10 DNA Technology

• Gene Annotation Levels are interpreting DNA/nucleic acid sequences; process of identifying functional elements along the sequence of a genome
• Gene annotation levels are necessary because the sequencing of DNA produces sequences of unknown functions.

  • After the genome is sequenced, it must be annotated to bring more logical information about its structural features and functional roles.
• Nucleotide-level annotation is the most successful method for complex genomes combine into gene prediction with sequence comparison with other organisms and expressed sequence databases.

  • A key part of this annotation is gene discovery.
• The primary goal of Protein-level annotation is to attribute function to the genome products.

  • For this form o annotation, databases of protein sequences, functional domains, and motifs are useful
• Process-level annotation is to understand the function of genes and their products in the context of cellular and organismal physiology.

  • The inconsistency of words employed by different model systems have been one of the roadblocks at this level of annotation
• Nucleotide-tools Annotation Tools

  • Genomic browser
  • UCSC Genome Browser
  • Ensembl Genome Browse
• Nucleotide-tools Annotation Tools

  • Genome alignment and assembly tools
  • Integrative Genomics Free Viewer
  • NCBI Assembly Viewer
  • Sequence Alignment Tool
• BLAST is a computational tool that finds the region of similarity between biological sequences.

  • Rapidly align and compare a query DNA sequence with a database of sequences which makes it a critical tool in ongoing genomic research.
  • Compares nucleotides or protein sequences to sequence databases.
  • Calculates the statistical significance
• Homology is the similarity attributed to descent from a common ancestor

  • Have the same function
• Similarity is the extent to which nucleotide or protein sequences are related

  • Very high similarities (70-99%)
• Identity is the extent to which 2 (nucleotide or amino acid) sequences are invariant

  • Identity percentage (specifically 100%)
• Conservation are changes at a specific position of an amino acids (less commonly, DNA) or sequence that preserves the physio-chemical properties of the original residue
• Ortholog are homologous sequences in different species (due to speciation)
• Paralog are homologous sequences in within a single species (due to duplication)
• Xenology are homologous sequences in different species due to a horizontal gene transfer (lateral gene transfer)
• Recombinant DNA Technology involves inserting a human gene into the genetic material of a common bacterium through a vector, plasmid

  • One of the ways in which we humans use certain types of organisms to benefit our own species.
  • Recombinant organism can produce protein encoded by the human gene
  • Examples: synthetic insulin, genetic plant modification
• To test primer specificity in RDT, we conduct cross reactivity

  • Something that we do to allow testing of the primer to other sequences that are closely related to it.
  • Sometimes use to sequences of species that are closely related to the species of interest.
• Ideal primer length in RDT: 15-30 bp
• Melting temperature is the estimate of a DNA-DNA hybrid stability.
• Too high of annealing temperature in RDT will produce insufficient primer template hybridization resulting in a very low PCR product yield.
• Too low annealing temp in RDT will lead to nonspecific products caused by high number of base pair mismatches like hairpin matches, dimerization, etc.
• Mismatched tolerance is found to have the strongest influence in PCR specificity.
• GC content in RDT should be around 40-60 and composed of G and C.
• 3 guanines or cytosines in the last 5 bases at the 3’ end is avoided in RDT
• RDT Digestion process:

  • Insertion of the gene of interest in the plasmid through the digestion of vector plasmid using restriction enzyme and ligation of the gene of interest
• Digestion in RDT is based on the principle that exposure of bacterial suspension to anionic detergent at high pH opens the cell wall, denatures chromosomes and proteins, and then release the plasmid into the supernatant.
• Restriction-digest-ligation protocols is used to transfer DNA fragments from one plasmid to another.

  • The restriction enzymes will digest the DNA at the corresponding restriction sites which now results in complementary ends of the target plasmids.
• Transformation in RDT is the process that involves the uptake of the naked DNA from the surrounding medium by competent cells
• Electroporation process in RDT where electrical pulses are introduced to washed cells, creating temporary pores that allow the entry of DNA molecule