replace faulty genes 
  • Synthesis of an entire new organism