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Nucleic Acid Hybridisation & Gene Mapping


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Nucleic Acid Hybridisation & Gene Mapping

  1. 1. Nucleic Acid Hybridisation <ul><li>Once a gene has been isolated from a complete genome as a piece of DNA; we may want to know from which chromosome gene it came from and where that chromosome is located; or from which cells of the organism the gene is transcribed; or to test a sample of human DNA for mutations in the gene suspected of causing an inherited disease </li></ul><ul><li>All these questions can be answered by taking advantage of the fundamental property of DNA : </li></ul>COMPLEMENTARY BASE PAIRING
  2. 2. <ul><li>Remember, the 2 strands of DNA are held together by HYDROGEN BONDING . These bonds can be broken by heating to 90 o c or altering the pH </li></ul><ul><li>These treatments release the single strands but DO NOT break the strong covalent bonds that link the nucleotides together </li></ul><ul><li>If the process is reversed i.e. slowly lowering the temperature and bringing the pH back to normal, the complementary strands will reform double helices - this is known as HYBRIDISATION </li></ul><ul><li>Using this technique, particular DNA sequences can be identified by hybridisation with the aid of a NUCLEIC ACID PROBE </li></ul>
  3. 3. <ul><li>Nucleic acid hybridization </li></ul><ul><li>(A) If the DNA helix is separated into two strands, the strands should reanneal, given the appropriate ionic conditions and time. </li></ul><ul><li>(B) Similarly, if DNA is separated into its two strands, RNA should be able to bind to the genes that encode it. If present in sufficiently large amounts compared with the DNA, the RNA will replace one of the DNA strands in this region </li></ul>
  4. 4. <ul><li>A Nucleic Acid Probe - a short, single-stranded DNA or RNA molecule that has been radioactively labelled ( e.g. 32-phosphate 32 P ) and is used to identify a complimentary nucleic acid sequence </li></ul>
  5. 5. Genetic Linkage Mapping <ul><li>Genetic maps are based on the recombination frequency between genetic markers during MEIOSIS [see Higher notes!] </li></ul><ul><li>These can be used to locate genes on particular chromosomes and establish the order of the genes and the approximate distance between them </li></ul><ul><li>This approach relies on having genetic markers that are detectable </li></ul><ul><li>Genetic markers are any gene that shows variation (different alleles). These include genes and other DNA sequences such as microsatellites , which are tandem repeats of units 2-4 bp in length. These units are also known as short tandem repeats and are distributed fairly evenly over the genome, and may even occur within genes. </li></ul><ul><li>Sometimes these are genes that cause disease, traced in a family by pedigree analysis </li></ul>
  6. 6. <ul><li>The marker alleles must be HETEROZYGOUS so that meiotic recombination can be detected </li></ul><ul><li>NB: if 2 genes are on different chromosomes - they are unlinked and will sort independently during meiosis </li></ul><ul><li>If 2 genes are on the same chromosomes they are physically linked and a crossover between them during Prophase I of meiosis can generate non-parental genotypes </li></ul><ul><li>The chance of a crossover occuring increases as linked genes become further apart. In fact, they may behave as if they are essentially unlinked </li></ul><ul><li>Genetic mapping is used to produce a picture of the locations of the marker loci on the chromosome. However, it doesn’t provide the precise distances between the genes </li></ul><ul><li>[Insert Genetic Linkage diagram] </li></ul>
  7. 7. Physical Mapping <ul><li>A physical map is a more detailed map of a genetic map </li></ul><ul><li>As with genetic maps, construction of a physical map requires markers that can be mapped to an exact location on the DNA </li></ul><ul><li>Physical maps of the genome can be constructed in a number of ways, all of which aim to generate a map in which the distances between markers are known with reasonable accuracy </li></ul>
  8. 8. Restriction Mapping <ul><li>Fragments of DNA are made by cutting with restriction enzymes or endonucleases </li></ul><ul><li>These are enzymes that cleave DNA at certain nucleotide sequences, thereby generating specific fragments </li></ul><ul><li>The recognition sequences where restriction enzymes are short (4,5 or 6 base pairs long) sequences that occur at defined positions in the DNA </li></ul><ul><li>Using a combination of these enzymes and measuring the size of fragments produced, the ‘puzzle’ can be pieced together to give the pattern of restriction enzyme recognition sites in the DNA </li></ul><ul><li>Defined fragments can then be identified either by their size or using a specific DNA probe to bind to its complementary map [ electrophoresis or nucleic acid hybridisation ] diagram </li></ul>
  9. 9. Restriction Mapping : An example <ul><li>The most straightforward method for restriction mapping is to digest samples of the DNA with a set of individual enzymes, and with pairs of those enzymes </li></ul><ul><li>The digests are then &quot;run out&quot; on an agarose gel to determine sizes of the fragments generated. If you know the fragment sizes, it is usually a fairly easy task to deduce where each enzyme cuts, which is what mapping is all about </li></ul>
  10. 10. Restriction Mapping : An example <ul><li>To illustrate these ideas, consider a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Within the vector, immediately flanking the unknown DNA are unique recognition sites for the enzymes Kpn I and BamH I. As illustrated in the diagram below, consider first separate digestions with Kpn I and BamH I : </li></ul>
  11. 11. <ul><ul><li>Digestion with Kpn I yields two fragments: 1000 bp and &quot;big&quot;. Since there is a single Kpn I site in the vector, the presence of a 1000 bp fragment tells you that there is also a single Kpn I site in the unknown DNA and that it is 1000 bp from the Kpn I in the vector. The &quot;big&quot; fragment consist of the vector plus the remaining 2000 bp of the unknown </li></ul></ul><ul><ul><li>Digestion with BamH I yields 3 fragments: 600, 2200 and &quot;big&quot;. The &quot;big&quot; fragment is again the vector plus a little bit (200 bp in this case) of unknown DNA. The presence of 600 and 2200 bp fragments indicate that there are two BamH I sites in the unknown. You can deduce immediately that one BamH I site is 2800 bp (600 + 2200) from the BamH I in the vector. The second BamH I site can be in one of two positions: 600 or 2200 bp from the BamH I site in the vector </li></ul></ul><ul><ul><li>At this point, there is no way to know which of these alternative positions is correct </li></ul></ul>
  12. 12. <ul><li>The trick to determining where the second BamH I site is located is to digest the plasmid with Kpn I and BamH I together </li></ul><ul><li>This so-called double digest yields fragments of 600, 1000 and 1200 bp (plus the &quot;big&quot; fragment). The 600 bp fragment is the same as obtained by digestion with BamH I alone. The 1000 and 1200 bp fragments tell you that Kpn I cut within the 2200 bp BamH I fragment observed when the plasmid was cut with BamH I alone </li></ul>You already know where Kpn I cuts in the unknown DNA, and you therefore now know the location of the second BamH I site!
  13. 13. <ul><li>Chromosome Walking </li></ul><ul><li>Used to locate genes or other DNA sequences on a physical map or to locate genes associated with disorders </li></ul><ul><ul><li>The marker DNA and target DNA must be linked </li></ul></ul><ul><ul><li>DNA probes used to locate and isolate multiple copies of DNA that have complementary sequences of DNA to the probe in libraries </li></ul></ul><ul><ul><li>2 libraries are made, one from cloned fragments of the marker and one from cloned fragments of the target DNA </li></ul></ul><ul><ul><li>Different restriction enzymes are used so that the fragments in each library are different but overlap </li></ul></ul>
  14. 14. Gel Electrophoresis <ul><li>Since nucleic acids are negatively charged, they migrate toward the positive pole in an electric field </li></ul><ul><li>When the electric field is applied through the gel, molecular sieving takes place. Shorter chains move faster than longer ones. Thus, the chains are spread out in the gel according to their size. </li></ul><ul><li>Double-stranded DNA can be visualized by adding ethidium bromide, a flat aromatic chemical that fits between base pairs in the double helix. Only when bound to DNA does the ethidium bromide fluoresce orange when irradiated with UV </li></ul>
  15. 15. DNA Sequencing <ul><li>The final stage of the genome project is to determine and assemble the actual DNA sequence itself. For this to happen: </li></ul><ul><ul><li>DNA fragments must be generated </li></ul></ul><ul><ul><li>The sequencing technology must be accurate and fast </li></ul></ul><ul><ul><li>Computer hardware/software must be available to analyse the data </li></ul></ul>
  16. 16. DNA Sequencing cont . <ul><li>The technique used for sequencing is the Dideoxy Chain Termination method as developed by F. Sanger in the 1970’s </li></ul><ul><li>This method relies on making a copy of the chosen DNA template </li></ul><ul><li>[See Student monograph for a more comprehensive explanation – pg.154 - 157] </li></ul>
  17. 17. Comparative Genome Analysis <ul><li>In addition to mapping the human genome, the genomes of other species are also being mapped. These include species important to biological research and agriculture such as the mouse, chicken, pig, cow, rice, wheat, Caenorhabditis elegans (nematode), Drosophila melanogaster (fruit fly), Saccharomyces cerevisiae (yeast), Escherichia coli , and other prokaryotes. </li></ul><ul><li>The genomes of some of these organisms, such as E. coli , yeast, the nematode and the fruit fly have now been completely mapped and sequenced. These maps can be used to locate homologous genes in the human genome and to help in determining gene function. </li></ul>
  18. 18. <ul><li>Comparative genome analysis is being used to find out more about evolution . The number of differences in an amino acid sequence can be used to calculate the time since two species diverged from a common ancestor. If there are lots of differences between the maps, it can be deduced that the species diverged longer ago than if there are only a few differences. This type of information is used alongside other methods of measuring the rate of evolution. </li></ul><ul><li>Gene maps can be used to predict gene order . If gene X is found next to gene Y and Z in one species, the likelihood is that it will be found next to the same two genes in another closely related species. Comparative maps will be used to find candidate genes for phenotypes mapped in species as diverse as chicken and human. </li></ul>