Manipulating Genomes

• How DNA sequencing got started

  Sanger first did it using radioactive labelling of bases and gel electrophoresis, done manually so took lots of time, radioactive bases swapped for fluorescent bases, led to automation and scaling up of process, led to capillary sequencing used in HGP
• Principles of DNA sequencing

  DNA mixed with different chemicals, PCR carried out in a thermal cycler, DNA polymerase builds up new strand with the nucleotides as part of PCR, addition of terminator base stops the replication, results in many different lengths of DNA, separated by length in capillary sequencing, fluorescent mark on terminator base used to identify the final base using 'lasers', order of bases shows sequence for complementary strand, used to find sequence of original strand
• Things that DNA is mixed with for sequencing

  Primers, Taq polymerase, normal nucleotide bases, terminator bases
• Capillary sequencing

  Gel electrophoresis in capillary tubes
• Current developments in DNA sequencing techniques

  High throughput sequencing
• How does high throughput sequencing work?

  Done on a flow cell, fragments of DNA attached slide, replicated in situ by PCR, clusters of identical DNA strands form, addition of fluorescent terminator bases can stop the reaction to take an image, clusters all sequenced at the same time
• Other terms for high-throughput sequencing

  Massively parallel, next-generation
• Bioinformatics

  Development of the software and computing tools needed to analyse biological data. The best type of science.
• Computational biology

  Studying biology using computational techniques. The best type of science
• Applications of DNA sequencing

  GWAs between individuals and species, determining the sequences of amino acids in polypeptides, synthetic biology
• Further detail on how DNA sequencing has allowed for genome wide comparison between individuals and species

  Show patterns of inherited DNA, show diseases that we are vulnerable to, affects epidemiology
• How has DNA sequencing allowed for the study of evolutionary relationships?

  Compare the sequences from different organisms, rate of mutation used to figure out when the organisms had a common ancestor
• Questions that DNA sequencing can help with

  Studying genotype-phenotype relationship, epidemiology, evolutionary relationships
• Fields that contribute to research into genotype-phenotype relationships

  Bioinformatics, computational biology, proteomics
• Reasons why the DNA sequence doesn't completely determine the amino acid sequence in a polypeptide

  Spliceosomes, protein modification
• Spliceosomes

  Enzyme complexes that join exons together in any order
• Protein modification

  Length might change to give other proteins
• Things that epidemiology covers

  Finding source of infection, identifying antibiotic resistant strains of bacteria, tracking the progress of an outbreak, identifying drug targets in a genome
• Uses of synthetic biology

  Genetic engineering, use of biological systems in industry, synthesis of new genes to replace faulty genes, synthesis of new organisms
• Example of production of new genes to replace faulty genes

  Replacing faulty genes in cystic fibrosis
• Example of synthesis of new organisms

  Genome of a bacterium made and put in a bacterium
• Introns

  Non-coding regions of DNA that are removed from mRNA before translation
• Satellite DNA

  Short sequences of DNA found in introns, centromeres and telomeres that are often repeated
• Other term for satellite DNA

  Variable tandem number repeats
• Microsatellite

  2 to 4 bases that are repeated 5 to 15 time
• Other term for micro satellite

  Short tandem repeats
• How are satellites inherited?

  Always in the same place on chromosomes, number of repeats varies between individuals, number of repeats inherited from parents
• DNA profiling

  Producing an image of the patterns in DNA
• Stages of DNA profiling

  Extract the sample, use PCR to get many copies, digest the sample with restriction endonucleases which cut at specific points in introns, fragments contain a mixture of mini and microsatellite regions, separate the fragments by electrophoresis, add radioactive or fluorescent probes, bind to complementary strands in hybridisation, take X-ray images or put under UV light to see the pattern
• Process of electrophoresis


  Agarose gel with wells in the bottom, fragments with known lengths in first and last wells, electric current put through the plate, DNA moves towards the positive electrode, rate of movement depends on size of DNA fragment, gel placed in alkaline buffer solution to denature the DNA fragments, bases exposed, transferred to nitrocellulose paper by Southern blotting
• Process of Southern Blotting

  Nitrocellulose paper placed on top of the cell, covered with sheets of absorbent paper, DNA drawn up in alkaline solution by capillary action, fixed in place by UV light
• Chemicals that go in a PCR machine

  DNA sample, excess of nucleotide bases, primers, Taq polymerase
• Process of PCR

  Temperature raises to 95 degrees celsius, denatures the DNA by breaking hydrogen bonds, decreases to 60 degrees celsius, primers anneal to the ends of the DNA, temperature raised to 72 degrees celsius, optimum temperature for Taq polymerase, adds bases to the primer to make complementary strands of DNA
• Uses of DNA profiling

  Forensics, paternity testing, immigration cases, identifying the species of an organism, showing evolutionary relationships, identifying individuals at risk of particular diseases
• How does DNA profiling help in identifying individuals at risk of particular diseases?

  Certain microsatellites and their patterns associated with cancers and heart disease
• Uses of PCR

  Forensics when tiny amounts of DNA available, lots of DNA made from small sample for DNA analysis
• Methods of isolating a gene for genetic engineering

  Restriction endonucleases, reverse transcriptase
• How to use restriction endonucleases to isolate a gene for genetic engineering

  Restriction endonucleases cut the DNA at a specific base sequence, leave sticky ends
• How to use reverse transcriptase to isolate a gene for genetic engineering

  Isolating the mRNA for the gene, reverse transcriptase makes the complementary DNA
• How to get the isolated gene into a vector for genetic engineering

  Use the same restriction endonuclease to cut open the plasmid, leaves sticky ends that are complementary to the original sticky ends, DNA ligase joints the two strands of the DNA