4. DNA Sequencing

• DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. By allowing genes to be isolated and read.
• The Sanger DNA Sequencing method:

  1. The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides (containing bases A, T, C, and G) and terminator bases with radioactive or fluorescent labels.
  2. The mixture is placed in a thermal cycler that rapidly changes temperature at programmed intervals in repeated cycles -at 96°C the double-stranded DNA separates into single strands.
  3. At 50°C the primers anneal to the DNA strand.
  4. At 60°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template.
• The Sanger DNA Sequencing method: (2)

  1. Each time a terminator base (or dideoxynucleotide) is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added. This is because terminator bases have a hydrogen atom instead of a hydroxyl group at the 3' carbon of the ribose ring, therefore they cannot form phosphodiester bonds with the next nucleotide.
• The Sanger DNA Sequencing method: (3)

  1. As the chain-terminating bases are present in lower amounts and are added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process.
  2. After many cycles, all of the possible DNA chains will be produced with the reaction stopped at every base.
  3. The DNA fragments are passed through a gel by electrophoresis. Smaller fragments travel further, so the fragments are sorted by length.
• The Sanger DNA Sequencing method: (4)


  The nucleotide base at the end of each fragment is read according to its radioactive label.

  • If the first one-base fragments has thymine at the end, then the first base in the sequence is T.
  • If the two-base fragments have cytosine at the end, then the sequence is TC.
  • If the three-base fragment ends with guanine, then the base sequence is TCG.
• The Sanger DNA Sequencing method: (5)

  1. Alternatively, the DNA fragments can be separated according to their length by capillary sequencing, which works like gel electrophoresis in minute capillary tubes.
  2. The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Lasers detect the different colours and thus the order of the sequence.
  3. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made. This is used to build up the sequence of the original DNA strand.
• The Sanger DNA Sequencing method: (6)

  1. The data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them.
• The implications of the Sanger method:

  • This method was efficient and safe but a very time-consuming and therefore costly process.
  • Recently, technological developments have led to new, automated, high-throughput sequencing processes.
• Pyrosequencing

  • It uses sequencing by synthesis, not by chain termination as in the Sanger method.
  • It involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting, by light emission, which base was added at each step.
• Pyrosequencing Method:


  1. A long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser.
  2. These lengths are then degraded into single-strandedDNA (ssDNA). These are the template DNAs and they are immobilised.
  3. A sequencing primer is added and the DNA is then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5' phosphosulfate (APS) and luciferin.
  4. Only one of the four possible activated nucleotides, ATP, TTP, CTP and GTP is added at any one time and any light generated is detected.
• Pyrosequencing method: (2)

  1. One activated nucleotide (a nucleotide with two extra phosphoryl groups), such as TTP (thymine triphosphate, is incorporated into a complementary strand of DNA using the strand to be sequenced as a template.
  2. As this happens, the two extra phosphoryls are released as pyrophosphate (PPi).
  3. In the presence of APS, the enzyme ATP sulfurylase, ATP, the enzyme luciferase and luciferin, oxyluciferin is made.
  4. This conversion generates visible light which can be detected by a camera.
• Pyrosequencing :

  
• How gene sequencing has allowed for genome-wide comparisons between individuals and between species:

  • All humans are genetically similar, we all have the same genes, but we have different alleles.
  • When the human genome was compared with those of other species, it became clear that few human genes are unique to us.
  • Genes that work well tend to be conserved by evolution.
  • Many of the differences between organisms are not because the organisms have totally different genes, but because some of their shared genes have been altered and now work in subtly different ways.
• How gene sequencing has allowed for genome-wide comparisons between individuals and between species: (2)


  • Genome analysis provides scientists with another tool to aid in species identification, by comparison to a standard sequence for the species. The challenge for scientists is to produce stock sequences for all the different species.
  • One useful technique is to identify particular sections of the genome that are common to all species but vary between them, so comparisons can be made - this technique is referred to as DNA barcoding.
• How gene sequencing has allowed the study of evolutionary relationships:

  • Comparing genomes of organisms thought to be closely related species has helped confirm their evolutionary relationships or has led to new knowledge about the relationships and, in some cases, to certain organisms being reclassified.
  • The DNA from bones and teeth of some extinct animals can be amplified and sequenced, so that the animals' evolutionary history can be verified.
  • The basic mutation rate of DNA can be calculated scientists can calculate how long ago two species diverged from a common ancestor.
• • Bioinformatics- the development of the software and computing tools needed to organise and analyse raw biological data to help us to make sense of the enormous quantities of data being generated.
• • Computational biology then uses this data to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances.
  • Computational biology is the study of biology using computational techniques, especially in the analysis of huge amounts of biodata.
  • For example, it is important for working out the 3D structures of proteins, and for understanding molecular pathways such as gene regulation, identifying genes linked to specific diseases in populations and in determining the evolutionary relationships between organisms.
• • Genomics- the field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes.
• Analysing the genomes of pathogens (Epidermiology)


  Sequencing the genomes of pathogens including bacteria, viruses, fungi, and protoctista has become fast and relatively cheap. This enables:

  • Doctors to find out the source of an infection, for example bird flu or MRSA in hospitals.
  • Doctors to identify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping prevent the spread of antibiotic resistance.
• Analysing the genomes of pathogens (Epidermiology) (2)

  • Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics, for example flu each winter.
  • Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines.
• How gene sequencing has allowed for the sequences of amino acids in polypeptides to be predicted:

  • Proteomics is the study and amino acid sequencing of an organism's entire protein complement.
  • However, if researchers have the organism's genome sequenced and know which gene codes for a specific protein, by using knowledge of which base triplets code for which amino acids, they can determine the primary structure of proteins. The researchers need to know which part of the gene codes for exons and which codes for introns.
  • However, some genes can code for many different proteins.
• How gene sequencing has allowed for the development of synthetic biology:

  • Synthetic biology- the ability to sequence the genome of organisms and understand how each sequence is translated into amino acids, along with the ever-increasing ability of computers to store, manipulate, and analyse the data, has led to the development of the new field of science.
  • Its ultimate goals may be to build engineered biological systems that store and process information, provide food, maintain human health and enhance the environment.

  
• Synthetic biology includes many different techniques. These include:

  • genetic engineering - this may involve a single change in a biological pathway or relatively major genetic modification of an entire organism.
  • use of biological systems or parts of biological systems in industrial contexts, for example, the use of fixed or immobilised enzymes and the production of drugs from microorganisms.
• Synthetic biology includes many different techniques. These include:


  • the synthesis of new genes to replace faulty genes, for example, in developing treatments for cystic fibrosis (CF), scientists have attempted to synthesise functional genes in the laboratory and use them to replace the faulty genes in the cells of people affected by CF.
  • the synthesis of an entire new organism. In 2010, scientists announced that they had created an artificial genome for a bacterium and successfully replaced the original genome with this new, functioning genome.