Plant molecular sys rainier

• Acquisition of Molecular Data


  1. Plant samples from which DNA is to be isolated may be acquired by various means

  2. Herbarium

  3. Live samples

  4. Pieces of leaves

  5. Can be frozen

  6. Extraction Buffer
• DNA Sequence Data


  Comparisons of homologous regions of DNA among the taxa under study yield the characters and character states that are used to infer relationships in phylogenetic analyses
• DNA Sequence Data


  1. Identify a particular region of DNA to be compared between species

  2. Polymerase Chain Reaction (PCR)
• Polymerase Chain Reaction (PCR)


  The invention of this technology was crucial to modern DNA sequencing, as it permitted rapid and efficient DNA amplification, the replication of thousands of copies of DNA
• Polymerase Chain Reaction (PCR)


  A solution is prepared, made up of (1) the isolated and purified DNA of a sample; (2) multiple copies of primers; (3) free nucleotides; (4) DNA polymerase molecules (5) buffer and salts
• Primers


  Short, single-stranded segments of DNA that are designed to be complementary to the beginning and end of the target sequence that will be amplified
• Primers


  • Usually around 20 nucleotides long, and are complementary to the beginning and end of the DNA fragment that will be amplified
• Parameters to consider when designing primers for PCR

  • Length
  • Melting temperature
  • GC (Guanine-Cytosine) content
• Examples of PCR primers for plants

  • 28KJ
  • Universal primers (ITS1 and ITS2 regions of plants)
  • Plant specific primers
  • Species specific primers
• Polymerase Chain Reaction (PCR)

  Uses of PCR: Cloning, Gene fragment amplification, DNA fragment modification, Pathogen detection, DNA fingerprinting, Gene expression, To infer evolutionary relationships
• DNA Sequencing Reaction


  1. Determines the order of nucleotides, or bases, in a DNA fragment

  2. Template DNA using PCR

  3. Primers, free nucleotides

  4. Dideoxynucleotides (dideoxyadenine, dideoxycytosine, dideoxyguanine, and dideoxythymine)
• Types of DNA Sequence Data in Plants

  • Nuclear (nDNA)
  • Chloroplast (cpDNA)
  • Mitochondrial (mtDNA)
• PCR


  Artificially duplicates DNA
• DNA Sequencing Reaction


  Determines the precise order of nucleotides in a DNA fragment
• PCR


  Detects the presence or absence of a gene, generates forensic DNA profiles, and creates sequencing templates
• DNA Sequencing Reaction


  Used for medical, criminal, and research purposes
• Analysis of DNA Sequence Data

  1. DNA sequence data is converted to characters and characters states to be used in phylogenetic analyses

  2. The sequences of a given length of DNA are aligned
• Analysis of DNA Sequence Data

  A character is equivalent to the nucleotide position, and a character state of that character is the specific nucleotide at that position
• Restriction Site Analysis


  • A restriction is a sequence of approximately 6 to 8 base pairs of DNA that binds to a given restriction enzymes
  • Restriction enzymes have been isolated from bacteria
  • Inactivate invading viruses by cleaving the viral DNA
• Restriction Fragment Length Polymorphism (RFLP)


  Differences between taxa in restriction sites, and therefore the lengths of fragments of DNA following cleavage with restriction enzymes
• Allozymes


  • Different molecular forms of an enzyme that correspond to different alleles of a common gene
  • Genetic markers used in phylogenetic relationships
• Allozymes


  1. Enzymes are extracted and placed on a medium through which electric current runs

  2. Enzymes will migrate toward one pole or the other depending on its charge

  3. Different allozymes of an enzyme will migrate differentially because they differ slightly in amino acid composition and therefore have somewhat different electrical charges

  4. Allozymes subjected to electrophoresis are identified with a stain specific to that enzyme and the bands marked by their relative position on the electrophoresis medium
• Allozymes


  Used to assess genetic variation within a population or species, but they can also used as data in phylogenetic analyses of closely related species within monophyletic genus
• Microsatellite DNA


  • Regions of DNA that contain short repeats of nucleotides
  • Short tandem repeats
• Microsatellite DNA


  Primers that flank the tandem repeats are constructed and then PCR is used to quickly generate multiple copies of the tandem repeat DNA, the length of which (for a given individual at a given locus or allele) can be determined by gel electrophoresis
• Allozymes


  Detect low levels of genetic variation
• Microsatellite


  Detect higher levels of genetic variation
• Allozyme electrophoresis


  Simple, versatile, and inexpensive technique for detecting genetic variation within and between populations
• Microsatellites


  Used for fine-scale genetic population studies, such as assessing genetic structure at the intraspecific level
• Random Amplified Polymorphic DNA (RAPDs)


  DNA typing method that uses polymerase chain reaction (PCR) to amplify random segments of genomic DNA
• Random Amplified Polymorphic DNA (RAPDs)


  Identical primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence
• Amplified Fragment Length Polymorphism (AFLPs)

  • A PCR-based technique that generates and compares unique DNA fingerprints for genomes
  • Useful when little is known about an organism's genome or genetics
• RAPD


  Amplifies genomic DNA with short oligonucleotide primers of a random sequence
• AFLP


  Selectively amplifies restriction fragments from a total restriction digest of genomic DNA
• RAPD


  • Analyzes genetic diversity of an individual
  • Can only study dominant genetic markers
  • Less reliable (simple, less reproducible)
  • Faster than AFLP
• AFLP


  • Can study both dominant and co-dominant genetic markers
  • More reliable (more reproducible)
  • Slower than RAPD