DNA Sequencing in Phylogeny


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DNA Sequencing in Phylogeny

  1. 1. Bikash Kar Nath MBM11005 M.Sc. 1st Semester. MBBT
  2. 2.  What is DNA sequencing?  What is Phylogeny?  DNA sequencing techniques.  Results of DNA sequencing.  Phylogenetic Interpretation of the results.  Construction of Phylogenetic Trees.  Applications.  Conclusion.
  3. 3.  DNA sequencing is a scientific approach involving various biochemical, biophysical and computational techniques to determine the order of the nucleotide bases- adenine, guanine, thymine & cytosine in a molecule of DNA.  DNA sequencing techniques are key tools in many fields. A large number of different sciences are receiving the benefits of these techniques, ranging from archaeology, anthropology, genetics, biotechnology, molecular biology, forensic sciences, among others.
  4. 4.  According to modern evolutionary theory, all organisms on earth have descended from a common ancestor, which means that any set of species, extant or extinct, is related. This relationship is called phylogeny, and is represented by phylogenetic trees, which graphically represent the evolutionary history related to the species of interest. Phylogenetic tree of various foxes Common Ancestor
  5. 5.  Frederick Sanger’s Chain termination or Dideoxy-nucleotide Sequencing.  Allan Maxam & Walter Gilbert’s Chemical Degradation Sequencing.  Lynx Therapeutics' Massively Parallel Signature Sequencing.  Polony sequencing.  Pyrosequencing.  Illumina sequencing.  Solid sequencing.  Ion semiconductor sequencing.  DNA nanoball sequencing.  Single Molecule SMRT sequencing &Single Molecule real time RNAP sequencing.  Nanopore DNA sequencing.
  6. 6. ddGTPddATP ddCTP ddTTP 6. One type of ddNTP per reaction 7.2. With region of known sequence 3’ 8. ddNTP incorporation - stops chain synthesis 3’ 3’ 3’ 3’ 3. Complementary primer 5’ 1. Unknown fragment 5’ Reaction 1 5. Four separate reactions 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 4. dNTP’s 35S labeled dATP or dCTP DNA Polymerase Reaction 4Reaction 3Reaction 2 (Read start be cloned for production of single-stranded DNA)
  7. 7. ddGTP ddATP ddTTP ddCTP Sequence of unknown fragment Shortest synthesized band = 5’ end of synthesized strand Longest synthesized band = 3’ end of synthesized strand 3’ A C A C T G T A G A G 5’
  8. 8. .  The DNA to be sequenced must first be end labeled at one 5’ end generally by kinase treatment with 32P ATP.  Labeled DNA is first precipitated to remove any salts which might interfere in the cleavage reactions.  Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The purines (A+G) are depurinated using formic acid, the guanines are methylated by DMS and the pyrimidines (C+T) are methylated using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the methylation of thymine for the C-only reaction.  The modified DNAs are then cleaved by hot piperidine at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule.  Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
  9. 9. Unknown DNA sequence
  10. 10.  Massively Parallel Signature Sequencing :- MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides. Subsequent development of less complex novel sequencing techniques made MPSS obsolete.  Polony sequencing :- It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing.  Pyrosequencing :-The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read- outs.  Single Molecule real time RNAP sequencing:-This method is based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types.
  11. 11.  The sample sequence is finally obtained as a graph with nucleotide peaks or as a false colour nucleotide base sequence.  Comparative analysis of DNA can be used as an important tool to analyze the phylogenetic relationship between organisms. Electrophoregram Multiple Sequence Alignment
  12. 12.  As obvious for a phylogenetic analysis DNA sequencing of more than a single species and its subsequent sequence alignment is necessary.  Since the species supposedly share a common ancestry hence mismatches can be interpreted as point mutations and gaps as indels (i.e. Insertion or deletion mutations) introduced in one or both lineages in the time since they diverged from one another.  It is still a long and expensive process to sequence the entire DNA of an organism (its genome) and this has been done for only a few species. However, it is quite feasible to determine the sequence of a defined area of a particular chromosome. At any location within such a sequence, the bases found in a given position may vary between organisms. The particular sequence found in a given organism is referred to as its haplotype, and a comparative analysis of these sequences can thus infer phylogenetic relationships.
  13. 13.  Does a high degree of similarity mean that two DNA sequences have the same meaning or function? “There are many scientists today who question the evolutionary paradigm and its atheistic philosophical implications”. “There are not many scientists today who question the evolutionary paradigm and its atheistic philosophical implications”. These sentences have 97% similarity and yet have almost opposite meanings! Thus Homology among DNA is often incorrectly concluded on the basis of sequence similarity. The terms "percent homology" and "sequence similarity" are often used interchangeably. As with anatomical structures, similarity might occur because of convergent evolution (giving rise to homoplastic organs), similarly high sequence similarity may occur with shorter sequences, because of chance. Such sequences are similar but not homologous. Hence establishment of phylogenetic relationship takes into account conserved sequences occurring in all the sample species & its extent of conservation.
  14. 14.  To build phylogenetic trees, statistical methods are applied to determine the tree topology and calculate the branch lengths that best describe the phylogenetic relationships of the aligned sequences in a dataset. Many different methods for building trees exist and no single method performs well for all types of trees and datasets.  Common approaches applied include the following:- 1. Distance-Matrix Methods:- They compute a matrix of pairwise “distances” between sequences that approximate evolutionary distance. 2. Discrete data methods:- They examine each column of a multiple sequence alignment dataset separately and search for the tree that best represents all this information. These methods produce a separate tree for each column in the alignment, so it is possible to trace the evolution for specific elements within a given sequence, such as catalytic sites or regulatory regions. 3. Maximum Likelihood:-The maximum likelihood method exhibits a probabilistic model of evolution for estimating nucleotide substitution.
  15. 15.  Thus a phylogenetic tree is obtained highlighting the relationship between various species and the extent to which they are related thereby tracing their evolutionary history. Phylogenetic Tree of the Canid family
  16. 16.  Tracing the evolution of Man.  Tracing the evolution of biologically vital proteins.  Tracing the evolution of infectious pathogens.  Increasing the efficacy & efficiency of drugs by sample testing on phylogenetically related species.  DNA sequencing and its use in phylogeny are increasingly being used in virology laboratories to study the transmission of viruses.
  17. 17.  Establishment of phylogenetic relationship between various species on the basis of DNA sequencing provides a detailed and reliable approach to tracing evolutionary history of those species as well as predicting their further evolutionary pattern. It is highly vital in understanding spontaneously evolving viruses and infectious pathogens & in an attempt to combat against their afflictions as well as increasing the value of economically important organisms.
  18. 18.  A review of DNA sequencing techniques: Lilian T. C. Franc:a, Emanuel Carrilho and Tarso B. L. Kist, Quarterly Reviews of Biophysics 35, 2 (2009), pp. 169–200. " 2009 Cambridge University Press.  Molecular Phylogenetics- An introduction to computational methods and tools for analyzing evolutionary relationships: Karen Dowell  Information from various internet sources.